WO2023125552A1 - 对靶标核酸进行检测的方法 - Google Patents

对靶标核酸进行检测的方法 Download PDF

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WO2023125552A1
WO2023125552A1 PCT/CN2022/142406 CN2022142406W WO2023125552A1 WO 2023125552 A1 WO2023125552 A1 WO 2023125552A1 CN 2022142406 W CN2022142406 W CN 2022142406W WO 2023125552 A1 WO2023125552 A1 WO 2023125552A1
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Prior art keywords
primer
probe
signal
region
stranded
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English (en)
French (fr)
Inventor
赵雨航
胡莞尔
王海鑫
黄秋萍
葛志琪
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Maccura Biotechnology Co Ltd
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Maccura Biotechnology Co Ltd
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Priority claimed from CN202211271772.XA external-priority patent/CN115651971A/zh
Application filed by Maccura Biotechnology Co Ltd filed Critical Maccura Biotechnology Co Ltd
Priority to EP22914805.1A priority Critical patent/EP4458984A4/en
Priority to JP2024539077A priority patent/JP2025501167A/ja
Priority to US18/724,658 priority patent/US20250066857A1/en
Priority to CN202280007182.8A priority patent/CN117043334A/zh
Publication of WO2023125552A1 publication Critical patent/WO2023125552A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to the field of nucleic acid sequence detection, in particular to a method for detecting target nucleic acids, more specifically, the present invention relates to a method for detecting single or multiple target nucleic acids using a universal probe on digital PCR.
  • PCR polymerase chain reaction
  • PCR is a molecular biology technique for the enzymatic replication of DNA without the use of living organisms.
  • PCR is commonly used in medical and biological research laboratories to undertake a variety of tasks, such as diagnosis of infectious diseases, gene cloning, phenotyping of experimental animals, transcriptome research, detection of genetic diseases, identification of genetic fingerprints, paternity testing, etc. Due to its unparalleled ability to replicate and be precise, PCR is considered by molecular biologists to be the method of choice for nucleic acid detection.
  • the real-time fluorescent quantitative PCR Real Time Quantitative PCR, qPCR
  • qPCR Real Time Quantitative PCR
  • Digital PCR digital PCR, dPCR
  • Digital PCR digital PCR, dPCR
  • digital PCR collects the fluorescence signal of each reaction unit independently after the amplification, and finally uses the principle of Poisson distribution and positive/negative reactions The ratio of cells yields the native copy number or concentration of the target molecule.
  • digital PCR can perform accurate absolute quantitative detection without relying on Ct values and standard curves, and has the advantages of high sensitivity and accuracy. Since digital PCR only judges the two amplification states of "presence/absence" when interpreting results, there is no need to detect the intersection point of the fluorescent signal and the set threshold line, and it does not depend on the identification of the Ct value at all, so the digital PCR reaction and Results Interpretation is greatly reduced by the influence of amplification efficiency, and the tolerance to PCR reaction inhibitors is greatly improved. In addition, the process of distributing the reaction system in digital PCR experiments can greatly reduce the concentration of background sequences that compete with target sequences locally. Therefore, digital PCR is particularly suitable for detecting rare mutations in complex backgrounds. Currently, it is mostly used in In liquid biopsy, the detection of rare mutation markers in the peripheral blood of tumor patients is realized.
  • the nucleic acid containing the target sequence to be tested is first amplified, and then the signals are collected at different temperatures, and the signals obtained at different temperatures may overlap (that is, different The target sequence generates signals at the same time), the traditional calculation method is to separate the overlapping signals through image processing or other signal calculation methods, and then calculate the signal value generated by each target sequence separately. This method is complex and prone to calculation errors.
  • the present invention provides a method for detecting target nucleic acids, which can detect one target or more than two target nucleic acids.
  • the method first amplifies the nucleic acid containing the target sequence to be detected, and then performs For signal acquisition, by setting the annealing temperature during amplification and the acquisition temperature during signal acquisition, a single signal can be obtained at different temperatures (only one target sequence can generate a signal at most in a single signal acquisition).
  • the problem to be solved by the present invention also includes how to detect and distinguish multiple different targets in a single-tube reaction, and avoid false positive signals due to primer dimers in the presence of multiple sets of primers .
  • the present invention provides a method for detecting a target nucleic acid, comprising the steps of:
  • the amplification reagent includes DNA polymerase and dNTPs
  • the annealing temperature used in the amplification is T1;
  • Operation 1 including:
  • the annealing temperature used in the amplification is T2;
  • Operation 2 including:
  • Operation 3 including:
  • the type of the target to be detected in the sample to be tested is one;
  • At most one signal collection contains the signal of the target.
  • one signal acquisition is performed after the first PCR amplification or after the first and second PCR amplifications, and the signal acquisition temperature increases sequentially (t2>t1).
  • performing 2 consecutive PCRs, or only performing 1 PCR can generate a double-stranded product that binds to the probe.
  • 2 PCRs and 2 signal acquisitions are performed, and the PCR and signal acquisition are performed alternately: the annealing temperature used in PCR amplification is sequentially decreased (T2 ⁇ T1), but the signal acquisition temperature is sequentially increased (t2 >t1).
  • the annealing temperature of the single-stranded pre-product formed by the target nucleic acid is lower than this temperature, and cannot combine with the probe to form a double-stranded product, and cannot cause a detection signal Change; after this PCR amplification, the signal is collected at another temperature, the single-stranded pre-product formed by the target nucleic acid and the double-stranded product of the probe are in a double-stranded state, and a detectable target nucleic acid signal is generated.
  • the difference in signal between the two acquisitions more accurately demonstrates the presence of the target nucleic acid.
  • the presence or absence of the target nucleic acid can be more accurately proved by using the above-mentioned PCR detection method: when the target nucleic acid exists, the signals of the two collections are different, and only one signal collection contains the signal of at most one target nucleic acid; If the target nucleic acid is not present, there is no signal for any target nucleic acid in both signal acquisitions.
  • T2 ⁇ T1 means that the annealing temperature of the second PCR is different from the annealing temperature of the first PCR.
  • the number of types of stranded products, compared with the number of types of double-stranded products formed with the probe after the first PCR amplification, can be increased by at most 1 type (no increase means that there is no corresponding target nucleic acid in the product to be tested) .
  • the temperature difference between T2 and T1 is more than 4°C, for example, a difference of 4-40°C, and for example, a difference of 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4- 12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • t2>t1 refers to the temperature of the second signal collection, which is different from the temperature of the first signal collection.
  • the temperature difference between the two makes the signal of the second signal collection different from the signal of the first signal collection.
  • the temperature difference between t2 and t1 is more than 6°C; for example, the difference is 6-46°C, and for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C °C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C, etc.
  • T2 ⁇ t1 means that the annealing temperature of the second PCR is different from the temperature of the first signal collection.
  • the temperature difference between T2 and t1 is more than 5°C, for example, the difference is 5-50°C, and for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5-18°C, 5- 15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C or 5-6°C, etc.
  • t2>T1 refers to the annealing temperature of the first PCR, which is different from the temperature of the second signal acquisition.
  • the temperature difference between t2 and T1 is more than 7°C, for example, the difference is 7-50°C, and for example, the difference is 7-40°C, 7-30°C, 7-20°C, 7-18°C, 7- 15°C, 7-12°C, 7-10°C or 7-8°C, etc.
  • the annealing temperature used in the first PCR amplification is T1
  • the collection temperature used in the first signal collection is t1; the T1 ⁇ t1 .
  • the annealing temperature is T1
  • one single-stranded pre-product specifically binds to the probe and extends.
  • the annealing temperature of the single-stranded pre-product is slightly lower than its melting temperature (usually, the annealing temperature of an oligonucleotide is usually lower than the melting temperature (Tm) of the oligonucleotide).
  • Signal collection is carried out at t1 (T1 ⁇ t1 ⁇ its melting temperature), at this time, the single-stranded pre-product and the probe are in a double-stranded state, causing the probe to produce a detectable signal change.
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe produces a detectable signal change.
  • t1 T1 ⁇ t1 ⁇ its melting temperature
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe produces a detectable signal change.
  • t1 T1 ⁇ t1 ⁇ its melting temperature
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe
  • the annealing temperature ranges from 40°C to 75°C, that is, 40°C ⁇ T2 ⁇ T1 ⁇ 75°C.
  • the signal collection temperature is 0-95 ° C, that is, 0 ° C ⁇ t1 ⁇ t2 ⁇ 90 ° C; usually, the selection of signal collection temperature can be made by those skilled in the art according to the actual conditions of the designed primer probe composition. The situation is determined. For example, through the design of the primer-probe composition, those skilled in the art can expect that the melting temperature of the reaction product representing the target nucleic acid is T. If the signal of the target nucleic acid is to be detected, the signal acquisition temperature is ⁇ T; If the signal of the target nucleic acid is detected, the signal collection temperature is >T.
  • the reaction conditions for the first PCR amplification described in step S200 include: pre-denaturation at about 85°C-about 105°C for about 0-about 15 minutes; denaturation at about 85°C-about 105°C for about 1 - About 60 seconds, about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles; preferably, when the target in the sample to be tested is RNA, the amplification reagent also includes reverse transcription Enzymes, the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 85°C-about 105°C pre-denaturation for about 0-about 15 minutes; About 85°C-about 105°C denaturation for about 1-about 60 seconds, about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles;
  • the second PCR amplification described in step S300 includes the following reaction conditions: about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 20°C-about 75°C annealing and extension for about 3-about 90 seconds, 1-20 cycles; or, about 20°C-about 75°C constant temperature incubation for about 10-about 1800 seconds.
  • the reaction conditions for the first PCR amplification described in step S200 include: pre-denaturation at about 90°C to about 96°C for about 5 minutes to about 15 minutes; denaturation at about 90°C to about 95°C for about 10 minutes - About 60 seconds, about 50°C-about 75°C annealing and extension about 30-about 90 seconds, 35-50 cycles; further preferably, when the target in the sample to be tested is RNA, the amplification reagent also includes reverse Recording enzyme, the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 90°C-about 96°C pre-denaturation for about 5-about 15 minutes ; About 90°C-about 95°C denaturation for about 10-about 60 seconds, about 50°C-about 75°C annealing and extension for about 30-about 90 seconds, 35-50 cycles;
  • the second PCR amplification described in step S300 includes the following reaction conditions: about 85°C-about 105°C denaturation for about 2-about 60 seconds, about 20°C-about 75°C annealing and extension for about 10-about 90 seconds, 1-20 cycles; or, about 20°C-about 75°C constant temperature incubation for about 10-about 1800 seconds.
  • the reaction system before performing the first PCR amplification on the reaction system, it also includes: distributing the reaction system to more than 500 reaction units, each reaction unit containing 1 sample to be tested The target nucleic acid of the target nucleic acid or the target nucleic acid that does not contain the sample to be tested; further preferably, the signal collection is to collect the fluorescent signal through the camera; the signal channel collection is to collect the fluorescent signal through the camera under the fluorescent signal channel.
  • the primer-probe composition includes: a probe (P) and a primer; after the primer specifically binds to the target in the sample to be tested, a pre-product is generated, and the pre-product contains A single-stranded pre-product that can specifically bind to the probe, the single-stranded pre-product specifically binds to the probe and extends ⁇ 0 bases to form a double-stranded product, the formation of the double-stranded product causes a change in the signal of the probe ;
  • the extension is an extension of 0-100 bases, even more preferably, the extension is an extension of 1-100 bases, and the upper limit of the number of bases extended is the entire length of the probe; and
  • the concentration of the primer is about 30 nM to about 1000 nM
  • the concentration of the probe (P) is about 100 nM to about 1200 nM.
  • the concentration unit nM refers to nmol/L.
  • the extension of 0 bases means that no extension occurs after the single-stranded pre-product specifically binds (hybridizes) with the probe (P).
  • the primer sequences and/or probe sequences in the primer set can realize the above-mentioned effect of "specific binding without extension” by designing the primer sequences and/or probe sequences in the primer set according to actual needs, for example, designing the first single Strand preproducts are bound at the 5' end of the probe, or, alternatively, the 3' end of the first single-stranded preproduct is designed to contain a region that does not pair complementary to the probe.
  • the probe (P) is a freely designed sequence that does not pair with any target sequence, including a probe signal detection region (H), and the probe is modified with a detection label; the primer Including forward primer (F) and/or reverse primer (R);
  • the forward primer (F) comprises a target sequence binding region specifically paired with the target sequence
  • the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region, or the reverse primer (R) is formed from The 5' end to the 3' end sequentially include an extension block region (M), a primer signal detection region (h) and a target sequence binding region;
  • the target sequence binding region of the reverse primer (R) is a sequence that specifically pairs and binds to the target sequence;
  • the primer signal detection region (h) of the reverse primer (R) is a segment that does not bind to any target sequence A paired binding sequence, the complementary sequence (h') of the primer signal detection region (h) is specifically paired with the probe signal detection region (H) of the probe (P);
  • the extension blocking region (M) is a freely designed sequence that is not identical or complementary to the sequence of any part of the probe (P) or any target sequence;
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • “specific pairing binding” or “specific binding” refers to the complementary base pairing binding of two single-stranded nucleic acid molecules with complementary sequences under certain conditions (suitable temperature and ionic strength, etc.). It is well known to those skilled in the art that two single-stranded nucleic acid molecules can be specifically bound only when they are not completely complementary to each other, and can also be specifically bound when there are a few base mismatches.
  • the forward primer (F) further includes a probe anchor region (A), and the forward primer (F) sequentially includes a probe anchor region from the 5' end to the 3' end (A) and the target sequence binding region; wherein, the probe anchor region (A) is a sequence that does not pair with any target sequence; further preferably, the probe anchor region (A) and the target sequence There is an interval of 0-20 bases between the binding regions; wherein, the number of bases in the interval can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20 or any combination thereof;
  • the probe (P) also includes a primer anchor region (A'), and the primer anchor region (A') is a sequence complementary to the probe anchor region (A) of the forward primer (F) ; Further preferably, there is an interval of 0-20 bases between the primer anchoring region (A') in the probe (P) and the probe signal detection region (H).
  • the structures of the forward primer and the reverse primer can be interchanged; for example, “the reverse primer (R) includes a primer signal detection region (h) and a target sequence binding region, and the Primer (F) contains the target sequence binding region”, the primer structure can be interchanged as “forward primer (F) contains the primer signal detection region (h) and the target sequence binding region, and the reverse primer (R) contains the target sequence binding region” or, “the reverse primer (R) includes a primer signal detection region (h) and a target sequence binding region, and the forward primer (F) includes a probe anchor region (A) and a target sequence binding region” primer structures can be interchanged into “the forward primer (F) includes a primer signal detection region (h) and a target sequence binding region, and the reverse primer (R) includes a probe anchor region (A) and a target sequence binding region”.
  • the target sequence binding region in the forward primer (F) is different from the target sequence binding region in the reverse primer (R), and cannot specifically bind.
  • At least one of the forward primers (F) includes a first forward primer and a second forward primer, and the first forward primer extends from the 5' end to The 3' end comprises a forward amplification product capture region (B) and a target sequence binding region in sequence; and, the second forward primer comprises a forward amplification product capture region (B), and the forward amplification product captures Region (B) is not paired with the target sequence or any fragment thereof; further preferably, there is a gap between the forward amplification product capture region (B) in the first forward primer and the target sequence binding region 0-20 base intervals.
  • the second forward primer also includes a probe anchor region (A), further preferably, the probe anchor region (A) and the probe anchor region (A) in the second forward primer There is an interval of 0-20 bases between the capture regions (B) of the forward amplification products.
  • At least one of the reverse primers (R) comprises a first reverse primer and a second reverse primer
  • the first reverse primer extends from the 5' end to The 3' end includes a reverse amplification product capture region (C) and a target sequence binding region in turn
  • the second reverse primer includes a primer signal detection region (h) and a reverse primer sequence from the 5' end to the 3' end an amplified product capture region (C), the reverse amplified product capture region (C) is not paired with the target sequence or any fragment thereof;
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the above-mentioned method for detecting target nucleic acid is a digital PCR detection method; in some embodiments of the present invention, for the above-mentioned method for detecting target nucleic acid, the reaction system is subjected to the first Before the first PCR amplification, it also includes: distributing the reaction system to more than 500 reaction units, each reaction unit containing a target nucleic acid of the sample to be tested or not containing the target nucleic acid of the sample to be tested; preferably, The signal collection is to collect the fluorescence signal through the camera; the signal channel collection is to collect the fluorescence signal through the camera under the fluorescence signal channel.
  • the primer probe composition can be a conventional primer probe composition in the art, for example, a primer probe composition containing a molecular beacon, or it can be provided by the following third aspect A primer probe composition for nucleic acid sequence detection.
  • the present invention provides a method for detecting more than two target nucleic acids, comprising the following steps:
  • the primer probe composition includes at least 1 probe (P), and at least 2 primers for different targets;
  • the amplification Reagents include DNA polymerase and dNTPs;
  • the annealing temperature adopted during the nth PCR amplification is Tn
  • the acquisition temperature adopted during the nth signal acquisition is tn, Tn ⁇ tn; Tn ⁇ T(n-1), and Tn ⁇ t(n -1); tn>t(n-1), and tn>T(n-1);
  • the n is an integer ⁇ 2, and the n ⁇ the number of target species to be detected in the sample to be tested.
  • a signal collection is performed after each PCR amplification, and the annealing temperature used in the PCR amplification is sequentially decreased (Tn ⁇ T(n-1)), but the signal collection temperature is sequentially increased (tn >t(n-1)). That is, when a certain temperature is used as the annealing temperature of PCR for PCR cycle amplification, the single-stranded pre-products whose annealing temperature is higher than this temperature can all combine with the probe to form double-stranded products and cause changes in the detection signal.
  • the low-temperature single-stranded pre-product is still in a single-stranded state; after this PCR amplification, the signal is collected at another temperature, and only one single-stranded pre-product and the double-stranded product of the probe are double-stranded state and produce a detectable target nucleic acid signal.
  • a part cannot anneal with the probe to form a double-stranded product in the PCR cycle amplification stage due to the lower annealing temperature; another part can be combined with the probe in the PCR cycle amplification stage due to the high annealing temperature.
  • Needle annealing forms double-stranded products, but because the melting temperature of these double-stranded products is lower, lower than the temperature of signal collection, the generated double-stranded products are also in a single-stranded state at the signal collection temperature.) Therefore, the above-mentioned PCR
  • the detection method can realize that the signal collected in each signal channel is only the signal of one target nucleic acid at most, and if the target nucleic acid does not exist, there will be no signal of any target nucleic acid.
  • Tn ⁇ T(n-1) refers to the annealing temperature of the nth PCR, which is different from the annealing temperature of the n-1th PCR.
  • the number of types of double-stranded products formed by a certain probe increases by at most 1 (no increase, refers to the number of types to be There is no corresponding target nucleic acid in the test product).
  • the temperature difference between Tn and T(n-1) is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4- 15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • tn>t(n-1) refers to the temperature of the nth signal collection, which is different from the temperature of the n-1th signal collection.
  • the temperature difference between the two makes the double-stranded product of the nth signal collection , which is different from the double-stranded product at the n-1th signal acquisition.
  • the double-stranded product during the n-1 signal acquisition is A
  • the double-stranded product during the n-th signal acquisition is B (during the n-time signal acquisition, since the melting temperature of the double-stranded product A is lower than that of the first
  • the temperature at the time of signal acquisition for n times, at this time, the double-stranded product A is in a single-chain state).
  • the temperature difference between tn and t(n-1) is more than 6°C; for example, the difference is 6-46°C, and for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C °C, 6-20°C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C, etc.
  • Tn ⁇ tn refers to the annealing temperature of the nth PCR, which is different from the temperature of the nth signal collection. The difference between the two temperatures makes it possible to obtain a specific double-stranded product after the nth PCR amplification.
  • the signal of the double-stranded product can be obtained when the signal is collected n times. In order to obtain the signal of the double-stranded product, the double-stranded product does not appear in a single-stranded state when the n-time signal is collected (under the condition of the signal collection temperature tn).
  • the difference between Tn and tn is more than 1°C, for example, the difference is 1-50°C, 1-40°C, 1-30°C, 1-25°C, 2-20°C, 2-15°C, 2-12°C °C, 2-10°C, 2-7°C, 2-6°C, 2-5°C, 3-5°C or 4-5°C, etc.
  • the signal collection temperature tn used is less than or equal to the melting temperature of the double-stranded product.
  • Tn ⁇ t(n-1) refers to the annealing temperature of the nth PCR, which is different from the temperature of the n-1th signal acquisition. The difference between the two temperatures makes the double temperature of the n-1th signal acquisition In the n-th PCR, the double-stranded product still exists, and at the same time, another double-stranded product different from the double-stranded product is produced.
  • the temperature difference between Tn and t(n-1) is more than 5°C, for example, the difference is 5-50°C, and for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5- 18°C, 5-15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C or 5-6°C, etc.
  • tn>T(n-1) refers to the annealing temperature of the n-1th PCR, which is different from the temperature of the nth signal collection.
  • the temperature difference between tn and T(n-1) is more than 7°C, for example, the difference is 7-50°C, and for example, the difference is 7-40°C, 7-30°C, 7-20°C, 7- 18°C, 7-15°C, 7-12°C, 7-10°C or 7-8°C, etc.
  • the number of types of probes is classified according to the number of types of markers they detect. Different types of probes have different modified detection labels (in some embodiments, the detection label is the detection group), which can realize signal collection under different signal channels (or fluorescent channels).
  • the primer-probe composition of the present invention contains two kinds of probes, and the detection group modified by one kind of probe is VIC and BHQ1, and the double-stranded product formed by specific binding with this kind of probe carries out signal in the VIC channel. Acquisition; the detection group modified by another probe is FAM and BHQ1, and the double-stranded product formed by specific binding to this probe is used for signal acquisition on the FAM channel.
  • a signal collection is performed after each PCR amplification, and each signal collection includes m signal channel collections, m is an integer ⁇ 1, and m is the detection base of the probe in the primer-probe composition.
  • the number of types of groups As mentioned above, after each PCR amplification, compared with the previous PCR amplification, the number of types of double-stranded products formed with the same probe increases by at most 1 type.
  • the number of types of probes in the primer-probe composition is m, then after each PCR amplification, compared with the previous PCR amplification, the number of types of double-stranded products formed with m types of probes increases by at most m types (The number of types of double-stranded products formed with each type of probe increases by up to 1 type).
  • the target nucleic acid signals corresponding to the increased m types of double-stranded products can be distinguished without crossing each other, thereby realizing multiple detection.
  • the at least one signal collection includes m signal channel collections, where m is the number of detection labels of probes in the primer-probe composition, and the product of n and m ⁇ The number of target species to be tested in the sample to be tested.
  • m is the number of detection labels of probes in the primer-probe composition
  • n and m The number of target species to be tested in the sample to be tested.
  • each signal acquisition is performed under 2 signal channels for 2 signal channel acquisitions.
  • there are 5 kinds of targets in the sample to be tested and the primer probe composition for the 5 kinds of targets contains 2 kinds of probes, the first probe detects 3 kinds of targets, and the second probe detects 3 kinds of targets.
  • each signal acquisition is 2 signal channel acquisitions under 2 signal channels. In this way, there are three signal acquisitions under a certain signal channel, but there are only two corresponding targets, and the signal acquired by a certain signal channel has no target, or the signals acquired by two adjacent signal channels are the same. .
  • the number of signal acquisitions is set based on the detection channel with the largest number of detection targets, so that the number of signal acquisitions is less than the number of target nucleic acid types, reducing the number of signal acquisitions.
  • the least number of signal acquisitions of other signal channels is not fully satisfied, the setting of the instrument program is more convenient, and those skilled in the art can choose according to actual needs.
  • those skilled in the art choose not to collect signals in channels without expected targets, thereby reducing the number of signal collections and simplifying the operation steps.
  • the annealing temperature used in the first PCR amplification is T1
  • the collection temperature used in the first signal collection is t1; the T1 ⁇ t1 .
  • the annealing temperature is T1
  • one single-stranded pre-product specifically binds to the probe and extends.
  • the annealing temperature of the single-stranded pre-product is slightly lower than its melting temperature (usually, the annealing temperature of an oligonucleotide is usually lower than the melting temperature (Tm) of the oligonucleotide).
  • Signal collection is carried out at t1 (T1 ⁇ t1 ⁇ its melting temperature), at this time, the single-stranded pre-product and the probe are in a double-stranded state, causing the probe to produce a detectable signal change.
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe produces a detectable signal change.
  • t1 T1 ⁇ t1 ⁇ its melting temperature
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe produces a detectable signal change.
  • t1 T1 ⁇ t1 ⁇ its melting temperature
  • signal collection can also be performed at t1 (t1 ⁇ T1), at this time, the single-stranded preproduct and the probe are also in a double-stranded state, so that the probe
  • the annealing temperature is: 40°C ⁇ Tn ⁇ T(n ⁇ 1) ⁇ 75°C (n is an integer ⁇ 2).
  • the collection temperature is: 0°C ⁇ t(n ⁇ 1) ⁇ tn ⁇ 90°C (n is an integer ⁇ 2).
  • the reaction conditions for the first PCR amplification of the reaction system include: about 85°C-about 105°C pre-denaturation for about 0-about 15 minutes; about 85°C-about 105°C denaturation for about 1-about 60 seconds, annealing at about 40°C-about 75°C and extension for about 3-about 90 seconds, 20-60 cycles; preferably, when the target in the sample to be tested is RNA, the amplification reagent also includes reverse Recording enzyme, the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 85°C-about 105°C pre-denaturation for about 0-about 15 minutes ; About 85°C-about 105°C denaturation for about 1-about 60 seconds, about 40°C-about 75°C annealing and extension for about 3-about 90 seconds, 20-60 cycles. .
  • the reaction conditions for the first PCR amplification of the reaction system include: about 90°C-about 96°C pre-denaturation for about 5-about 15 minutes; about 90°C-about 95°C denaturation for about 10-about 60 seconds, annealing at about 50°C-about 75°C and extension for about 30-about 90 seconds, 35-50 cycles; preferably, when the target in the sample to be tested is RNA, the amplification reagent also includes reverse Recording enzyme, the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 90°C-about 96°C pre-denaturation for about 5-about 15 minutes ; About 90°C-about 95°C denaturation for about 10-about 60 seconds, about 50°C-about 75°C annealing and extension for about 30-about 90 seconds, 35-50 cycles.
  • the reaction conditions for the first PCR amplification of the reaction system include: about 85°C-about 105°C pre-denaturation for about 0-about 15 minutes; about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 40°C-about 75°C annealing and extension about 3-about 90 seconds, about 20-about 60 cycles; about 85°C-about 105°C denaturation about 1-about 60 seconds, about 20°C- Annealing and extending at about 75°C for about 3-90 seconds, 1-20 cycles; alternatively, the reaction conditions for the first PCR amplification of the reaction system include: about 85°C-about 105°C pre-denaturation about 0- About 15 minutes; about 85°C-about 105°C denaturation about 1-about 60 seconds, about 40°C-about 75°C annealing and extension about 3-about 90 seconds, 20-60 cycles; about 20°C-about 75°C constant temperature Incubate for about 10 to about 1800 seconds;
  • the amplification reagent also includes reverse transcriptase
  • the reaction conditions for performing the first PCR amplification on the reaction system include: reverse transcription at about 30°C to about 65°C 2-about 30 minutes; about 85°C-about 105°C pre-denaturation about 0-about 15 minutes; about 85°C-about 105°C denaturation about 1-about 60 seconds, about 40°C-about 75°C annealing and extension about 3- about 90 seconds, 20-60 cycles; about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 20°C-about 75°C annealing and extension for about 3-about 90 seconds, 1-20 cycles; or,
  • the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 85°C-about 105°C pre-denaturation for about 0-about 15 minutes; about 85°C °C-about 105°C denaturation for about
  • the reaction conditions for the first PCR amplification of the reaction system include: about 90°C-about 96°C pre-denaturation for about 5-about 15 minutes; about 90°C-about 95°C denaturation for about 10-about 60 seconds, about 50°C-about 75°C annealing and extension for about 30-about 90 seconds, about 35-about 50 cycles; about 90°C-about 95°C denaturation about 10-about 60 seconds, about 20°C- Annealing and extension at about 75°C for about 30-90 seconds, 1-20 cycles; alternatively, the reaction conditions for the first PCR amplification of the reaction system include: about 90°C-about 96°C pre-denaturation for about 5- About 15 minutes; about 90°C-about 95°C denaturation for about 10-about 60 seconds, about 50°C-about 75°C annealing and extension for about 30-about 90 seconds, 35-50 cycles; about 20°C-about 75°C constant temperature Incubate for about 10 to about 1000 seconds;
  • the amplification reagent also includes reverse transcriptase
  • the reaction conditions for performing the first PCR amplification on the reaction system include: reverse transcription at about 30°C to about 65°C 2-about 30 minutes; about 90°C-about 96°C pre-denaturation about 5-about 15 minutes; about 90°C-about 95°C denaturation about 10-about 60 seconds, about 50°C-about 75°C annealing and extension about 30- about 90 seconds, 35-50 cycles; about 90°C-about 95°C denaturation for about 10-about 60 seconds, about 20°C-about 75°C annealing and extension for about 30-about 90 seconds, 1-20 cycles; or,
  • the reaction conditions for the first PCR amplification of the reaction system include: about 30-about 65°C reverse transcription for about 2-about 30 minutes; about 90°C-about 96°C pre-denaturation for about 5-about 15 minutes; about 90°C °C-about 95°C denaturation for about 10-about 60 seconds,
  • other PCR amplifications after the first PCR amplification include the following reaction conditions: about 85°C-about 105°C denaturation for about 1-about 60 seconds, about 20°C-about 75°C Annealing and extending for about 3-about 90 seconds, 1-20 cycles; or, about 20°C-about 75°C constant temperature incubation for 10-1800 seconds.
  • other PCR amplifications after the first PCR amplification include the following reaction conditions: about 90°C-about 95°C denaturation for about 10-about 60 seconds, about 20°C-about 75°C Annealing and extending for about 30-about 90 seconds, 1-20 cycles; or, about 20°C-about 75°C constant temperature incubation for 10-1000 seconds.
  • the primers specifically binds to the target to produce a pre-product
  • the pre-product contains a single-stranded pre-product that can specifically bind to the probe (P).
  • the pre-product is specifically combined with the probe (P) and extended to form a double-stranded product, and the formation of the double-stranded product causes a change in the signal of the probe; preferably, the extension is an extension of 0-100 bases, more preferably Extending 1-100 bases, the upper limit of the number of bases extended is the entire length of the probe; and/or, the concentration of the primer is about 30nM-about 1000nM, and the concentration of the probe (P) is about 100nM - about 1200nM. If multiple targets share one probe, the amount of the probe will be more, for example, 1300nM, 1400nM, 1500nM, 1600nM, 1700nM, 1800nM, 1900nM, 2000nM or more.
  • the extension of 0 bases means that no extension occurs after the single-stranded pre-product specifically binds (hybridizes) with the probe (P).
  • the primer sequences and/or probe sequences in the primer set can realize the above-mentioned effect of "specific binding without extension” by designing the primer sequences and/or probe sequences in the primer set according to actual needs, for example, designing the first single Strand preproducts are bound at the 5' end of the probe, or, alternatively, the 3' end of the first single-stranded preproduct is designed to contain a region that does not pair complementary to the probe.
  • the at least two primers for different targets include any of the following:
  • the different forward primers (F) each independently comprise target sequence binding regions that specifically pair and bind to different target sequences;
  • the probe (P) is a freely designed sequence that does not pair with any target sequence, including a probe signal detection region (H), and the probe is modified with a detection label;
  • the reverse primer (R) comprises a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region; wherein,
  • the target sequence binding regions of the different reverse primers (R) are sequences that specifically pair and bind to different target sequences; the primer signal detection regions (h) of the different reverse primers (R) are all a segment that does not Any target sequence that binds in a pair, but has a part or all of the same sequence as the probe signal detection region (H) of the probe (P); preferably, the sequence of the primer signal detection region (h) of a different reverse primer (R) different from each other.
  • the primer signal detection region (h) of the reverse primer (R) has part or all of the same sequence as the probe signal detection region (H) of the probe (P), which means that the reverse primer (R The complementary sequence of the primer signal detection region (h) of ) is specifically paired with the probe signal detection region (H) of the probe (P).
  • the ratio of the forward primer and the reverse primer is not limited.
  • the content of the primer not containing the signal detection region h of the primer is more than that of other primers containing the signal detection region h of the primer, preferably the content of the primer not containing the signal detection region h of the primer is greater than that of the other primers containing the signal detection region h 1.2-20 times of the content of the primer, more preferably the content of the primer without the primer signal detection region h is 2 times, 2.5 times, 3 times, 4 times, 5 times of the content of other primers containing the primer signal detection region h , 6 times, 7 times, 8 times, 9 times or 10 times.
  • At least one of the reverse primers (R) sequentially includes an extension block region (M) and a primer signal detection region (h) from the 5' end to the 3' end and the target sequence binding region;
  • the extension blocking region (M) is a freely designed sequence that is not identical or complementary to the sequence of any part of the probe (P) or any target sequence; if there are other kinds of reverse primers, the The 5' end of the other reverse primers (R) does not contain an extension block (M).
  • the other kinds of reverse primers include a primer signal detection region (h) and a target sequence binding region, and the primer signal detection region (h) is located at The 3' end of the target sequence binding region; in some embodiments, if there are other kinds of reverse primers, the other kinds of reverse primers (R) include the primer signal detection region (h ) and the target sequence binding region.
  • At least one forward primer (F) also includes a probe anchor region (A), and the probe anchor region (A) in the forward primer (F) A) Located at the 5' end of the target sequence binding region; wherein, the probe anchor region (A) is a sequence that does not pair with any target sequence; the target sequence binding region of different forward primers (F) The sequences are different; further preferably, there is an interval of 0-20 bases between the probe anchor region (A) and the target sequence binding region in the forward primer (F); wherein, the number of bases in the interval can be 0 , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any combination thereof;
  • the probe (P) is a sequence that does not pair with any target sequence, which includes a primer anchor region (A') and a probe signal detection region (H); the primer anchor region (A') is A sequence complementary to the probe anchor region (A) of the forward primer (F); further preferably, the primer anchor region (A') and the probe signal detection region in the probe (P) There are 0-20 base intervals between (H). Wherein, the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • At least one forward primer (F) sequentially includes a probe anchoring region (A) and a target sequence binding region from the 5' end to the 3' end; wherein, the The probe anchor region (A) is a sequence that does not pair with any target sequence; the sequences of the target sequence binding regions of different forward primers (F) are different; preferably, the probe anchor in the forward primer (F) There is an interval of 0-20 bases between the fixed region (A) and the target sequence binding region; wherein, the number of bases in the interval can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or any combination thereof;
  • the probe (P) is a sequence that does not pair with any target sequence, which includes a primer anchor region (A') and a probe signal detection region (H); the primer anchor region (A') is A sequence reversely complementary to the probe anchor region (A) of the forward primer (F); preferably, the primer anchor region (A') and the probe signal detection in the probe (P) Regions (H) are separated by 0-20 bases.
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • At least one of the forward primers (F) includes a first forward primer and a second forward primer, and the first forward primer extends from the 5' end to the The 3' end comprises a forward amplification product capture region (B) and a target sequence binding region in sequence; and, the second forward primer comprises a forward amplification product capture region (B), and the forward amplification product captures Region (B) is not paired with the target sequence or any fragment thereof, and further preferably, there is a gap between the forward amplification product capture region (B) in the first forward primer and the target sequence binding region 0-20 base intervals.
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the second forward primer also includes a probe anchor region (A), preferably, the probe anchor region (A) and the forward amplification product in the second forward primer There are 0-20 base intervals between capture regions (B).
  • At least one of the reverse primers (R) comprises a first reverse primer and a second reverse primer
  • the first reverse primer extends from the 5' end to The 3' end includes a reverse amplification product capture region (C) and a target sequence binding region in turn
  • the second reverse primer includes a primer signal detection region (h) and a reverse primer sequence from the 5' end to the 3' end an amplified product capture region (C), the reverse amplified product capture region (C) is not paired with the target sequence or any fragment thereof;
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the above-mentioned method for detecting more than two target nucleic acids is a digital PCR detection method; in some embodiments of the present invention, before performing the first PCR amplification on the reaction system , further comprising: distributing the reaction system to more than 500 reaction units, each reaction unit containing a target nucleic acid of a sample to be tested or not containing a target nucleic acid of a sample to be tested; preferably, the signal collection is The fluorescence signal is collected by the camera; the signal channel collection is the collection of the fluorescence signal by the camera under the fluorescence signal channel.
  • the primer probe composition can be a conventional primer probe composition in the art, for example, a primer probe composition containing a molecular beacon, or it can be provided by the following third aspect A primer probe composition for nucleic acid sequence detection.
  • the present invention provides a primer probe composition for nucleic acid sequence detection, including a first probe and a first primer mixture;
  • the first primer mixture includes at least two primer sets, and different primer sets specifically bind to different target nucleic acids;
  • the primer set in the first primer mixture specifically binds to its corresponding target nucleic acid
  • a pre-product is generated, and the pre-product contains a single-stranded pre-product that specifically binds to the first probe, and the single-stranded pre-product is combined with the
  • the first probe specifically binds and extends ⁇ 0 bases to form a double-stranded product, and the formation of the double-stranded product causes a detectable signal change;
  • Different primer sets in the first primer mixture and their corresponding target nucleic acid produce different single-stranded pre-products; when the annealing temperature of the first single-stranded pre-product and the first probe is higher than the annealing temperature of the second single-stranded pre-product and the At the annealing temperature of the first probe, the melting temperature of the double-stranded product generated by the first single-stranded preproduct and the first probe is lower than the double-stranded product generated by the second single-stranded preproduct and the first probe The melting temperature of the product.
  • the first probe refers to a type 1 probe with the same modified detection label, and its base sequence may be the same or different. That is, the number of types of probes is classified according to the number of types of markers they detect.
  • the signal generated by one probe can be collected in one detection channel (or fluorescent channel); the signal generated by two probes needs to be collected in two different detection channels.
  • the same detection labels do not mean that the modified detection groups are exactly the same.
  • the detection groups include fluorescent groups and quenching groups. As long as the fluorescent groups are the same, probes can also be The generated signal can be collected in one detection channel (or fluorescence channel).
  • At least 2 primer sets means that there are at least 2 types of primer sets, and each primer set targets different types of target nucleic acids, that is, each primer set can specifically bind to its corresponding target nucleic acid.
  • one primer set can specifically bind to one type of target nucleic acid regardless of type, then this type of target nucleic acid without type can also be considered as one type of target nucleic acid.
  • the primer set is a dual primer situation, ie, includes a pair of upstream primer and downstream primer for the same target nucleic acid.
  • the primer set is a single primer, that is, it contains only one kind of primer for a certain target nucleic acid, and the primer can specifically bind to its corresponding target nucleic acid but cannot specifically bind to other types of target nucleic acid.
  • different types of primer sets may share primers, for example, two primer sets include the same primer.
  • the same primer refers to the same sequence of the primer.
  • the annealing temperature of an oligonucleotide is usually about 5°C lower than the melting temperature (Tm) of the oligonucleotide, although it can be determined to be about 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0°C or lower, or any range therebetween, inclusive.
  • Tm melting temperature
  • the annealing temperature and melting temperature of oligonucleotides are related to factors such as the length and composition of oligonucleotides.
  • Those skilled in the art can adjust the annealing temperature and melting temperature of oligonucleotides according to actual needs, for example, increasing the GC content of oligonucleotides, making the length of oligonucleotides longer, can obtain higher melting temperature.
  • the single-stranded pre-product specifically binds to the probe and extends ⁇ 0 bases
  • the melting temperature of the double-stranded product formed by the group is higher.
  • the single-stranded pre-product obtained after the primer set specifically binds to the target nucleic acid is different.
  • the first annealing temperature between the first single-stranded pre-product and the probe
  • the annealing temperature of the probe (hereinafter referred to as “the second annealing temperature”), that is, the first annealing temperature is higher than the second annealing temperature; Adjust the length of different single-stranded pre-products combined with the probe and extend, so as to realize the melting temperature of the double-stranded product generated by the first single-stranded pre-product and the probe (herein referred to as "first melting temperature"), low
  • the melting temperature of the double-stranded product generated by the second single-stranded pre-product and the probe (hereinafter referred to as "the second melting temperature”), that is, the first melting temperature is lower than the second melting temperature.
  • the expressions "higher than” and “lower than” are used to describe that there is a difference between two temperatures, and the existence of the difference can make different kinds of single-stranded pre-products collect signals at a specific temperature. Realize different states of single-stranded or double-stranded, for example, at a certain signal temperature, the first single-stranded pre-product is in a single-stranded state, while the second single-stranded pre-product is in a double-stranded state when combined with the probe.
  • "higher than” is used to describe that the difference between two annealing temperatures is more than 4°C; for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C , 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • "lower than” is used to describe the difference between two melting temperatures of more than 6°C; for example, the difference is 6-46°C, and for example, the difference is 6-40°C, 6-35°C, 6-30°C , 6-25°C, 6-20°C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C, etc.
  • the primer-probe composition by setting high and low differences in the annealing temperature and melting temperature, it can be realized that when signal collection is performed at a certain temperature, there is at most one single-stranded pre-product and the probe in double chain state, therefore, at most one target nucleic acid signal is produced (the "most” refers to that if there is no target nucleic acid in the sample to be tested, no signal will be generated; if there is, only the target nucleic acid signal, There will be no simultaneous occurrence of other target nucleic acid signals). Therefore, it is achieved that the signals collected at the same temperature do not overlap.
  • the primer-probe composition there are 1 probe (the first probe) and 2 primer sets, and the 2 primer sets are respectively the first primer set and the second primer set; the first primer set and the second primer set are respectively combined with the first target nucleic acid and the second target nucleic acid to generate the first single-stranded pre-product and the second single-stranded pre-product respectively, and the annealing temperature of the first single-stranded pre-product and the probe is The first annealing temperature, the annealing temperature of the second single-stranded pre-product and the probe is the second annealing temperature; the double-stranded product produced by the first single-stranded pre-product and the probe is the first double-stranded product, and its melting temperature is the first Melting temperature, the double-stranded product produced by the second single-stranded pre-product and the probe is the second double-stranded product, and its melting temperature is the second melting temperature.
  • the first signal acquisition is performed at a temperature of t1 (t1 ⁇ first melting temperature), at this time, only the first double-stranded product is in a double-stranded state, and the signal generated is the signal of the first target nucleic acid; then after PCR cycles are performed at an annealing temperature of T2 (the second annealing temperature ⁇ T2), the second annealing temperature is generated Two double-stranded products, and the reaction system also contains the first double-stranded product; then the second signal acquisition is performed at a temperature of t2 (first melting temperature ⁇ t2 ⁇ second melting temperature), at this time, only the second double-stranded product The strand product is in a double-stranded state, and the signal generated is the signal of the second target nucleic acid (since t2 > the first melting temperature, the first double
  • the single-stranded pre-products whose annealing temperature is higher than this temperature can all combine with the probe to form a double-stranded Products, the single-stranded pre-products whose annealing temperature is lower than this temperature are still in the single-stranded state; when a certain temperature is used for signal acquisition, only one single-stranded pre-product and the double-stranded product of the probe are in the double-stranded state, And produce a detectable target nucleic acid signal.
  • the primer probe composition for target nucleic acid detection
  • the primer probe composition includes a first primer set for the first target nucleic acid, the first primer set and the first target nucleic acid
  • a first pre-product is generated, which contains a first single-stranded pre-product that specifically binds to the probe, and the first single-stranded pre-product specifically binds to the probe and extends 0
  • the first double-stranded product is formed after the base, and the formation of the first double-stranded product causes a detectable signal change;
  • the annealing temperature of the first single-stranded pre-product and the probe is higher than that of other single-stranded pre-products and the same
  • the annealing temperature of the probe; the melting temperature of the first single-stranded pre-product and the first double-stranded product generated by the probe is lower than the melting temperature of other single-stranded pre-products and the double-stranded product generated by the same probe.
  • the first single-stranded pre-product produced by a primer set specifically binds to the probe and does not extend.
  • first primer set a primer set for a certain target nucleic acid specifically binds to the probe and does not extend.
  • first primer set a primer set for a certain target nucleic acid specifically binds to the probe and does not extend.
  • first is only used to describe and realize the purpose of distinction, to distinguish the defined objects, and not to limit the order or primary or secondary in any way, and not to limit the target nucleic acid, primer set, pre-product, single-stranded pre-product , or the number of species of double-stranded products.
  • the above-mentioned primer probe composition for target nucleic acid detection also includes a second probe and a second primer mixture; the second probe and the The base sequences of the first probes are different, and the modified detection labels are also different; the second primer mixture includes at least one primer set, and different primer sets specifically bind to different target nucleic acids.
  • another probe is included in the primer probe composition.
  • the number of types of probes is classified according to the number of types of markers they detect. Different types of probes require different detection channels for signal acquisition due to their different detection labels. In some embodiments, different detection labels do not mean that the modified detection groups are completely different.
  • the detection groups include fluorescent groups and quenching groups, as long as the fluorescent groups are different (quenching groups can be The same or different), it is also possible to realize the signal acquisition of the signals generated by the two probes in the two detection channels (or fluorescent channels).
  • the number of types of probes increases, which can realize signal detection for different target nucleic acids under different channels, and can realize more multiple target nucleic acid detection.
  • the primer probe composition for target nucleic acid detection also includes a third probe and a third primer mixture, a fourth probe and a fourth primer mixture, a third Five probe and fifth primer mixes, sixth probe and sixth primer mixes, or more probe and primer mixes.
  • the detection labels modified by different probes are different, and the base sequences are also different; the primer sets in different primer mixtures are also different. That is to say, in order to realize more multiple target nucleic acid detection, those skilled in the art can design more primer sets corresponding to the target nucleic acid and more probes corresponding to the primer sets as required.
  • the primer group in the second primer mixture specifically binds to its corresponding target nucleic acid to produce a pre-product
  • the pre-product is Contains a single-stranded pre-product that specifically binds to the second probe, and forms a double-stranded product after the single-stranded pre-product specifically binds to the second probe and extends ⁇ 0 bases, and the formation of the double-stranded product causes Detectable signal changes;
  • different primer sets in the second primer mixture and their corresponding target nucleic acid produce different single-stranded pre-products; when the annealing temperature of the third single-stranded pre-product and the second probe is higher than that of the fourth single-stranded pre-product At the annealing temperature of the second probe, the melting temperature of the double-stranded product produced by the third single-stranded pre-product and the second probe is lower than the double-stranded product produced by the fourth single-stranded pre-product and the second probe. The melting temperature of the chain product.
  • the setting of the relationship between the above-mentioned annealing temperature and melting temperature can realize that at a certain detection temperature of a certain detection channel, the signal collected is at most the signal of one target nucleic acid (the "most” refers to that if the sample to be tested If there is no such target nucleic acid, no signal will be generated; if there is, only the signal of this target nucleic acid will be present, and no other target nucleic acid signals will appear at the same time).
  • the appearance of "the fourth single-stranded pre-product" in this embodiment is not limited to only four kinds of target nucleic acids in this embodiment. In some embodiments, more than 4 kinds of target nucleic acids are also included, and 4 or more kinds of single-stranded pre-products are produced.
  • the second primer mixture includes 3 or more primer sets, the annealing temperature and melting temperature of the single-stranded pre-products generated by these primer sets and their corresponding target nucleic acids, and the third single-stranded pre-product, the first Four single-stranded pre-products are similar, that is, the lower the annealing temperature required for the single-stranded pre-product produced by a certain primer set and its target nucleic acid to specifically bind to the probe, the single-stranded pre-product specifically binds to the probe and extends ⁇ The melting temperature of the double-stranded product formed by 0 bases is higher.
  • the single-stranded pre-products whose annealing temperature is higher than the temperature can be combined with the second probe to form double-stranded products, and the annealing temperature is higher than that of the single-stranded pre-product.
  • the low-temperature single-stranded pre-product is still in a single-stranded state; when a certain temperature is used for signal collection, only one single-stranded pre-product and the double-stranded product of the second probe are in a double-stranded state, and can generate Detected target nucleic acid signal.
  • the melting temperature of the first single-stranded pre-product and the double-stranded product generated by the first probe is equal to or close to that of the The melting temperature of the double-stranded product produced by the third single-stranded pre-product and the second probe; preferably, the annealing temperature of the first single-stranded pre-product and the first probe is equal to or close to the third single-stranded The annealing temperature of the preproduct and the second probe.
  • two or more types of probes are included in the primer-probe composition.
  • the number of types of probes is classified according to the number of types of markers they detect. Different types of probes require different detection channels for signal acquisition due to their different detection labels.
  • the expressions "equal to” and “close to” are used to describe that there is no difference between two temperatures, or the difference is small. Moreover, due to the small difference or no difference, different kinds of single-stranded pre-products can achieve the same state of single-chain or double-chain when signal collection is performed at a specific temperature. For example, at a specific temperature, both the first single-chain preproduct and the third single-chain preproduct are in a single-chain state, or the first single-chain preproduct and the third single-chain preproduct are both in a double-chain state.
  • the types of probes they bind to when they assume a double-stranded state are different.
  • “equal to” and “close to” are used to describe that the difference between two annealing temperatures/two melting temperatures is within 4°C; for example, the difference is 0-4°C, preferably, the difference is 0.02°C, more preferably ground, a difference of 001°C.
  • first”, “second”, “third” and so on are only used for descriptive purposes to distinguish the defined objects, and do not limit the order or priority in any way, nor are they used to limit the single The number of species of strand preproducts or probes.
  • the appearance of "the third single-stranded pre-product” in this embodiment is not intended to limit that there are only 3 target nucleic acids and 3 single-stranded pre-products in this embodiment.
  • the “third” in this example is only used to distinguish from “first”, that is, the "first single-stranded pre-product” and “third single-stranded pre-product” belong to different species, and the corresponding target nucleic acid species are different .
  • first probe and second probe in this embodiment is not intended to limit the existence of only two types of probes in this embodiment, and is only used to distinguish between "first probe” and
  • the “second probe” belongs to different types of probes, and the detection groups modified by the two probes are different, and the base sequences are also different.
  • This embodiment also includes the situation of 3 or more probes. In order to realize detection of more multiple target nucleic acids, those skilled in the art can design more kinds of probes as needed.
  • the above melting temperature By setting the above melting temperature to be the same or similar, it can be realized that when signal collection is performed at a certain temperature, more than two single-stranded pre-products and the probe can be in a double-stranded state. However, since at this temperature, the types of probes bound by different single-stranded pre-products in double-stranded state are different, signal acquisition can be performed at the same temperature but in different detection channels to realize the detection of different probes. The target nucleic acid corresponding to the single-stranded pre-product is identified. In this way, by adding detection channels (fluorescence channels), more multiple nucleic acid detections can be realized.
  • the signal collection temperature that is, at a certain signal collection temperature, it is only necessary to switch the detection channel to detect different targets; it can also reduce the number of PCR cycles, that is, in a certain PCR After the cycle, the number of types of double-stranded products formed with each probe increases by at most 1 compared to the previous PCR amplification (if there are 2 probes, the number of double-stranded products increases by 2).
  • the melting temperature Tm1 of the double-stranded product produced by the first single-stranded preproduct and the first probe is not equal to or not close to the melting temperature of the double-stranded product produced by the third single-stranded preproduct and the second probe Tm2, by adjusting the temperature of signal collection to make it lower than Tm1 and Tm2, then at the signal collection temperature, the two double-stranded products are still in a double-stranded state.
  • the first probe or the second probe is a sequence that does not specifically bind to any target nucleic acid, which includes a probe Signal detection region (H), the sequences of the probe signal detection regions (H) of different probes are different from each other;
  • the primer set includes a first primer and a second primer, the first primer includes a target sequence binding region 1; the second primer includes a primer signal detection region (h) and a target sequence binding region 2, and the primer signal detection region (h) is located at the 5' end of the target sequence binding region 2; the primer signal detection region (h) is a section that does not specifically bind to any target nucleic acid and has a part with the probe signal detection region (H) of the corresponding probe or all the same sequences; the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other.
  • the target sequence binding region 1 and the target sequence binding region 2 specifically bind to different positions of the target nucleic acid respectively.
  • the primer signal detection region (h) has part or all of the same sequence as the probe signal detection region (H) of the probe, which means that the reverse complementary sequence (h) of the primer signal detection region (h) '), capable of specifically binding to part or all of the sequence of the probe signal detection region (H) of the probe.
  • the above-mentioned first primer and second primer specifically combine with the target nucleic acid to generate a pre-product
  • the pre-product contains a reverse complementary sequence (h') having a primer signal detection region (h)
  • the single-stranded pre-product, the reverse complementary sequence (h') of the single-stranded pre-product is specifically combined with the probe signal detection region (H) of the probe and extended ⁇ 0 bases to form a double-stranded product, so The formation of the double-stranded product causes a detectable signal change.
  • the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other, that is, the bases of the sequences of the primer signal detection regions (h) of the second primers in different primer sets Different and/or different in length, so that the sequence of the reverse complementary sequence (h') of different single-stranded preproducts is different, and different single-stranded preproducts specifically bind to the probe and extend 0, or > 0 bases to form Different double-stranded products, the melting temperature of different double-stranded products can be separated from each other; or, different single-stranded pre-products are respectively combined with different types of probes and extended ⁇ 0 bases to form different double-stranded products , the melting temperatures of different double-stranded products cannot be separated from each other, but due to the different types of probes of different double-stranded products, they can be distinguished through different detection channels. That is, different types of target nucleic acids can be distinguished by melting temperature and/or detection channels to achieve multiple detection
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the first probe or the second probe further comprises a primer anchoring region (A'); the first primer mixture Or the first primer of at least one primer set in the second primer mixture also includes a probe anchor region (A), and the probe anchor region (A) is located at the 5' end of the target sequence binding region 1;
  • the probe anchor region (A) does not specifically bind to any target nucleic acid but specifically binds to the primer anchor region (A').
  • the first primer and the second primer in the above primer set specifically combine with the target nucleic acid to produce a pre-product, which contains a reverse complementary primer with a probe anchoring region (A) and a primer signal detection region (h).
  • different primer sets Different from the single-stranded pre-product produced by its target nucleic acid, different single-stranded pre-products have different annealing temperatures generated by specific binding to the probe, and the melting temperature of the double-stranded product formed after specific binding and extension ⁇ 0 bases
  • the melting temperature of the double-stranded product formed by the group is higher.
  • the sequences of the target sequence binding regions 1 of the first primers in different primer sets are different; the sequences of the probe anchor regions (A) of the first primers in different primer sets can be the same or different; preferably , there is an interval of 0-20 bases between the probe anchor region (A) of the first primer and the target sequence binding region 1 .
  • the number of bases at intervals can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or the extent of its composition.
  • the second primer in at most one primer set also includes an extension resistance A blockage region (M), the extension blockage region (M) is located at the 5' end of the primer signal detection region (h), and the extension blockage region (M) and its complementary sequence are not combined with any probe or any Target nucleic acid specific binding.
  • the above-mentioned first primer and the second primer containing the extension blocking region (M) respectively specifically bind to the target nucleic acid to generate a pre-product
  • the pre-product contains a reverse complementary sequence with a primer signal detection region (h) ( h')
  • the reverse complementary sequence (h') of the single-stranded pre-product specifically binds to the probe and extends 0 bases to form a double-stranded product (due to the existence of the extension block,
  • the single-stranded pre-product specifically binds to the probe without elongation), thereby causing the probe to produce a detectable signal change.
  • the resulting single-stranded pre-product does not extend after specifically binding to the probe (ie, extends 0 bases).
  • the primer sequences and/or probe sequences in the primer set can realize the above-mentioned effect of "specific binding without extension” by designing the primer sequences and/or probe sequences in the primer set according to actual needs, for example, designing the first single Strand preproducts are bound at the 5' end of the probe, or, alternatively, the 3' end of the first single-stranded preproduct is designed to contain a region that does not pair complementary to the probe.
  • First primer mix "second primer mix”, etc. are used for descriptive purposes only to distinguish defined objects. Both represent a set of primer mixtures that specifically bind to the probe, including a variety of primer sets, the second primer in these primer sets contains the reverse complementary sequence (h) of the signal detection region (h) of the primer '), all of which can specifically bind to the same probe. However, the types of probes to which the primer sets specifically bind differ between the "first primer mix” and the "second primer mix”.
  • the second primer in one primer set contains an extension block region (M), and this primer set is specific to the target nucleic acid.
  • M extension block region
  • first primer and second primer are only used for descriptive purposes to distinguish defined objects, and do not limit the order or priority in any way.
  • the structures of the first primer and the second primer of the primer set can be interchanged, for example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a target Sequence binding region; as another example, the first primer comprises a primer signal detection region (h) and a target sequence binding region, and the second primer comprises a probe anchor region (A) and a target sequence binding region.
  • the "first primer” is also called “forward primer”
  • the "second primer” is also called "reverse primer”.
  • the probe is a freely designed sequence that does not pair with any target nucleic acid, and the probe is modified with a detection label.
  • the detection label includes a first detection group and a second detection group, and the first detection group and the second detection group produce a signal change through a change in distance; preferably, the first detection group
  • the interval between the detection group and the second detection group is 3-250 angstroms; preferably, the interval is 3-201 angstroms; more preferably, the interval is 3-140 angstroms; and/or, the first detection group is A fluorescent reporter group, the second detection group is a quenching group or other modification groups capable of producing signal changes with the first detection group through fluorescence resonance energy transfer.
  • the positions of the first detection group and the second detection group on the probe make the reverse complementary sequence (h') of the single-stranded pre-product specifically bind to the probe (P) and extend ⁇ After 0 bases, a double-stranded product is formed, as long as the formation of the double-stranded product can lead to a change in the position of the first detection group and the second detection group, it can cause the probe to produce a detectable signal change.
  • the positions of the first detection group and the second detection group may be interchanged.
  • the probe when there is no target nucleic acid to be detected, no single-stranded pre-product specifically binds to the probe, and the probe is in a single-stranded state or other secondary structures.
  • the first detection group and the second detection group The distance between the groups is relatively close, and the efficiency of fluorescence resonance energy transfer is high; if the first primer contains a probe anchor region (A), when there is no target nucleic acid to be detected, even if the primer anchor region of the probe (A') is complementary to the probe anchor region (A) in the first primer, but other parts of the probe are in a single-stranded state or other secondary structures.
  • the first detection group and the second detection group The distance between them is still relatively short, and the efficiency of fluorescence resonance energy transfer is relatively high.
  • the first primer and the second primer specifically bind to the target sequence and extend to generate a double-stranded amplification product, wherein one single-stranded amplification product specifically binds to the probe to form a double-stranded product,
  • the distance between the first detection group and the second detection group becomes longer, and the efficiency of fluorescence resonance energy transfer decreases, so that the fluorescence signal changes and can be detected by the instrument.
  • the present invention provides a PCR detection kit, including the primer-probe composition for target nucleic acid detection in the third aspect.
  • the kit also includes amplification reagents for amplification, such as DNA polymerase and dNTPs.
  • the amplification reagent further includes reverse transcriptase.
  • Amplification reagents also include some reagents that promote PCR reactions, such as KCl, MgCl 2 , Tris-HCl, dithiothreitol (DTT), (NH 4 ) 2 SO 4 , etc.
  • the PCR detection kit provided by the present invention is a digital PCR detection kit, which includes the primer-probe composition for target nucleic acid detection according to the third aspect.
  • the present invention provides a multiple time-sharing nucleic acid detection device, comprising
  • the reaction liquid containing part is used to hold several micro liquids containing samples and reagents;
  • a temperature regulating part for regulating the temperature of the micro liquid in the reaction liquid containing part
  • a signal detection part used to detect the signal generated by the micro-liquid in the reaction solution containing part
  • the control part controls the temperature adjustment part to adjust the temperature of the reaction liquid containing part in the first temperature adjustment mode, so that only the first part of the micro liquids in the several micro liquids generates effective signals;
  • the reaction solution container performs signal detection to obtain a first signal;
  • the control unit controls the temperature adjustment unit to adjust the temperature of the reaction liquid container in the second temperature adjustment mode, so that the micro liquids contained in the same reaction liquid container are only different from the first part
  • the second part of the micro-liquid generates an effective signal; the signal detection part is controlled to detect the signal of the reaction solution containing part to obtain a second signal.
  • the microfluid is a mixture containing a sample and a reagent.
  • the above-mentioned first temperature adjustment mode is different from the second temperature adjustment mode.
  • the difference means that the minimum temperatures adopted by the two temperature adjustment modes are different.
  • the minimum temperature employed in the first thermostat mode is higher than the minimum temperature employed in the second thermostat mode.
  • control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquid in the reaction solution storage unit to the temperature t2, and controls the signal detection unit to control the temperature of the reaction solution storage unit. performing signal detection to obtain a first signal
  • the control unit controls the temperature adjustment unit to adjust the temperature of the micro liquid in the reaction solution storage unit to temperature t3, and controls the signal detection unit to detect the signal of the reaction solution storage unit to obtain the second Signal,
  • the temperature difference between t2 and t3 is more than 6°C; for example, the difference is 6-46°C, and for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C °C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C, etc.
  • the reagent includes a first reagent and a second reagent
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the second reagent in the second part of the micro-liquid combines with the sample to generate a signal.
  • the first temperature adjustment mode includes an incubation phase and a signal collection phase
  • the temperature regulating part periodically changes the temperature of the micro liquid in the reaction liquid containing part so that the reaction liquid containing part repeatedly drops from a high temperature to a low temperature T2, or the temperature regulating part makes the The reaction solution containing part maintains a constant temperature T2,
  • the signal detection part detects the signal of the reaction solution containing part to obtain a first signal.
  • the second temperature adjustment mode includes an incubation phase and a signal collection phase
  • the temperature regulating part periodically changes the temperature of the micro liquid in the reaction liquid containing part so that the reaction liquid containing part repeatedly drops from a high temperature to a low temperature T3, or the temperature regulating part makes the The reaction solution containing part maintains a constant temperature T3,
  • the signal detection part detects the signal of the reaction solution containing part to obtain a second signal
  • the temperature difference between T2 and T3 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4- 12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • the temperature difference between t2 and T3 is more than 5°C, for example, the difference is 5-50°C, and for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5-18°C, 5- 15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C or 5-6°C, etc.
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the second reagent in the second part of the micro-liquid combines with the sample to generate a signal
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part in the first temperature adjustment mode and the second adjustment mode:
  • the control unit controls the temperature adjustment unit to periodically change the temperature of the micro-liquids in the reaction liquid container so that the reaction liquid container repeatedly drops from a high temperature to a low temperature T1 so that only The fourth part of the micro-fluid produces an effective signal, and the fourth part of the micro-liquid is different from the first part of the micro-liquid and the second part of the micro-liquid, wherein T1>T2.
  • the temperature difference between T1 and T2 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4- 12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • the reagent further includes a fourth reagent
  • the fourth reagent in the fourth part of the micro-liquid combines with the sample to generate a signal
  • the fourth reagent in the fourth part of the micro-liquid combines with the sample to generate a signal
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the fourth reagent in the fourth part of the micro-liquid combines with the sample to generate a signal
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the second part of the micro-liquid combines with the sample to generate a signal.
  • the second reagent in part of the microfluid combines with the sample to generate a signal.
  • the signal detection part is controlled to perform signal detection on the reaction solution containing part to obtain a fourth signal.
  • the control part controls the temperature regulating part The temperature of the liquid is adjusted to t1 or maintained at T1,
  • the fourth reagent in the fourth part of microfluid remains combined with the sample to continue to generate signals.
  • At most one unit of the sample is dispensed into the microfluidics.
  • the signal is a fluorescent signal
  • the signal detection unit is a camera
  • control unit controls the signal detection unit to perform signal detection on the reaction solution storage unit at the position where the microfluid is spread on the reaction solution storage unit.
  • control part controls the temperature adjusting part to adjust the temperature of the reaction liquid containing part at the position where the micro liquid is spread on the reaction liquid containing part.
  • the present invention provides a detection method for detecting several micro-liquids containing samples and reagents, comprising steps:
  • the temperature difference between t2 and t3 is more than 6°C; for example, the difference is 6-46°C, and for example, the difference is 6-40°C, 6-35°C, 6-30°C, 6-25°C, 6-20°C °C, 6-18°C, 6-15°C, 6-12°C, 6-10°C, 6-8°C or 6-7°C, etc.
  • the reagent includes a first reagent and a second reagent
  • the first reagent in the first part of the micro-liquid combines with the sample to generate a signal
  • the second reagent in the second part of the micro-liquid combines with the sample to generate a signal.
  • the first temperature adjustment mode includes an incubation phase and a signal collection phase
  • signal detection is performed on several micro-liquids to obtain a first signal.
  • the second temperature adjustment mode includes an incubation phase and a signal collection phase
  • signal detection is performed on several micro-liquids to obtain a second signal
  • the temperature difference between T2 and T3 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4- 12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • the temperature difference between t2 and T3 is more than 5°C, for example, the difference is 5-50°C, and for example, the difference is 5-40°C, 5-30°C, 5-20°C, 5-18°C, 5- 15°C, 5-12°C, 5-10°C, 5-8°C, 5-7°C or 5-6°C, etc.
  • the first reagent in the first part of the micro-liquids combines with the sample to generate a signal
  • the first reagent in the first part of microfluids combines with the sample to generate a signal
  • the second reagent in the second part of microfluids combines with the sample to generate a signal
  • the temperature difference between T1 and T2 is more than 4°C, for example, the difference is 4-40°C, and for example, the difference is 4-30°C, 4-20°C, 4-18°C, 4-15°C, 4- 12°C, 4-10°C, 4-8°C, 4-6°C or 4-5°C, etc.
  • the reagent further includes a fourth reagent
  • the fourth reagent in the fourth part of the micro-liquids combines with the sample to generate a signal
  • the fourth reagent in the fourth part of the microliquids combines with the sample to generate a signal
  • the first reagent in the first part of the microliquids combines with the sample to generate a signal
  • the fourth reagent in the fourth part of the micro-liquids combines with the sample to generate a signal
  • the first reagent in the first part of the micro-liquids combines with the sample to generate a signal
  • the second reagent in the second part of the micro-liquids combines with the sample to generate a signal.
  • the second reagent binds to the sample to generate a signal.
  • signal detection is performed on several microfluidics to obtain the fourth signal.
  • the temperature of several micro-liquids is adjusted to t1 or kept at T1
  • the fourth reagent in the fourth part of microfluids remains combined with the sample to continue to generate signals.
  • At most one unit of the sample is dispensed into the microfluidics.
  • the signal is a fluorescent signal
  • the signal detection of several micro-liquids is carried out by a camera.
  • the detection method and primer probe composition of the present invention have the advantages of:
  • the detection signals do not cross each other: the detection method and the primer-probe composition provided by the present invention, for each signal collection of each fluorescent channel, there is at most only one detection signal of the target nucleic acid, and the detection result is more intuitive and accurate. Avoid overlapping of fluorescent signals of more than two target nucleic acids, resulting in detection errors;
  • the detection method and primer-probe composition provided by the present invention can simultaneously perform two-dimensional analysis of fluorescence channel and melting temperature in a digital PCR single-tube reaction, that is, use the same fluorescent channel, through the melting temperature characteristic , can detect different targets (target nucleic acids); or use different fluorescent channels to realize the target type detection of the product of the number of fluorescent channels and the melting temperature characteristic;
  • each fluorescent channel can use only one probe, which can be distinguished by the different melting temperatures of different single-stranded pre-products and probes, which greatly reduces the fluorescent background in digital PCR reactions and improves Reaction sensitivity; at the same time, the number of fluorescent probes in the reagent is reduced, which greatly reduces the cost of the reagent, which is conducive to large-scale promotion and application;
  • the melting temperature can be adjusted: the method provided by the invention is to utilize the secondary amplification product (target nucleic acid and primer set amplification to generate a single-stranded pre-product, which is "primary amplification product”; single-stranded pre-product and probe After specific binding, a double-stranded product is formed, which is the "secondary amplification product") to distinguish between different melting temperatures, so by adjusting the length or sequence composition of the primer signal detection region (h), or adjusting the primer signal detection region (h ) is reverse complementary to the full signal detection region (H) of the probe (P), thereby increasing or decreasing the melting temperature of the double-stranded product formed with the probe (P), thereby adjusting different signal acquisition temperatures;
  • the primer probe design method of the present invention has two parts (first primer, second primer) complementary pairing with the target sequence (target nucleic acid) (the probe is not complementary paired with the target nucleic acid), Compared with the Taqman hydrolysis probe method, three parts (first primer, second primer and Taqman probe) are required to be paired with the target sequence.
  • first primer, second primer and Taqman probe are required to be paired with the target sequence.
  • the probe design method is more inclusive and less difficult to design;
  • the primer probe composition and detection method provided by the present invention consume less samples, are particularly suitable for the detection of rare samples, can greatly increase the concentration of samples added to the detection reaction, and improve detection sensitivity.
  • the single-tube reaction provides the possibility for subsequent extraction-free one-tube detection;
  • the method of the present invention has very low requirements on the length of the target sequence.
  • the target type is a short-segment nucleic acid, such as a free nucleic acid
  • a shorter target sequence length has higher sensitivity in detection
  • the method of the present invention can be applied to nucleic acid detection of various sample types, including serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples , formalin-fixed paraffin-embedded tissue (FFPE), etc.
  • sample types including serum samples, plasma samples, whole blood samples, sputum samples, swab samples, lavage fluid samples, fresh tissue samples , formalin-fixed paraffin-embedded tissue (FFPE), etc.
  • FFPE formalin-fixed paraffin-embedded tissue
  • the multiple time-sharing nucleic acid detection device and detection method of the present invention has the advantage that different nucleic acids to be tested can generate effective signals at different times, that is, the signals of different nucleic acids to be tested can be isolated to avoid In the event of signal overlap and detection errors, time-sharing and high-precision detection of multiple nucleic acids is realized.
  • Fig. 1 is a kind of primer probe design schematic diagram (for 3 kinds of targets) among the present invention
  • the probe (P) has only the primer anchor region (A') opposite to the probe anchor region (A) of the forward primer (F1/2/3) Complementary, the rest of the probe does not interact with any sequence, at this time the distance between the first detection group modified on the probe (P) and the second detection group is d1; during PCR amplification, at a higher Annealing at the first annealing temperature (T1), the forward primer (F1) and the reverse primer (R1) respectively specifically bind to the target sequence 1 and extend to generate a double-stranded pre-product, one of which is a single-stranded pre-product (S1) From the 5' end to the 3' end are the probe anchor region (A1), the target sequence, the reverse complementary sequence (h1') of the primer signal detection region, and the reverse complementary sequence (M') of the extension block region; The forward primer (F2) and the reverse primer (R2) specifically bind to the target sequence 2 and extend to generate
  • the first signal acquisition is performed at a lower first signal acquisition temperature (t1).
  • the probe signal detection region (H) of the probe (P) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded pre-product (S1), and the primer of the probe (P)
  • the anchor region (A1') is reverse-complementary paired with the probe anchor region (A1) at the 5' end of the single-stranded pre-product (S1) to obtain a double-stranded product (D1) formed with the probe (P), while the single-stranded
  • the reverse complementary sequence (M') of the extension block region at the 3' end of the pre-product (S1) is not complementary to any part of the probe (P) or target sequence; at this time, all or part of the sequence of the probe (P) consists of a single
  • the chain becomes a double-stranded state, the distance between the first detection group and the second detection group changes, and a signal is generated, which can
  • the probe (P ) of the probe signal detection region (H) is complementary to the reverse complementary sequence (h3') of the primer signal detection region (h3') at the 3' end of the single-stranded pre-product (S3), and the primer anchor region (A3') of the probe is paired with the single
  • the probe anchor region (A3) at the 5' end of the strand pre-product (S3) is reverse-complementary paired, and the 3' end of the single-stranded pre-product (S3) can continue to extend to the 5' end of the probe (P) to obtain a probe Part or all of the reverse complementary sequence of (P), that is, to complete the secondary amplification using the single-stranded pre-product (S3) as a primer and the probe (P) as a template to obtain the amplification formed with the probe (P) Double-stranded product (D3), at this time, all or part of the sequence of the probe (P) changes from single-stranded to double-stranded, and the distance between
  • the hybrid double-stranded product (D1) formed by the strand pre-product (S1) and the probe (P) cannot form a double-stranded structure because the third signal collection temperature (t3) is higher than the melting temperature of the hybrid double-stranded product (D1);
  • the amplified double-stranded product (D2) formed by the strand pre-product (S2) and the probe (P) cannot form a double-stranded structure because the third signal collection temperature (t3) is higher than the melting temperature of the amplified double-stranded product (D2) ; and the amplification double-stranded product (D3) formed by the single-stranded pre-product (S3) and the probe (P) forms a double-stranded state, the distance between the first detection group and the second detection group becomes farther, and the fluorescence Resonance energy transfer is less efficient, resulting in a change in the fluorescent signal that can be detected by the instrument.
  • FIG. 2A is a signal diagram of the digital PCR detection reaction of Example 1 at the first signal collection temperature (60° C.);
  • FIG. 2B is a signal diagram of the digital PCR detection reaction of Example 1 at the second signal collection temperature (78° C.).
  • FIG. 3A is a signal diagram of the digital PCR detection reaction of Example 2 at the first signal collection temperature (60° C.);
  • FIG. 3B is a signal diagram of the digital PCR detection reaction of Example 2 at the second signal collection temperature (78° C.).
  • FIG. 4A is a signal diagram of the digital PCR detection reaction of Example 3 at the first signal collection temperature (60° C.);
  • FIG. 4B is a signal diagram of the digital PCR detection reaction of Example 3 at the second signal collection temperature (78° C.).
  • FIG. 5A is a signal diagram of the digital PCR detection reaction of Example 4 at the first signal collection temperature (56° C.);
  • FIG. 5B is a signal diagram of the digital PCR detection reaction of Example 4 at the second signal collection temperature (72° C.).
  • FIG. 6A is a signal diagram of the digital PCR detection reaction of Example 5 at the first signal acquisition temperature (55°C);
  • FIG. 6B is a signal diagram of the digital PCR detection reaction of Example 5 at the second signal acquisition temperature (65°C);
  • FIG. 6C is a signal diagram of the digital PCR detection reaction in Example 5 at the third signal collection temperature (73° C.).
  • Figure 7A is the signal diagram of the digital PCR detection reaction of Example 6 at the first signal acquisition temperature (72°C), from which it can be seen that there are positive droplets that are different from the background;
  • Figure 7B is the digital PCR detection of Example 6 The signal diagram of the reaction at the second signal collection temperature (82° C.), it can be seen from the diagram that they are all negative droplets.
  • Fig. 8A is the signal diagram collected at the first signal collection temperature (60°C) and the VIC fluorescence channel in Example 7, and it can be seen that there are positive droplets different from the background;
  • Fig. 8B is the second signal in Example 7 In the signal diagram collected by the collection temperature (68°C) and the VIC fluorescence channel, positive droplets that are different from the background can be seen, and the positive droplets appearing under this temperature and channel are consistent with the first signal collection temperature (60°C) VIC channel The positions of the positive droplets that appear are different;
  • FIG. 8C is a signal diagram collected at the second signal collection temperature (68° C.) and the FAM fluorescence channel in Example 7.
  • FIG. 9A is a signal diagram of embodiment 8 at the first signal collection temperature (60° C.).
  • FIG. 9B is a signal diagram of Example 8 at the second signal collection temperature (78° C.).
  • FIG. 10A is a signal diagram of Example 9 at the first signal collection temperature (60° C.).
  • FIG. 10B is a signal diagram of Example 9 at the second signal collection temperature (78° C.).
  • FIG. 11A is a signal diagram of embodiment 10 at the first signal collection temperature (60° C.).
  • FIG. 11B is a signal diagram of Example 10 at the second signal collection temperature (78° C.).
  • FIG. 12A is a signal diagram of Example 11 at the first signal collection temperature (60° C.).
  • FIG. 12B is a signal diagram of Example 11 at the second signal collection temperature (78° C.).
  • FIG. 13A is a signal diagram of embodiment 1 at the first signal collection temperature (61° C.).
  • FIG. 13B is a signal diagram of Example 12 at the second signal collection temperature (68° C.).
  • FIG. 13C is a signal diagram of Example 12 at the third signal collection temperature (76° C.).
  • Fig. 14A is a schematic diagram of a multiple time-sharing nucleic acid detection device disclosed in one embodiment of the present application
  • Fig. 14B is a schematic diagram of a multiple time-sharing nucleic acid detection device disclosed in another embodiment of the present application
  • Fig. 14C is a schematic diagram of a multiple time-sharing nucleic acid detection device disclosed in another embodiment of the present application Structural diagram of an orifice plate composed of multiple reaction liquid storage parts.
  • Fig. 15A is a graph showing the temperature change of the controller controlling the temperature regulating part to adjust the temperature of the reaction liquid container disclosed in one embodiment of the present application
  • Fig. 15B is a graph showing the controller controlling the temperature regulating part regulating the reaction liquid containing part disclosed in another embodiment of the present application
  • the temperature change curve of temperature
  • FIG. 15C is the temperature change curve of the controller controlling the temperature adjustment part to adjust the temperature of the reaction liquid container disclosed in another embodiment of the application
  • FIG. 15D is the controller control disclosed in another embodiment of the application A temperature change graph in which the temperature adjustment unit adjusts the temperature of the reaction liquid storage unit.
  • Fig. 16A is a graph showing the temperature change of the controller controlling the temperature regulating part to adjust the temperature of the reaction solution containing part disclosed in another embodiment of the present application
  • Fig. 16C is a temperature change curve diagram of the controller disclosed in another embodiment of the present application to control the temperature adjustment part to adjust the temperature of the reaction solution containing part
  • Fig. 16D is a controller disclosed in another embodiment of the present application A temperature change graph for controlling the temperature adjustment unit to adjust the temperature of the reaction liquid storage unit.
  • a fully automatic digital PCR analysis system and reagent consumables are used for detection and data analysis (wherein, what are used in embodiments 1-7 is the D600 fully automatic digital PCR analysis system and reagent consumables of Mike Biological Co., Ltd.; embodiment 8-12 used the OS-500 digital PCR all-in-one machine and reagent consumables of Beijing Da Microbe Technology Co., Ltd.).
  • This digital PCR analysis system is to disperse the reaction system containing samples, primer probe compositions and amplification reagents into four Amplification and detection are carried out in the reaction wells (each reaction well contains multiple small droplets, and each small droplet is a reaction unit).
  • the accompanying drawings of the following examples provide one of the reaction units. A plot of the droplet results for a well.
  • Forward primer F1 (SEQ ID NO: 3), is a specific primer designed for the target sequence of EGFR exon 21 L858R mutation in non-small cell lung cancer.
  • the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchor region (A1), and the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1), That is, the primer anchor region (A1'), reverse complementary;
  • Reverse primer R1 (SEQ ID NO: 2), is a specific primer designed for the L858R mutation target sequence of EGFR exon 21 in non-small cell lung cancer.
  • the target sequence binding region, the 1st to 5th bases at the 5' end are the extension block (M), the 6th to 27th bases at the 5' end are the primer signal detection area (h3), and the probe P1 ( The 13th to 34th base sequences of the 5' end of SEQ ID NO: 1) are identical.
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 ; mM refers to mmol/L (the same below).
  • Sample preparation Use non-small cell lung cancer EGFR exon 21 L858R mutation DNA as a positive sample, and use the reverse primer (R1) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence , to verify the detection ability of a single mutation. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 60°C; denaturation at 94°C for 15 seconds, Annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 78°C for the second signal acquisition.
  • Figure 2A is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation-positive sample amplified by the reverse primer (R1) at the first signal acquisition temperature (60°C), and it can be seen that there is a difference Positive droplets in the background.
  • Figure 2B is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation-positive sample amplified by the reverse primer (R1) at the second signal collection temperature (78°C). Negative droplets.
  • Forward primer F1 (SEQ ID NO: 3), is a specific primer designed for the target sequence of EGFR exon 21 L858R mutation in non-small cell lung cancer.
  • the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchor region (A1), and the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1), That is, the primer anchor region (A1'), reverse complementary;
  • Reverse primer R2 (SEQ ID NO: 4), is a specific primer designed for the L858R mutation target sequence of EGFR exon 21 in non-small cell lung cancer.
  • the full length of R2 is 33bp, and the first to 20th base of its 3' end In the target sequence binding region, the 1st to 10th bases at the 5' end are primer signal detection regions (h2), which are identical to the 1st to 10th bases at the 3' end of probe P1 (SEQ ID NO: 1).
  • Reagent components concentration 2 ⁇ PCR Reaction Buffer 1 ⁇ DNA Polymerase 2U Forward primer (F1) 500nM Reverse primer (R2) 100nM Probe (P1) 400nM
  • nucleic acid template 130 copies Ultra-pure water Add to 20 ⁇ L
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use non-small cell lung cancer EGFR exon 21 L858R mutation DNA as a positive sample, and use the reverse primer (R2) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence , to verify the detection ability of a single mutation. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 60°C; denaturation at 94°C for 15 seconds, Annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 78°C for the second signal acquisition.
  • Figure 3A is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 exon L858R mutation-positive samples amplified by the reverse primer (R2) at the first signal collection temperature (60°C), and it can be seen that they are all negative droplet.
  • Figure 3B is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation-positive sample amplified by the reverse primer (R2) at the second signal collection temperature (78°C), and it can be seen that there is a difference Positive droplets in the background.
  • Forward primer F1 (SEQ ID NO: 3), is a specific primer designed for the target sequence of EGFR exon 21 L858R mutation in non-small cell lung cancer.
  • the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchor region (A1), and the 1st to 10th base sequence at the 5' end of the probe P1 (SEQ ID NO: 1), That is, the primer anchor region (A1'), reverse complementary;
  • Reverse primer R2 (SEQ ID NO: 4), is a specific primer designed for the L858R mutation target sequence of EGFR exon 21 in non-small cell lung cancer.
  • the full length of R2 is 33bp, and the first to 20th base of its 3' end In the target sequence binding region, the 1st to 10th bases at the 5' end are primer signal detection regions (h2), which are identical to the 1st to 10th bases at the 3' end of probe P1 (SEQ ID NO: 1).
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use the non-small cell lung cancer EGFR exon 21 L858R mutation DNA as a positive sample, and use the reverse primer (R2) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 21 L858R mutation target sequence , to verify the detection ability of a single mutation. Pure water was used as no template control (NTC).
  • NTC no template control
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used are: 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing and extension for 30 seconds, a total of 45 cycles; 60°C for the first signal acquisition; 40°C constant temperature incubation for 5 minutes ; 78°C for the second signal acquisition.
  • Figure 4A is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation-positive sample amplified by the reverse primer (R2) at the first signal collection temperature (60°C).
  • Negative droplets droplets that are on the edge and/or appear larger, a problem with the experimental procedure, not a positive droplet).
  • Figure 4B is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 21 L858R mutation-positive sample amplified by the reverse primer (R2) at the second signal acquisition temperature (78°C), and it can be seen that there is a difference Positive droplets in the background.
  • Forward primer F1-4 (SEQ ID NO: 6), a specific primer designed for the target sequence of the gene VIM mutation resistant to carbapenem antibiotics, the full length of F1-4 is 37bp, and the 3' end of the 1st to 23rd bases are the target sequence binding region, the 1st to 11th bases at the 5' end are the probe anchor region (A), and the 1st to 11th bases at the 5' end of the probe P2-4 (SEQ ID NO:5) 11 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R1-4 (SEQ ID NO: 7), is a specific primer designed for the gene VIM mutation target sequence resistant to carbapenem antibiotics, the full length of R1-4 is 55bp, and the 3' end of the 1st to 25th bases are the target sequence binding region, the 1st to 5th bases at the 5' end are the signal blocking region (M), the 6th to 27th bases at the 5' end are the primer signal detection region (h1), and The 14th to 35th base sequences of the 5' end of the probe P2-4 (SEQ ID NO:5) are identical;
  • Forward primer F2-4 (SEQ ID NO: 8), a specific primer designed for the target sequence of the gene OXA-48 mutation resistant to carbapenem antibiotics, F2-4 full length 33bp, its 3' end first
  • the 1st to 19th bases are the target sequence binding region
  • the 1st to 11th bases at the 5' end are the probe anchoring region (A)
  • the 5' end of the probe P2-4 (SEQ ID NO:5) 1 to 11 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R2-4 (SEQ ID NO: 9), is a specific primer designed for the target sequence of the gene OXA-48 mutation resistant to carbapenem antibiotics.
  • the full length of the R2-4 primer is 37bp, and its 3' end is 1 to 24 bases are the target sequence binding region, and the 1st to 10th bases at the 5' end are identical to the 1st to 10th bases at the 3' end of the probe P2-4 (SEQ ID NO: 5).
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use the nucleic acid of the gene VIM mutation resistant to carbapenem antibiotics and the nucleic acid of the gene OXA-48 mutation resistant to carbapenem antibiotics as positive samples, and use reverse primers with different signal detection region sequences ( R1-4 and R2-4) were used to detect the carbapenem-resistant gene VIM mutation and the carbapenem-resistant gene OXA-48 mutation target sequence to verify the ability to detect double mutations. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 56°C; denaturation at 94°C for 15 seconds, Annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 72°C for the second signal acquisition.
  • Figure 5A is the droplet diagram of this embodiment at the first signal acquisition temperature (56°C), and it can be seen that there are positive droplets different from the background, which are positive droplets of the VIM mutation of the gene resistant to carbapenem antibiotics ( Droplets that are on the edge and/or appear larger are a manipulation problem, not a positive drop).
  • Figure 5B is the droplet diagram of this embodiment at the second signal collection temperature (72°C). It can be seen that there are positive droplets that are different from the background, and they are positive droplets of the gene OXA-48 mutation resistant to carbapenem antibiotics. Droplets, and the positions of the positive droplets that appear at this temperature are different from the positive droplets that appear at the first signal collection temperature (56°C).
  • the detection signals of different target sequences can be obtained at different signal detection temperatures (56°C, 72°C) by using the primer-probe composition and the digital PCR detection method of the present invention. , and do not cross each other.
  • the forward primer F1-5 (SEQ ID NO: 11) is a specific primer designed for the IMP mutation target sequence of the gene resistant to carbapenem antibiotics.
  • the full length of F1-5 is 42bp, and the 3' end of the The first to 24 bases are the target sequence binding region, the 1st to 15th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P1-5 (SEQ ID NO: 10) 1 to 15 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • the reverse primer R1-5 (SEQ ID NO: 12) is a specific primer designed for the IMP mutation target sequence of the gene resistant to carbapenem antibiotics.
  • the full length of R1-5 is 56bp, and the 3' end of the The first to 23 bases are the target sequence binding region, the 1st to 5th bases at the 5' end are the signal blocking region (M), and the 6th to 30th bases at the 5' end are the primer signal detection region (h1) , which is identical to the 17th to 41st base sequence of the 5' end of probe P1-5 (SEQ ID NO: 10);
  • the forward primer F2-5 (SEQ ID NO: 13) is a specific primer designed for the mutated target sequence of the gene OXA-48 resistant to carbapenem antibiotics.
  • the full length of F2-5 is 37bp, and its 3' end
  • the 1st to 19th bases are the target sequence binding region
  • the 1st to 15th bases at the 5' end are the probe anchor region (A)
  • the 5' of the probe P1-5 SEQ ID NO:10
  • the 1st to 15th nucleotide sequence at the end, that is, the primer anchor region (A') is reverse complementary;
  • Reverse primer R2-5 (SEQ ID NO: 14), is a specific primer designed for the target sequence of the gene OXA-48 mutation resistant to carbapenem antibiotics.
  • the full length of the R2-5 primer is 38bp, and its 3' end is 1 to 24 bases are the target sequence binding region, and the 1st to 11th bases at the 5' end are identical to the 15th to 25th bases at the 3' end of the probe P1-5 (SEQ ID NO: 10).
  • the forward primer F3-5 (SEQ ID NO: 15) is a specific primer designed for the gene KPC mutation target sequence resistant to carbapenem antibiotics.
  • the full length of F3-5 is 39bp, and its 3' end has a first
  • the first to 21 bases are the target sequence binding region
  • the 1st to 15th bases at the 5' end are the probe anchoring region (A)
  • Reverse primer R3-5 (SEQ ID NO: 16), is a specific primer designed for the gene KPC mutation target sequence resistant to carbapenem antibiotics, the R3-5 primer is 33bp in length, and its 3' end first to The 22 bases are the target sequence binding region, and the 1st to 8th bases at the 5' end are identical to the 1st to 8th bases at the 3' end of the probe P1-5 (SEQ ID NO: 10).
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation nucleic acid samples of carbapenem-resistant gene IMP mutation, carbapenem-resistant gene OXA-48 mutation nucleic acid sample and carbapenem-resistant gene KPC mutation nucleic acid sample
  • a positive sample use reverse primers (R1-5/R2-5/R3-5) with different signal detection region sequences to detect the IMP mutation of the gene resistant to carbapenem antibiotics, and the gene resistant to carbapenem antibiotics
  • the gene OXA-48 mutation and the carbapenem-resistant gene KPC mutation target sequence were detected to verify the detection ability of the triple mutation. Pure water was used as a no-template control (NTC).
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 55°C; denaturation at 94°C for 15 seconds, 52°C annealing and extension for 20 seconds, a total of 5 cycles; 65°C for the second signal acquisition. Denaturation at 94°C for 15 seconds, annealing and extension at 40°C for 20 seconds, a total of 5 cycles; 73°C for the third signal acquisition.
  • Figure 6A shows the carbapenem-resistant gene IMP mutation in this example, the carbapenem-resistant gene OXA-48 mutation and the carbapenem-resistant gene KPC mutation positive samples in the first signal
  • a temperature 55°C
  • positive droplets that are different from the background can be seen, which are positive droplets of the gene IMP mutation resistant to carbapenem antibiotics.
  • Figure 6B shows the carbapenem-resistant gene IMP mutation in this example, the carbapenem-resistant gene OXA-48 mutation and the carbapenem-resistant gene KPC mutation-positive samples in the second signal
  • a temperature 65°C
  • positive droplets that are different from the background can be seen, which are positive droplets of the gene OXA-48 mutation resistant to carbapenem antibiotics, and the positive droplets that appear at this temperature
  • the position of the droplet is different from that of the positive droplet at the first signal acquisition temperature (55° C.).
  • Figure 6C shows the carbapenem-resistant gene IMP mutation in this example, the carbapenem-resistant gene OXA-48 mutation and the carbapenem-resistant gene KPC mutation positive samples in the third signal
  • the droplet diagram at the collection temperature (73°C) is the positive droplet of the gene KPC mutation resistant to carbapenem antibiotics, and the positive droplet at this temperature is consistent with the first signal collection temperature (55°C) and the second signal collection temperature (55°C). The location of the positive droplet at the signal collection temperature (65°C) was different.
  • Forward primer F2-6 (SEQ ID NO: 18), is a specific primer designed for the L858R mutation target sequence of EGFR exon 21 in non-small cell lung cancer.
  • the 19 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A2), and the 5' end of the probe P1-6 (SEQ ID NO:5) at the 1st 1 to 10 base sequences, i.e. the primer anchor region (A2'), reverse complementary;
  • Reverse primer R2-6 (SEQ ID: 17), is a specific primer designed for non-small cell lung cancer EGFR exon 21 L858R mutation target sequence, the full length of R2-6 is 33bp, and its 3' end is 1st to 20th bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the primer signal detection region (h2), and the 1st to 10th bases at the 3' end of probe P1-6 (SEQ ID NO:5) 10 nucleotide sequences are the same.
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use non-small cell lung cancer EGFR exon 21 L858R mutation as a positive sample, and pure water as a no-template control (NTC).
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used are: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, and a total of 45 cycles; denaturation at 94°C for 15 seconds, annealing and extension at 40°C for 20 seconds, and a total of 45 cycles. 10 cycles; 72°C for the first signal acquisition; 82°C for the second signal acquisition.
  • FIG. 7A is a droplet image at the first signal collection temperature (72° C.), and it can be seen that there are positive droplets that are different from the background.
  • FIG. 7B is a droplet diagram at the second signal collection temperature (82° C.), and it can be seen that all the droplets are negative.
  • a triple detection primer-probe system for detecting carbapenem-resistant gene KPC mutation, carbapenem-resistant gene IMP mutation and carbapenem-resistant gene VIM mutation such as Table 13 shows:
  • Forward primer F1-7 (SEQ ID NO: 20), is a specific primer designed for the target sequence of the gene KPC mutation resistant to carbapenem antibiotics, the full length of F1-7 is 36bp, and the 1st to 3rd
  • the 21 bases are the target sequence binding region
  • the 1st to 12th bases at the 5' end are the probe anchoring region (A)
  • the 5' end of the probe P1-7 (SEQ ID NO: 19) at the 1st 1 to 12 base sequences i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R1-7 (SEQ ID: 21), a specific primer designed for the gene KPC mutation target sequence resistant to carbapenem antibiotics, the full length of R1-7 is 52bp, and its 3' end is 1st to 22nd bases are the target sequence binding region, the 1st to 5th bases at the 5' end are the signal blocking region (M), and the 6th to 27th bases at the 5' end are the primer signal detection region (h1) , which is identical to the 14th to 35th base sequence of the 5' end of the probe P1-7 (SEQ ID NO: 19);
  • Forward primer F2-7 (SEQ ID NO: 22), is a specific primer designed for the IMP mutation target sequence of the gene resistant to carbapenem antibiotics.
  • the 24 bases are the target sequence binding region, the 1st to 12th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P1-7 (SEQ ID NO: 19) at the 1st 1 to 12 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R2-7 (SEQ ID NO: 23), is a specific primer designed for the gene IMP mutation target sequence resistant to carbapenem antibiotics, the R2-7 primer is 37bp in length, and its 3' end first to The 23rd base is the target sequence binding region, the 1st to 11th bases at the 5' end and the 1st to 11th bases at the 3' end of the probe P1-7 (SEQ ID NO: 19) The sequence is the same.
  • Forward primer F3-7 (SEQ ID NO: 24), is a specific primer designed for the target sequence of the gene VIM mutation resistant to carbapenem antibiotics, the full length of F3-7 is 36bp, the 1st to 3rd The 23 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P2-7 (SEQ ID NO: 1) 1 to 10 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R3-7 (SEQ ID NO: 19), is a specific primer designed for the gene VIM mutation target sequence resistant to carbapenem antibiotics, the R3-7 primer is 38bp in length, and the 3' end of the The 25th base is the target sequence binding region, the 1st to 10th bases at the 5' end and the 1st to 10th bases at the 3' end of the probe P2-7 (SEQ ID NO: 1) The sequence is the same.
  • the gene KPC mutation of mycocene antibiotics specifically binds and extends to generate a double-stranded pre-amplification product, and the 5' end to the 3' end of a single-stranded pre-amplification product (S1) is the probe anchor region (A1 ), target sequence, reverse complementary sequence (h1') of primer signal detection region, reverse complementary sequence (M') of extension block region; forward primer (F2-7) and reverse primer (R2-7) Respectively bind specifically to the target sequence 2 (IMP mutation of carbapenem-resistant gene) and extend to generate double-stranded pre-amplification products, one of which is a single-stranded pre-amplification product (S2) from the 5' end to the 3' end
  • the sequence is the probe anchor region (A2), the target sequence, and the reverse complementary sequence (h2') of the primer signal detection region; the forward primer (F3-7) and the reverse primer (R3-7) are respectively compatible
  • the probe signal detection region (H) of the probe (P1-7) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplified product (S1), and the probe (
  • the primer anchoring region (A1') of P1-7) is paired with the probe anchoring region (A1) at the 5' end of the single-stranded preamplified product (S1) in reverse complementarity to obtain the formation of the probe (P1-7).
  • the double-stranded product (D1) hybridizes, and the reverse complement (M') of the extension block region at the 3' end of the single-stranded preamplified product (S1) is not complementary to any part of the probe (P1-7) or target sequence ;
  • the partial sequence of the probe (P1-7) changes from a single strand to a double strand state, the distance between the first detection group and the second detection group changes, and a signal is generated, which can be detected by the instrument.
  • the annealing temperature of the sequence (h3') is lower than the first annealing temperature (T1) and the first signal collection temperature (t1), so it does not extend in reverse complementarity with the probe signal detection region (H) of the probe (P);
  • the second amplification cycle of the single-stranded pre-amplification products (S2 and S3) is carried out, and the cycle condition is high temperature melting, and at a lower second amplification cycle.
  • Annealing at two annealing temperatures (T2), at this time, the probe signal detection region (H) of the probe (P1-7) and the 3' end of the single-stranded preamplified product (S2)
  • the reverse complementary sequence (h2') of the primer signal detection region is complementary, and the primer anchor region (A2') of the probe is reversed to the probe anchor region (A2) at the 5' end of the single-stranded preamplified product (S2)
  • the 3' end of the single-stranded pre-amplification product (S2) can continue to extend to the 5' end of the probe (P) to obtain all the reverse complementary sequences of the probe (P1-7), that is, to complete the single-stranded
  • the pre-amplification product (S2) is used as a primer, and the secondary amplification using the probe (P1-7) as a template obtains the amplified double-stranded product (D2) formed with the probe (P1-7).
  • the probe The partial sequence of (P1-7) changes from single-stranded to double-stranded state, and the distance between the first detection group and the second detection group changes to generate a signal; at this time, the probe of the probe (P2-7)
  • the signal detection region (H) is complementary to the reverse complementary sequence (h3') of the primer signal detection region (h3') at the 3' end of the single-stranded preamplified product (S3), and the primer anchor region (A3') of the probe is matched with the single-stranded preamplified product (S3).
  • the probe anchor region (A3) at the 5' end of the amplification product (S3) is reverse-complementary paired, and the 3' end of the single-stranded pre-amplification product (S3) can continue to extend to the 5' end of the probe (P2-7) , to obtain all the reverse complementary sequences of the probe (P2-7), that is, to complete the secondary amplification using the single-stranded pre-amplified product (S3) as a primer and the probe (P2-7) as a template, and obtain the The amplified double-stranded product (D3) formed by the needle (P2-7), at this time, the partial sequence of the probe (P2-7) changes from single-stranded to double-stranded, and the difference between the first detection group and the second detection group The distance between them changes, generating a signal.
  • Probe 400nM Carbapenem-resistant gene KPC mutation nucleic acid template 150 copies Gene IMP mutation nucleic acid template resistant to carbapenem antibiotics 80 copies Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics 120 copies Ultra-pure water Add to 20 ⁇ L
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation using the gene KPC mutation, gene IMP mutation and gene VIM mutation of carbapenem antibiotics as positive samples, the three samples were mixed and tested to verify the detection ability of the triple target. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected using D600 fully automatic digital PCR analysis system and reagent consumables from Mike Biological Co., Ltd.):
  • the amplification and lighting programs used are: 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing and extension for 30 seconds, a total of 45 cycles; 60°C for the first signal acquisition (VIC channel); 94°C Denaturation for 15 seconds, 52°C annealing and extension for 20 seconds, a total of 5 cycles; 68°C for the second signal acquisition (including two signal channel acquisitions, respectively VIC channel and FAM channel).
  • Figure 8A is the droplet image collected at the first signal collection temperature (60°C) and the VIC fluorescence channel in this example. It can be seen that the positive droplet is different from the background, which is the gene KPC mutation resistant to carbapenem antibiotics. Positive droplet.
  • Figure 8B is the droplet image collected at the second signal collection temperature (68°C) and the VIC fluorescence channel in this example. It can be seen that the positive droplet is different from the background, which is the gene IMP resistant to carbapenem antibiotics. Mutate the positive droplet, and the position of the positive droplet that appears at this temperature and channel is different from the position of the positive droplet that appears in the VIC channel at the first signal collection temperature (60°C).
  • Figure 8C is the droplet image collected at the second signal collection temperature (68°C) and the FAM fluorescence channel in this example. It can be seen that the positive droplet is different from the background, which is the gene VIM resistant to carbapenem antibiotics. Mutate positive droplets, and the positive droplets appearing under this temperature and channel are different from the positive droplets appearing in the VIC channel of the first signal acquisition temperature (60°C) and the second signal acquisition temperature (68°C) VIC channel.
  • Forward primer F1-8 (SEQ ID NO:27), is a specific primer designed for non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence, the full length of F1-8 is 29bp, and the 1st to 3' end
  • the 16 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A1), and the 5' end of the probe P1-8 (SEQ ID NO:25) 1 to 10 base sequences, i.e. the primer anchor region (A1'), reverse complementary;
  • Reverse primer R1-8 (SEQ ID NO: 26), is a specific primer designed for the H773_V774insH mutation target sequence of exon 20 of non-small cell lung cancer EGFR.
  • the 21 bases are the target sequence binding region, the 1st to 5th bases at the 5' end are the extension block (M), and the 6th to 27th bases at the 5' end are the primer signal detection region (h3 ), which is identical to the 26th to 47th base sequence of the 3' end of the probe P1-8 (SEQ ID NO: 25).
  • the principle of detection using the above-mentioned primer probes is as follows: when the target is specifically amplified, annealing is performed at a higher first temperature (T1), and the forward primer (F1-8) and the reverse primer (R1-8) are annealed. ) respectively specifically combined with the target sequence 1 and extended to generate double-stranded pre-amplification products, in which the 5' end to the 3' end of a single-stranded pre-amplification product (S1) is the probe anchor region (A1), and the target Sequence, reverse complementary sequence (h1') of the primer signal detection region, reverse complementary sequence (M') of the extension block region; Signal Acquisition.
  • the probe signal detection region (H) of the probe (P1-8) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplified product (S1), and the probe (
  • the primer anchor region (A1') of P1-8) is reverse-complementary paired with the probe anchor region (A1) at the 5' end of the single-stranded preamplified product (S1) to obtain the formation of the probe (P1-8).
  • the double-stranded product (D1) hybridizes, and the reverse complement (M') of the extension block region at the 3' end of the single-stranded preamplified product (S1) is not complementary to any part of the probe (P1-8) or target sequence ; At this time, all or part of the sequence of the probe (P) changes from a single strand to a double strand state, the distance between the first detection group and the second detection group changes, and a signal is generated, which can be detected by the instrument.
  • the cycle condition is high temperature melting, and annealing at a lower second annealing temperature (T2) (T2 ⁇ T1, and T2 ⁇ t1)) program , and collect the signal (t2>t1, and t2>T1) at the second signal collection temperature (t2), at this time the hybrid double-stranded product formed by the single-stranded pre-amplification product (S1) and the probe (P1-8) (D1) Since the second signal collection temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1), a double-stranded structure cannot be formed, and the distance between the first detection group and the second detection group changes, so that The fluorescent signal changes and can be detected by the instrument.
  • T2 ⁇ T1, and T2 ⁇ t1 the cycle condition is high temperature melting, and annealing at a lower second annealing temperature (T2) (T2 ⁇ T1, and T2 ⁇ t1)) program , and collect the signal (t2>t1, and t2>T1) at the second signal collection temperature
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA as a positive sample, and use the reverse primer (R1) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence , to verify the detection capability of a single mutation. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected by OS-500 digital PCR all-in-one machine and reagent consumables from Beijing Da Microbe Technology Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 60°C; denaturation at 94°C for 15 seconds, Annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 78°C for the second signal acquisition.
  • Figure 9A is the droplet diagram of the H773_V774insH mutation-positive sample of non-small cell lung cancer EGFR exon 20 exon 20 amplified by the reverse primer (R1-8) at the first signal acquisition temperature (60°C). It can be seen There are positive droplets that are distinguished from the background.
  • Figure 9B is the droplet diagram of the H773_V774insH mutation-positive sample of non-small cell lung cancer EGFR exon 20 exon 20 amplified by the reverse primer (R1-8) at the second signal acquisition temperature (78°C), and it can be seen All are negative droplets.
  • Forward primer F1-9 (SEQ ID NO:27), is a specific primer designed for the H773_V774insH mutation target sequence of exon 20 of non-small cell lung cancer EGFR.
  • the 16 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A1), and the 5' end of the probe P1-9 (SEQ ID NO:25) 1 to 10 base sequences, i.e. the primer anchor region (A1'), reverse complementary;
  • Reverse primer R2-9 (SEQ ID NO: 28), is a specific primer designed for the H773_V774insH mutation target sequence of exon 20 of non-small cell lung cancer EGFR.
  • the 21 bases are the target sequence binding region, the 1st to 8th bases at the 5' end are the primer signal detection region (h2), and the 1st base at the 3' end of the probe P1-9 (SEQ ID NO:25) Up to 8 base sequences are identical.
  • the principle of detection using the above-mentioned primer probes is as follows: when the target is specifically amplified, annealing is performed at a higher first temperature (T1), and the forward primer (F1-9) and the reverse primer (R2-9) are annealed.
  • the 5' end to the 3' end of a single-stranded pre-amplification product (S2) is the probe anchor region (A1), and the target sequence , the reverse complementary sequence (h2') of the signal detection region of the primer;
  • the first signal acquisition (t1>T1) is performed at a lower first signal acquisition temperature (t1), due to the single-stranded preamplified product (S2)3
  • the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 'end is less than the first annealing temperature (T1) and the first signal acquisition temperature (t1), so it does not match the probe signal of the probe (P1-9)
  • the detection region (H) is extended in reverse complementarity; after the first signal acquisition is completed at the first signal acquisition temperature (t1), the cycle condition is high temperature melting, and at a lower second annealing temperature (T2) Under the annealing (T2 ⁇ T
  • the amplified double-stranded product ( D2) A double-chain state is formed, the distance between the first detection group and the second detection group becomes longer, and the efficiency of fluorescence resonance energy transfer is lower, so that the fluorescence signal changes and can be detected by the instrument.
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA as a positive sample, use the reverse primer (R2-9) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence Assays were performed to validate the detectability of single mutations. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected by OS-500 digital PCR all-in-one machine and reagent consumables from Beijing Da Microbe Technology Co., Ltd.):
  • the amplification and lighting programs used were: pre-denaturation at 95°C for 2 minutes; denaturation at 94°C for 10 seconds, annealing and extension at 56°C for 30 seconds, a total of 45 cycles; first signal acquisition at 60°C; denaturation at 94°C for 15 seconds, Annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 78°C for the second signal acquisition.
  • Figure 10A is the droplet diagram of the H773_V774insH mutation-positive sample of the single-plex PCR non-small cell lung cancer exon 20 exon H773_V774insH amplified by the reverse primer (R2-9) at the first signal acquisition temperature (60°C). All are negative droplets.
  • Figure 10B is the droplet diagram of the single-plex PCR non-small cell lung cancer EGFR exon 20 H773_V774insH mutation-positive sample amplified by the reverse primer (R2-9) at the second signal acquisition temperature (78°C). There are positive droplets that are distinguished from the background.
  • Forward primer F1-10 (SEQ ID NO:27), is a specific primer designed for the H773_V774insH mutation target sequence of exon 20 of non-small cell lung cancer EGFR.
  • the 16 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A1), and the 5' end of the probe P1-10 (SEQ ID NO:25) 1 to 10 base sequences, i.e. the primer anchor region (A1'), reverse complementary;
  • Reverse primer R2-10 (SEQ ID NO: 28), is a specific primer designed for the H773_V774insH mutation target sequence of exon 20 of non-small cell lung cancer EGFR, the full length of R2-10 is 32bp, and the 1st to 3rd The 21 bases are the target sequence binding region, the 1st to 8th bases at the 5' end are the primer signal detection region (h2), and the 1st base at the 3' end of the probe P1-10 (SEQ ID NO:25) Up to 8 base sequences are identical.
  • the principle of detection using the above primer probes is as follows: when the target is specifically amplified, annealing at a higher first temperature (T1), the forward primer (F1-10) and the reverse primer (R2-10 ) specifically combined with the target sequence 2 and extended to generate double-stranded pre-amplified products, wherein the 5' end to the 3' end of a single-stranded pre-amplified product (S3) is the probe anchor region (A1), and the target Sequence, the reverse complementary sequence of the signal detection region of the primer (h2'); the first signal acquisition (t1>T1) was performed at a lower first signal acquisition temperature (t1), due to the single-stranded preamplification product (S3)
  • the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 3' end is lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), so it does not interact with the probe of the probe (P1-10)
  • the signal detection region (H) is
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation Use non-small cell lung cancer EGFR exon 20 H773_V774insH mutation DNA as a positive sample, and use the reverse primer (R2-10) with the signal detection region sequence to detect the non-small cell lung cancer EGFR exon 20 H773_V774insH mutation target sequence Assays were performed to validate the detectability of single mutations. Pure water was used as no template control (NTC).
  • NTC no template control
  • Digital PCR amplification and signal acquisition (detected by OS-500 digital PCR all-in-one machine and reagent consumables from Beijing Da Microbe Technology Co., Ltd.):
  • the amplification and lighting programs used are: 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing and extension for 30 seconds, a total of 45 cycles; 60°C for the first signal acquisition; 40°C constant temperature incubation for 5 minutes ; 78°C for the second signal acquisition.
  • Figure 11A is the droplet diagram of the positive sample of H773_V774insH mutation in exon 20 of EGFR non-small cell lung cancer EGFR exon 20 amplified by the reverse primer (R2-10) at the first signal acquisition temperature (60°C). It can be seen All are negative droplets (droplets that are on the edge and/or appear to be relatively large are experimental problems, not positive droplets).
  • Figure 11B is the droplet diagram of the positive sample of H773_V774insH mutation in exon 20 of EGFR non-small cell lung cancer EGFR exon 20 amplified by the reverse primer (R2-10) at the second signal acquisition temperature (78°C). It can be seen There are positive droplets that are distinguished from the background.
  • the dual detection primer probe system for detecting influenza A virus (Influenza A virus, IAV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is shown in Table 21:
  • Forward primer F1-11 (SEQ ID NO: 30), is a specific primer designed for the target sequence of respiratory syncytial virus, the full length of F1-11 is 42bp, and the 1st to 29th bases of its 3' end are the target sequence In the binding region, the 1st to 10th bases at the 5' end are the probe anchor region (A), and the 1st to 10th bases at the 5' end of the probe P1-11 (SEQ ID NO: 25) , that is, the primer anchor region (A'), reverse complementary;
  • Reverse primer R1-11 (SEQ ID NO: 29), is a specific primer designed for the target sequence of respiratory syncytial virus, the full length of R1-11 is 54bp, and the 1st to 24th bases at the 3' end are the target sequence In the binding region, the 1st to 6th bases at the 5' end are the signal blocking region (M), and the 7th to 27th bases at the 5' end are the primer signal detection region (h1), and the probe P1- The 26th to 46th base sequences of the 3' end of 11 (SEQ ID NO:25) are identical;
  • Forward primer F2-11 (SEQ ID NO: 31), is a specific primer designed for the target sequence of influenza A virus, the full length of F2-11 is 32bp, and the 1st to 20th bases at the 3' end are the target sequence In the binding region, the 1st to 10th bases at the 5' end are the probe anchor region (A), and the 1st to 10th bases at the 5' end of the probe P1-11 (SEQ ID NO: 25) , that is, the primer anchor region (A'), reverse complementary;
  • Reverse primer R2-11 (SEQ ID NO: 32), is a specific primer designed for the target sequence of influenza A virus.
  • the full length of the R2-11 primer is 31bp, and the 1st to 19th bases at the 3' end are the target In the sequence binding region, the 1st to 8th bases at the 5' end are identical to the 1st to 8th bases at the 3' end of the probe probe P1-11 (SEQ ID NO: 25).
  • the principle of detection using the above primer probes is as follows: when the target is specifically amplified, annealing is performed at a higher first temperature (T1), and the forward primer (F1-11) and the reverse primer (R1-11) are annealed. ) respectively specifically combined with the target sequence 1 (respiratory syncytial virus) and extended to generate double-stranded pre-amplified products, in which a single-stranded pre-amplified product (S1) was anchored from the 5' end to the 3' end in sequence Region (A1), target sequence, reverse complementary sequence (h1') of primer signal detection region, reverse complementary sequence (M') of extension block region; forward primer (F2-11) and reverse primer (R2 -11) Respectively bind specifically to the target sequence 2 (influenza A virus) and extend to generate double-stranded pre-amplified products, in which a single-stranded pre-amplified product (S2) is probed from the 5' end to the 3' end in turn Anchor region (A
  • the probe signal detection region (H) of the probe (P1-11) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplified product (S1), and the probe (
  • the primer anchor region (A1') of P1-11) is reverse-complementary paired with the probe anchor region (A1) at the 5' end of the single-stranded pre-amplification product (S1), and the formation of the probe (P1-11) is obtained.
  • the double-stranded product (D1) hybridizes, and the reverse complement (M') of the extension block region at the 3' end of the single-stranded preamplified product (S1) is not complementary to any part of the probe (P1-11) or target sequence ;
  • the partial sequence of the probe (P1-11) changes from a single strand to a double strand state, the distance between the first detection group and the second detection group changes, and a signal is generated, which can be detected by the instrument.
  • the annealing temperature of the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded preamplified product (S2) is lower than the first annealing temperature (T1) and the first signal collection temperature (t1), Therefore, it does not extend in reverse complementarity with the probe signal detection region (H) of the probe (P1-11); after the first signal acquisition is completed at the first signal acquisition temperature (t1), the single-stranded pre-amplification product is performed (S2) Secondary amplification cycle, the cycle condition is high temperature melting, and annealing (T2 ⁇ T1, and T2 ⁇ t1)) at a lower second annealing temperature (T2), at this time the probe (P1- 11)
  • the probe signal detection region (H) is complementary to the reverse complementary sequence (h2') of the primer signal detection region at the 3' end of the single-stranded preamplification product (S2), and the primer anchor region (A2') of the probe ) and the probe anchor region (A
  • the temperature is raised to a higher second signal collection temperature (t2) to collect signals (t2>t1, and t2> T1)
  • the amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P1-11) forms a double-stranded state
  • the distance between the first detection group and the second detection group The farther the distance, the lower the efficiency of fluorescence resonance energy transfer, so that the fluorescence signal changes and can be detected by the instrument; at this time, the hybrid double-stranded product formed by the single-stranded pre-amplification product (S1) and the probe (P1-11) (D1)
  • a double-stranded structure cannot be formed because the second signal collection temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1).
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation use influenza A virus (Influenza A virus, IAV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) as positive samples, use reverse primers (R1-11 and R2- 11) Detect the target sequences of Influenza A virus (IAV) and Respiratory Syncytial Virus (RSV) to verify the detection ability of double virus infection. Pure water was used as a no-template control (NTC).
  • IAV Influenza A virus
  • RSV Respiratory Syncytial Virus
  • Digital PCR amplification and signal acquisition (detected by OS-500 digital PCR all-in-one machine and reagent consumables from Beijing Da Microbe Technology Co., Ltd.):
  • the amplification and lighting programs used are: 55°C reverse transcription for 15 minutes; 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing extension for 30 seconds, a total of 45 cycles; 60°C for the first signal acquisition ; Denaturation at 94°C for 15 seconds, annealing and extension at 40°C for 20 seconds, a total of 10 cycles; 78°C for the second signal acquisition.
  • Figure 12A is the droplet diagram of the positive samples of Influenza A virus (Influenza A virus, IAV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) at the first signal collection temperature (60°C) in this embodiment, which can be seen If there are positive droplets that are different from the background, they are influenza A virus positive droplets (droplets that are on the edge and/or appear to be relatively large are problems of experimental operation, not positive droplets).
  • Influenza A virus Influenza A virus, IAV
  • RSV respiratory syncytial virus
  • Figure 12B is the droplet diagram of Influenza A virus (Influenza A virus, IAV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) positive samples at the second signal collection temperature (78°C) in this embodiment, you can see
  • the positive droplets that are different from the background are positive droplets for respiratory syncytial virus, and the positive droplets that appear at this temperature are different from the positive droplets that appear at the first signal collection temperature (60° C.).
  • the detection signals of different target sequences can be respectively obtained at different signal detection temperatures (t1, t2) by using the primer-probe composition and the digital PCR detection method of the present invention, and Do not cross each other.
  • Forward primer F1-12 (SEQ ID NO: 35), is a specific primer designed for the target sequence of influenza A virus (Influenza A virus, IAV), the full length of F1-12 is 33bp, and the 1st to 3rd ends of the 3' end
  • the 20 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P1-12 (SEQ ID NO:33) 1 to 10 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R1-12 (SEQ ID NO: 34), is a specific primer designed for the target sequence of influenza A virus (Influenza A virus, IAV), the full length of R1-12 is 47bp, and the 1st to 3rd ends
  • the 20 bases are the target sequence binding region, the 1st to 5th bases at the 5' end are the signal blocking region (M), and the 6th to 24th bases at the 5' end are the primer signal detection region (h1 ), identical to the 30th to 48th base sequence of the 3' end of the probe P1-12 (SEQ ID NO:33);
  • Forward primer F2-12 (SEQ ID NO: 37), is a specific primer designed for the target sequence of influenza B virus (Influenza B virus, IBV), the full length of F2-12 is 33bp, and the 1st to 3rd ends of the 3' end
  • the 20 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P1-12 (SEQ ID NO:33) 1 to 10 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R2-12 (SEQ ID NO: 36) is a specific primer designed for the target sequence of influenza B virus (Influenza B virus, IBV).
  • the 21st base is the target sequence binding region, the 1st to 11th bases at the 5' end and the 17th to 27th bases at the 3' end of the probe P1-12 (SEQ ID NO:33) The sequence is the same.
  • Forward primer F3-12 (SEQ ID NO: 39), is a specific primer designed for the target sequence of respiratory syncytial virus (Respiratory Syncytial Virus, RSV), the full length of F3-12 is 35bp, and the 1st to 3rd ends of the 3' end
  • the 22 bases are the target sequence binding region, the 1st to 10th bases at the 5' end are the probe anchoring region (A), and the 5' end of the probe P1-12 (SEQ ID NO:33) 1 to 10 base sequences, i.e. the primer anchor region (A'), reverse complementary;
  • Reverse primer R3-12 (SEQ ID NO: 38), is a specific primer designed for the target sequence of respiratory syncytial virus (Respiratory Syncytial Virus, RSV), the R3-12 primer is 29bp in full length, and the first to The 19th base is the target sequence binding region, the 1st to 7th bases at the 5' end and the 1st to 7th bases at the 3' end of the probe probe P1-12 (SEQ ID NO:33) The sequence is the same.
  • the principle of detection using the above primer probes is as follows: when the target is specifically amplified, annealing is performed at a higher first temperature (T1), and the forward primer (F1-12) and the reverse primer (R1-12 ) respectively specifically combined with the target sequence 1 (respiratory syncytial virus) and extended to generate double-stranded pre-amplified products, in which a single-stranded pre-amplified product (S1) was anchored from the 5' end to the 3' end in sequence Region (A1), target sequence, reverse complementary sequence (h1') of primer signal detection region, reverse complementary sequence (M') of extension block region; forward primer (F2-12) and reverse primer (R2 -12) Respectively bind specifically to the target sequence 2 (influenza A virus) and extend to generate a double-stranded pre-amplified product, in which a single-stranded pre-amplified product (S2) is a probe from the 5' end to the 3' end in turn Anchor region (A2), target sequence
  • the probe signal detection region (H) of the probe (P1-12) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplified product (S1), and the probe (
  • the primer anchor region (A1') of P1-12) is reverse-complementary paired with the probe anchor region (A1) at the 5' end of the single-stranded pre-amplification product (S1), and the formation of the probe (P1-12) is obtained.
  • the double-stranded product (D1) hybridizes, and the reverse complement (M') of the extension block region at the 3' end of the single-stranded preamplified product (S1) is not complementary to any part of the probe (P1-12) or target sequence ;
  • the partial sequence of the probe (P1-12) changes from a single strand to a double strand state, the distance between the first detection group and the second detection group changes, and a signal is generated, which can be detected by the instrument.
  • the annealing temperature of the sequence (h3') is lower than the first annealing temperature (T1) and the first signal acquisition temperature (t1), so it does not reversely complement the probe signal detection region (H) of the probe (P1-12) Extension; after the first signal acquisition is completed at the first signal acquisition temperature (t1), the second amplification cycle of the single-stranded pre-amplification product (S2) is performed, and the cycle condition is high temperature melting, and at a lower second Annealing (T2 ⁇ T1, and T2 ⁇ t1)) at two annealing temperatures (T2), at this time, the probe signal detection region (H) of the probe (P1-12) is 3' from the single-stranded preamplified product
  • the distance between the first detection group and the second detection group changes to generate a signal; at this time, the single-stranded pre-amplified product (S1)
  • the hybrid double-stranded product (D1) formed with the probe (P1-12) cannot form a double-stranded structure because the second signal collection temperature (t2) is higher than the melting temperature of the hybrid double-stranded product (D1); and because The annealing temperature of the reverse complementary sequence (h3') of the primer signal detection region at the 3' end of the single-stranded preamplification product (S3) is lower than the second annealing temperature (T2), so it does not interact with the probe of the probe (P1-12)
  • the signal detection region (H) is extended in reverse complementarity; after the second signal acquisition is completed at the second signal acquisition temperature (t2), the second amplification cycle of the single-stranded pre-amplification product (S3) is carried out, and the cycle condition
  • the probe anchor region (A3) is reverse-complementary paired, and the 3' end of the single-stranded preamplified product (S3) can continue to extend to the 5' end of the probe (P1-12) to obtain the probe (P1-12) Part or all of the reverse complementary sequence, that is, to complete the secondary amplification using the single-stranded pre-amplification product (S3) as the primer and the probe (P1-12) as the template, and obtain the formation of the probe (P1-12)
  • the amplified double-stranded product (D3) at this time, all or part of the sequence of the probe (P1-12) changes from single-stranded to double-stranded, and the distance between the first detection group and the second detection group changes , to generate a signal; after the cyclic amplification at the third annealing temperature (T3), the temperature is raised to a higher third signal acquisition temperature (t3) for the second signal acquisition (t3>t2, and t3>T2 ), at this time, the hybrid double
  • 2X PCR Reaction Buffer includes: 3mM MgCl 2 , 30mM Tris-HCl at pH 8.3, 0.5mM dNTP and 70mM (NH 4 ) 2 SO 4 .
  • Sample preparation use influenza A virus (Influenza A virus, IAV), influenza B virus (Influenza B virus, IBV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) as positive samples, and use the detection area with different signals
  • the reverse primers (R1-12/R2-12/R3-12) of the sequence are effective against influenza A virus (Influenza A virus, IAV), type B influenza virus (Influenza B virus, IBV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) target sequences were detected to verify the detection ability of triple virus infection. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Digital PCR amplification and signal acquisition (detected by OS-500 digital PCR all-in-one machine and reagent consumables from Beijing Da Microbe Technology Co., Ltd.):
  • the amplification and lighting programs used are: 55°C reverse transcription for 15 minutes; 95°C pre-denaturation for 2 minutes; 94°C denaturation for 10 seconds, 56°C annealing extension for 30 seconds, a total of 45 cycles; 61°C for the first signal acquisition ; Denaturation at 94°C for 15 seconds, annealing and extension at 52°C for 20 seconds, a total of 5 cycles; 68°C for the second signal acquisition. Denaturation at 94°C for 15 seconds, annealing and extension at 40°C for 20 seconds, a total of 5 cycles; 76°C for the third signal acquisition.
  • Fig. 13A is the first signal collection temperature of influenza A virus (Influenza A virus, IAV), influenza B virus (Influenza B virus, IBV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) positive samples in this embodiment (61°C) droplet diagram, you can see positive droplets different from the background, which are positive droplets for influenza A virus.
  • influenza A virus Influenza A virus, IAV
  • influenza B virus Influenza B virus, IBV
  • RSV respiratory syncytial virus
  • Fig. 13B is the second signal collection temperature of influenza A virus (Influenza A virus, IAV), influenza B virus (Influenza B virus, IBV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) positive samples in this embodiment (68°C), you can see positive droplets that are different from the background, which are positive droplets of influenza B virus, and the positive droplets that appear at this temperature are the same as the first signal collection temperature (61°C) The location of the positive droplet that appears varies.
  • influenza A virus Influenza A virus, IAV
  • influenza B virus Influenza B virus, IBV
  • respiratory syncytial virus Respiratory Syncytial Virus, RSV
  • Fig. 13C is the third signal collection temperature of influenza A virus (Influenza A virus, IAV), influenza B virus (Influenza B virus, IBV) and respiratory syncytial virus (Respiratory Syncytial Virus, RSV) positive samples in this embodiment
  • the droplet diagram at (76°C) is a positive droplet for respiratory syncytial virus, and the positive droplet at this temperature is consistent with the first signal collection temperature (61°C) and the second signal collection temperature (68°C) Positive droplet positions vary.
  • the detection signals of different target sequences can be respectively obtained at different signal detection temperatures (t1, t2, t3) by using the primer-probe composition and the digital PCR detection method of the present invention , and do not cross each other.
  • first, second and the like used in this application may be used to describe various elements herein, but these elements are not limited by these terms. These terms are only used to distinguish one element from another element.
  • first movement information could be termed second movement information, and, similarly, second movement information could be termed first movement information, without departing from the scope of the present application.
  • Both the first movement information and the second movement information are movement information, but they are not the same movement information.
  • the term “multiple” used in the embodiments of the present application refers to two or more than two.
  • the multiple time-sharing nucleic acid detection device of this embodiment is used to detect the sample to be tested.
  • the sample to be tested may include one component to be tested, or two, three, four, five or even more components to be tested.
  • multiple detection of the components to be tested can be realized by the multiple time-sharing nucleic acid detection device of this embodiment.
  • the multiple time-sharing nucleic acid detection device in this embodiment includes a reaction liquid storage unit 1 , a temperature adjustment unit 2 , a signal detection unit 3 , and a control unit 4 .
  • the reaction liquid container 1 is used to accommodate the sample containing the components to be tested and the reagents capable of reacting with the components to be tested, and provides a place for various reactions between the components to be tested and the reagents.
  • the temperature adjusting part 2 is used to adjust the temperature of the mixed solution in the reaction solution containing part 1, so that the component to be measured and the reagent react differently at different temperatures.
  • the signal detection unit 3 is used to detect a signal generated by a specific substance in the mixed liquid after a specific reaction, and the signal may be an optical signal or an electrical signal.
  • the controller 4 is used to control the temperature adjusting part 2 to adjust the temperature of the mixed liquid in the reaction liquid containing part 1 in different temperature modes, and control the signal detecting part 3 to detect the signal generated by the specific substance in the mixed liquid.
  • the sample is body fluid from a human body or an animal body, and the sample includes different components to be tested, and the components to be tested can be nucleic acid or protein.
  • the technical solution is described by taking the component to be detected as nucleic acid.
  • nucleic acid extraction and purification operations can be performed to extract nucleic acids. This operation can be performed manually or with a specialized nucleic acid extraction instrument.
  • nucleic acid extraction and purification operations may not be performed.
  • the sample contains different test nucleic acid molecules, for example, the first test component (the first test nucleic acid), the second test component (the second test nucleic acid), the third test component (the third test nucleic acid ), the fourth component to be tested (the fourth nucleic acid to be tested), the fifth component to be tested (the fifth nucleic acid to be tested) and the like.
  • the first test component the first test nucleic acid
  • the second test component the second test nucleic acid
  • the third test component the third test nucleic acid
  • the fourth component to be tested the fourth nucleic acid to be tested
  • the fifth component to be tested the fifth nucleic acid to be tested
  • the reagent includes an amplification substance for amplifying nucleic acid and a labeling substance for labeling nucleic acid.
  • the amplification substance can amplify different nucleic acid molecules.
  • the labeling substance can combine with nucleic acid at a certain temperature to generate a detectable signal.
  • the temperature ranges for different nucleic acids to bind to the labeled substances are different, and the temperature ranges for the separation from the labeled substances after binding are also different.
  • the primers in the amplification substances for amplifying different nucleic acids to be tested are different, and the marker substances for marking different nucleic acids to be tested can be the same or different. In this application, the same marker substance is used to mark different nucleic acids to be tested.
  • the microfluid may be a tiny droplet formed in an emulsion, or may be a tiny amount of liquid accommodated in micropores on a container. Contained in each microfluidic is at most one unit of nucleic acid molecule and sufficient reagents to amplify and label the plurality of target nucleic acids.
  • each first nucleic acid to be tested and each second nucleic acid to be tested are assigned In different microfluids, there are sufficient reagents in one microfluid, which can meet the needs of the first nucleic acid to be detected in the amplification and labeling microfluid, and can also meet the needs of the second nucleic acid to be detected in the amplification and labeling microfluid. nucleic acid testing needs.
  • the process of making micro-liquid can be the way of high-frequency vibration to form micro-liquid: absorb the sample and a sufficient amount of reagent through the micro-pipe, extend the micro-pipe under the oily liquid and vibrate at high frequency, during the vibration process, the liquid in the micro-pipe
  • the micro-liquid of the sample and reagent is discharged at a certain speed, so that the micro-droplet is thrown out by vibration, that is, the micro-liquid, which is incompatible with the oily liquid and does not react.
  • each micro-division has at most 1 unit of nucleic acid molecule.
  • the reaction solution containing part is a box-shaped container with an upward opening, and the nucleic acid to be tested and the reagent are added to the box-shaped container through the liquid filling mechanism (that is, the high-frequency vibration as described above to form micro-liquid), Nucleic acid to be detected and reagents can also be added to the boxed container by laboratory workers.
  • the liquid filling mechanism that is, the high-frequency vibration as described above to form micro-liquid
  • Nucleic acid to be detected and reagents can also be added to the boxed container by laboratory workers.
  • the reaction solution containing part is a chamber connected with the microfluidic channel, and the nucleic acid to be tested and the reagent are driven into the chamber by the microfluidic driving structure.
  • the microfluid containing the nucleic acid to be tested and reagents is wrapped in about 20,000 microfluids, and each microfluid contains one or no nucleic acid molecule to be detected There are several components to be tested, which are all dispensed into different microfluidics (ideally, there may be more than one nucleic acid molecule to be tested in a small microfluidic).
  • the reaction solution container is used as a boxed container for illustration, and an oily liquid is injected into the reaction solution container.
  • the oily liquid is not compatible with or reacts with samples and reagents.
  • the micro-liquid is formed in the oily liquid by means of frequency vibration, and the micro-liquid is flatly spread on the bottom of the reaction liquid container under the action of its own gravity.
  • the orifice plate 100 is composed of a plurality of reaction solution containing parts 1 , and the bottom of the reaction solution containing part 1 is in the shape of a flat plate, so that a plurality of micro-liquids can be spread on the bottom of the reaction solution containing part 1 .
  • the temperature regulating part 2 is arranged below, above or on the side of the reaction solution containing part 1, and can provide heat to the reaction solution containing part 1, and can also absorb the heat of the reaction solution containing part 1, thereby The function of heating and cooling the micro liquid in the reaction solution containing part 1 is realized.
  • the temperature adjustment unit 2 can change the temperature of the minute liquid in the reaction liquid storage unit 1 within the range of 4°C to 105°C.
  • FIG. 14A when the reaction solution container 1 is a box-shaped container with an upward opening, a special place for placing the reaction solution container can be provided on the temperature adjustment part, and the temperature of the reaction solution container 1 needs to be adjusted. , place it on the placement slot.
  • the temperature adjustment part 2 when the reaction solution containing part 1 is a chamber connected with the microchannel, the temperature adjustment part 2 can be arranged in a reasonable position of the reaction solution containing part 1 in consideration of heating efficiency and other factors.
  • the temperature adjustment part 2 is arranged under the orifice plate 100 composed of a plurality of reaction solution containing parts 1, and is in contact with the flat orifice plate 100 for heating and cooling operations, so that each of the orifice plate 100
  • the microfluid in the reaction liquid container 1 changes in temperature as the temperature of the orifice plate 100 rises and falls.
  • the signal detection part 3 is arranged near the reaction solution containing part 1, and according to the difference of the signal generated by the micro-liquid in the reaction solution containing part 1, the signal detecting part can be a device for detecting optical signals (such as fluorescent signals) (camera or optoelectronic device) or a device capable of detecting electrical signals.
  • the signal detection unit is a camera, it is possible to take a picture of the signal spread in the reaction solution storage unit.
  • the signal detection part 3 can be moved relative to the reaction solution containing part 1.
  • the controller controls the driving mechanism to move the signal detecting part 3 to a position close to the reaction solution containing part 1 (for example, as shown in FIG. 14A above).
  • the signal detection unit 3 may also be fixedly arranged relative to the reaction solution storage unit.
  • control unit 4 is communicably connected to the temperature adjustment unit 2 and the signal detection unit 3 .
  • the control unit may include a first control unit and a second control unit (not shown in the figure), the first control unit is communicatively connected with the temperature adjustment unit to control the temperature adjustment unit to operate in different modes, and the second control unit It is communicatively connected with the signal detection part to control the signal detection part to collect the signal from the micro liquid in the reaction solution containing part.
  • the control unit 4 can also be a single component, which can simultaneously control the temperature adjustment unit and the signal detection unit.
  • the test sample contains the first test component (the first test nucleic acid) and the second test component (the second test nucleic acid).
  • first test association produced by the first test nucleic acid Product (after the nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage, the corresponding product to be tested is produced, usually, the product to be tested is a nucleic acid), It can be combined with the labeling substance in the reagent within the first binding temperature range; the second test related product produced by the second test nucleic acid can be combined with the labeling substance in the reagent within the second binding temperature range, and the first binding The maximum temperature of the temperature range and the second bonding temperature range are different.
  • the highest temperature in the second combining temperature range is lower than the highest temperature in the first combining temperature range.
  • the first correlation product to be detected can bind to the marker substance when the temperature is lower than or equal to temperature A1
  • the second correlation product to be detected can bind to the marker substance when the temperature is lower than or equal to temperature A2, wherein temperature A2 is lower than temperature A1.
  • the first associated product to be detected can be separated from the labeled substance in the reagent within the first separation temperature range
  • the second associated product to be detected can be separated from the labeled substance in the reagent within the second separated temperature range.
  • the first separated temperature range and The lowest temperature of the second separation temperature range is different.
  • the lowest temperature in the second separation temperature range is higher than the lowest temperature in the first separation temperature range.
  • the first associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature B1
  • the second associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature B2, wherein the temperature B2 is higher than the temperature B1.
  • the mixed solution of the sample and the reagent including the amplification substance and the labeling substance
  • the controller performs the following control:
  • the horizontal axis represents time
  • the vertical axis represents temperature value.
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid container, so that the nucleic acid to be detected in the sample produces an associated
  • the associated product to be detected (the nucleic acid to be tested comprises the first nucleic acid to be tested and the second nucleic acid to be tested, but exists in different micro-liquids); in this process, the micro-liquids will experience multiple temperature cycles, and in each temperature cycle , the micro-liquid will change to several different temperatures, and stay at this temperature for a certain period of time, and the number of cycles can be multiple, such as 20-60, for example, about 40.
  • the microfluid will reach a high temperature state (temperature t5), the double-stranded nucleic acid becomes a single-stranded nucleic acid, and then the microfluid reaches a low temperature state (temperature T1), and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be respectively at different temperatures, the extension temperature being higher than the annealing temperature).
  • the number of different nucleic acids to be tested and/or related products to be tested in the sample increases (that is, the amount of nucleic acids to be tested and/or related products to be tested in each microfluid increases, However, the type of nucleic acid to be tested remains the same), and it is easier to be detected.
  • the annealing temperature T1 (low temperature T1) can take a value within the first range, but cannot coincide with the subsequent T2 range (the second range), and the values in the first range are greater than the values in the second range.
  • the microfluid before performing 40 temperature cycles, can be heated to temperature t0 to pre-denature the nucleic acid to be tested in the sample for a period of time (about 2 minutes). Pre-denaturing for a period of time is to change the nucleic acid from a double-stranded state to a single-stranded state.
  • the controller can control the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid container to temperature t1 and maintain it for a certain period of time, as shown in Figure 15A to Figure 15D, this interval is Interval 1, and the controller
  • the signal detection unit can be controlled to perform a detection operation once, and the signal detected this time is used as a blank signal.
  • the temperature t1 can be higher or lower than the annealing temperature T1; after the cycle is completed, the temperature adjustment part can also keep the temperature of the micro-liquid at the state of temperature T1, and the controller controls the signal detection part to perform a detection action. Since the signal obtained by this detection is a blank signal, in order to improve efficiency, this detection action can also be omitted.
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid container in the first temperature adjustment mode.
  • the micro-liquid produces a first association to be detected that can be combined with the labeling substance.
  • the product, the labeled substance combined with the first related product to be detected will generate a signal, and in this state, the controller controls the signal detection part to detect the signal of the reaction solution containing part to obtain the first signal.
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected or the second nucleic acid to be detected, After the adjustment of the first temperature adjustment mode, only the microfluid containing the first nucleic acid to be detected can generate a signal. If multiple microfluids overlap, the signal sent by this microfluid is an invalid signal; a single microfluid that does not overlap The signal sent out is an effective signal, and this type of micro-liquid is the first part of the micro-liquid, that is, only the first part of the micro-liquid produces an effective signal.
  • the amplifying substance for amplifying the first nucleic acid to be detected and the labeling substance for marking the first associated product to be detected in the microfluid are the first reagent.
  • the first temperature adjustment mode includes an incubation stage and a signal collection stage, in which a specific type of associated product to be detected that can be combined with a marker substance is produced in the incubation stage, and certain types of associated products to be detected in a specific type are realized in the signal collection stage Without combining with the labeled substance, the remaining related product to be detected remains bound to the labeled substance, and completes the signal detection of the signal detection part to the reaction solution containing part. If after incubation in the incubation stage of the first temperature regulation mode, only one associated product to be detected binds to the labeled substance, then in the signal collection stage, the state of binding of the associated product to be detected to the labeled substance is maintained.
  • the temperature regulating part In the incubation stage, in order to realize the binding of the first associated product to be detected with the labeling substance, the temperature regulating part periodically changes the temperature of the micro-liquid in the reaction solution containing part so that the reaction solution containing part repeatedly drops from a high temperature to a low temperature T2, or The temperature adjusting part maintains the reaction liquid containing part at a constant temperature T2, and the temperature T2 is lower than the annealing temperature T1.
  • T2 the temperature of the micro-liquid in the reaction solution containing part
  • the first associated product to be detected in the first part of the micro-liquid combines with the first reagent to generate a signal.
  • the temperature T2 can take values within the second range, but cannot coincide with the subsequent T3 range (third range), and the values in the second range are all greater than the values in the third range, and the values in the second range are all greater than the values in the third range. less than the value in the first range.
  • the control part controls the temperature adjustment part to periodically change the temperature of the micro liquid in the reaction solution containing part.
  • temperature in each temperature cycle, the first associated product to be tested produced by the first nucleic acid to be tested is combined with the labeled substance at T2, and melts with the labeled substance at the high temperature t5.
  • the quantity of the first associated product to be detected bound by the marker substance reaches an ideal maximum, and the number of cycles is about 15, for example, 10 cycles.
  • the controller controls the signal detection part to perform signal detection on the microfluid so as to obtain the first signal.
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part to temperature t2 (temperature t2 has not reached the temperature that can separate/melt the first related product to be tested from the labeled substance), so that the first related product to be tested and the labeled substance are kept in a combined state for a certain period of time. This period of time is interval 2.
  • the second controller controls the signal detection part to detect the signal of the reaction liquid storage part to obtain the first signal.
  • the first signal includes the effective signal generated by the first part of the microfluid, and if there is an overlapping microfluid, it also includes the invalid signal generated by the overlapping microfluid, which will be excluded in the subsequent analysis process.
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution container to maintain the temperature at T2 for a certain period of time to control the temperature of the micro-liquid. Incubation is carried out, and the time may be 10-1800 seconds. After the incubation time is over, the signal detection part is controlled to perform signal detection on the reaction solution containing part to obtain the first signal.
  • control part can also control the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part to temperature t2, so that the first to-be-tested The nucleic acid and the labeling substance remain in a combined state for a certain period of time.
  • This period of time is Interval 2.
  • the control controller controls the signal detection unit to perform signal detection on the reaction solution storage unit to obtain the first signal.
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part in the second temperature adjustment mode. After the adjustment in the second temperature adjustment mode, only the second related product to be detected and the reagent are included in the pre-amplification product. When the labeled substance in the reaction solution is combined, the labeled substance combined with the second related product to be detected will generate a signal. In this state, the controller controls the signal detection part to detect the signal of the reaction solution containing part to obtain the second signal.
  • this nucleic acid molecule may be the first nucleic acid to be detected, or the second nucleic acid to be detected, after the second temperature
  • this nucleic acid molecule may be the first nucleic acid to be detected, or the second nucleic acid to be detected, after the second temperature
  • only the microfluid where the second nucleic acid to be tested is located can generate a signal. If multiple microfluids overlap, the signal sent by this microfluid is an invalid signal; the signal sent by a single microfluid that does not overlap is Effective signal, this type of micro-liquid is the second part of the micro-liquid, that is, only the second part of the micro-liquid produces an effective signal.
  • the amplification substance for amplifying the second nucleic acid to be detected in the microfluid and the labeling substance for marking the second nucleic acid to be detected are the second reagents.
  • the second temperature adjustment mode includes an incubation stage and a signal collection stage.
  • the incubation stage the combination of specific types of related products to be tested and labeled substances is realized.
  • a related product to be detected remains in a combined state with the labeled substance, and the signal detection of the signal detection part to the reaction solution containing part is completed.
  • the temperature regulating part periodically changes the temperature of the micro-liquid in the reaction solution containing part so that the reaction solution containing part repeatedly drops from a high temperature to a low temperature T3, or
  • the temperature adjustment unit maintains the reaction liquid storage unit at a constant temperature T3, which is lower than the temperature T2.
  • the temperature T3 can take values within the third range, but cannot coincide with the subsequent T4 range (the fourth range), and the values in the third range are all greater than the values in the fourth range, and the values in the third range are all greater than the values in the fourth range. less than a value in the second range.
  • the control part controls the temperature adjustment part to periodically change the micro liquid in the reaction solution containing part.
  • the second associated product to be tested produced by the second nucleic acid to be tested combines with the labeled substance at T3, and melts with the labeled substance at the high temperature t5.
  • the number of the second associated product to be detected combined with the labeled substance reaches an ideal maximum, and the number of cycles is about 15, for example, 10 cycles.
  • the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part to temperature t3 (temperature t3 reaches the point where the first associated product to be detected is separated from the labeled substance. /melting temperature, does not reach the temperature that can separate/melt the second associated product to be measured from the labeled substance), and separate/melt the first associated product to be measured from the labeled substance (the first part of the microliquid in the first part)
  • the related product to be tested is separated/melted from the labeled substance, and the signal due to the combination disappears), so that the second related product to be tested and the labeled substance remain in a combined state for a certain period of time.
  • the device controls the signal detection part to detect the signal of the reaction liquid storage part to obtain the second signal.
  • the second signal includes effective signals generated by the second part of the micro-liquids, and if there are overlapping micro-liquids, it also includes invalid signals generated by the overlapping micro-liquids, and such invalid signals will be excluded in the subsequent analysis process.
  • the first associated product to be detected will also combine with the marker substance. Therefore, in each temperature cycle, the first associated product to be detected and the second associated product to be detected will undergo a process of binding and separating from the marker substance.
  • the control signal detection unit performs signal detection on the reaction solution storage unit to obtain the second signal.
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part to maintain the temperature at T3 for a certain period of time to control the temperature of the micro-liquid. Incubation is carried out, and the time may be 10-1800 seconds. After the incubation time is over, the signal detection part is controlled to perform signal detection on the reaction solution containing part to obtain a second signal.
  • both the first related product to be tested and the second related product to be tested will combine with the labeling substance, and after the incubation (that is, the signal detection stage), the control part controls the temperature regulation part to transfer the microbes in the reaction solution containing part to The temperature of the liquid is adjusted to temperature t3 and maintained for a certain period of time.
  • the first related product to be tested is melted/separated from the labeled substance; , the first related product to be tested is separated/unchained from the marker substance and maintains the separated/unchained state, and the second related product to be tested is still combined with the labeled substance.
  • the controller controls the signal detection part to The reaction solution container performs signal detection to obtain a second signal.
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part in the first temperature adjustment mode, so that the nucleic acid in the micro-liquid is amplified by the amplification substance in the reagent, and the quantity At the same time, its product can be combined with the labeling substance in the reagent.
  • the microfluid will go through multiple temperature cycles, and in each temperature cycle, the microfluid will change to multiple different temperatures and stay at this temperature for a certain period of time, with about 40 cycles.
  • the annealing temperature of the amplification process is temperature T2.
  • the annealing temperature has reached the temperature at which the first associated product to be detected (for example, the amplification product of the first nucleic acid to be detected) can be combined with the labeling substance, therefore, at this first temperature
  • the amplification of the nucleic acid to be tested and the combination of the amplified product and the marker substance are simultaneously realized.
  • the first part of the microfluid containing the first nucleic acid to be tested will generate a signal.
  • the controller controls the signal
  • the detection part detects the signal of the reaction liquid containing part to obtain the first signal.
  • the specific method may be that after 40 cycles in the first temperature adjustment mode, the controller controls the temperature regulating part to set the temperature of the micro liquid in the reaction liquid containing part to Adjust to temperature t2 to keep the first related product to be tested in a combined state with the labeled substance for a certain period of time. This period of time is interval 2. In interval 2, the controller controls the signal detection part to perform signal detection on the reaction solution containing part to obtain first signal. Of course, it is also possible to keep the temperature of the micro-liquid in the reaction solution container at T2 after the cycle is completed, and the controller controls the controller to control the signal detection part to perform signal detection on the reaction solution container to obtain the first signal.
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid container in the second temperature adjustment mode. This control is the same as that in Embodiment 13 and will not be repeated here.
  • the sample also includes a third component to be tested (the third nucleic acid to be tested), and it should be noted that the third nucleic acid to be tested or the third associated product to be tested (the third nucleic acid to be tested by polymerase chain reaction After operations such as (PCR) and/or nucleic acid hybridization and/or nuclease cleavage etc., produce the 3rd related product to be tested corresponding to it, usually, the 3rd related product to be tested is nucleic acid) can be in certain temperature range and reagent In this embodiment, the highest temperature at which the third nucleic acid to be tested or the third related product to be tested is combined with the labeled substance is lower than the temperature at which the second nucleic acid to be tested or the second related product to be tested is combined with the labeled substance.
  • the third nucleic acid to be tested or the third associated product to be tested the third nucleic acid to be tested by polymerase chain reaction After operations such as (PCR) and/or nucleic acid hybridization and/or
  • the third nucleic acid to be detected or the third associated product to be detected can be combined with the labeling substance when the temperature is lower than or equal to temperature A3, and the temperature A3 is lower than the temperature A2 (the second nucleic acid to be detected or the second associated product to be detected at temperature When it is lower than or equal to the temperature A2, it can combine with the marker substance).
  • the third nucleic acid to be detected or the third associated product to be detected can be separated from the labeled substance in the reagent within a certain temperature range.
  • the minimum temperature at which the third nucleic acid to be detected or the third associated product to be detected is separated from the labeled substance It is higher than the minimum temperature at which the second nucleic acid to be detected or the second associated product to be detected is separated from the label substance.
  • the third nucleic acid to be detected or the third associated product to be detected can be separated from the labeling substance when the temperature is higher than or equal to temperature B3, and the temperature B3 is higher than the temperature B2 (the second nucleic acid to be detected or the second associated product to be detected at temperature It can be separated from the marker substance when it is higher than or equal to the temperature B2).
  • the controller controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part in the second temperature adjustment mode, and the controller controls the signal detection part to obtain the second signal, the controller also performs the following control:
  • the controller controls the temperature adjustment part to adjust the temperature of the micro liquid in the reaction liquid containing part in the third temperature adjustment mode. After the adjustment in the third temperature adjustment mode, only the third nucleic acid to be tested or the third related product to be tested and The labeling substance in the reagent is combined, and the labeling substance generates a signal due to the combination. In this state, the controller controls the signal detection part to detect the signal of the reaction solution storage part to obtain a third signal.
  • this nucleic acid molecule may be the first nucleic acid to be detected or the second nucleic acid to be detected or the third nucleic acid to be detected.
  • the signal sent by this microfluid is an invalid signal, and the signal sent by a single microfluid that does not overlap
  • the signal is an effective signal, and this type of micro-liquid is the third part of the micro-liquid, that is, only the third part of the micro-liquids produces an effective signal.
  • the amplification substance for amplifying the third nucleic acid to be detected and the labeling substance for marking the third nucleic acid to be detected in the microfluid are the third reagent.
  • the third temperature adjustment mode includes an incubation stage and a signal collection stage.
  • the incubation stage the combination of a specific type of nucleic acid to be tested or its corresponding associated product to be tested and a labeling substance is realized
  • the signal collection stage certain types of certain types of The nucleic acid to be tested or its corresponding related product to be tested is separated from the labeling substance, and the remaining nucleic acid to be tested or its corresponding related product to be tested is kept in combination with the labeling substance, and the signal detection part is separated from the reaction solution containing part. signal detection.
  • the temperature regulating part periodically changes the temperature of the micro-liquid in the reaction solution containing part so that the reaction solution containing part repeatedly changes from The high temperature is lowered to the low temperature T4, or the temperature adjustment part keeps the reaction liquid storage part at a constant temperature T4, and the temperature T4 is lower than the temperature T3.
  • the temperature of the microfluid in the reaction solution containing part is T4
  • the third nucleic acid to be detected or the third associated product to be detected in the microfluid containing the third nucleic acid to be detected combines with the third reagent to generate a signal.
  • the temperature T4 can take values within the fourth range, but it cannot coincide with the acceptable range (fifth range) of the subsequent T5 (if there is a fourth temperature adjustment mode), and the values in the fourth range are greater than those in the fifth range Values, the values in the fourth range are all smaller than the values in the third range.
  • the control part controls the temperature adjustment part to periodically change the temperature of the micro liquid in the reaction solution containing part. Temperature, in each temperature cycle, the third nucleic acid to be detected or the third associated product to be detected is combined with the labeling substance at a low temperature T4, and is melted/separated from the labeling substance at a high temperature t5, after repeated cycles , the quantity of the third test nucleic acid or the third test-associated product combined with the labeling substance reaches an ideal maximum, and the number of cycles is about 15 (for example, 9 cycles, 10 cycles, etc.).
  • the control part controls the temperature adjustment part to adjust the temperature of the micro liquid in the reaction solution containing part to temperature t4 (the temperature t4 reaches the first nucleic acid to be detected (or the first nucleic acid to be tested). detection associated product) and the temperature at which the second nucleic acid to be detected (or the second associated product to be detected) is separated/melted from the marker substance, and the temperature for separating/melting the third nucleic acid to be detected/the third associated product to be detected from the marker substance has not been reached.
  • the first nucleic acid to be detected (or the first associated product to be detected) is separated/melted from the labeling substance (in the microfluid containing the first nucleic acid to be detected, the first nucleic acid to be detected/the first nucleic acid to be detected
  • the associated product is separated/melted from the labeled substance, and the signal due to the combination disappears)
  • the second nucleic acid to be tested/the second associated product to be tested is separated/melted from the labeled substance (in the microfluid containing the second nucleic acid to be tested,
  • the second nucleic acid to be detected/the second associated product to be detected is separated/melted from the marker substance, and the signal due to the combination disappears)
  • the third nucleic acid to be detected/the third associated product to be detected is kept in a combined state with the marker substance, and maintained For a certain period of time, the period of time is interval four, in which the controller controls the signal detection unit to perform signal detection on the reaction solution container to obtain a third signal.
  • the first nucleic acid to be detected (or the associated product to be detected) and the second nucleic acid to be detected (or the second nucleic acid to be detected) associated product) will also bind to the labeled substance. Therefore, in each temperature cycle, the first nucleic acid to be detected (or the first associated product to be detected), the second nucleic acid to be detected (or the second associated product to be detected) and the third nucleic acid to be detected (or the third associated product to be detected) products) will go through the process of binding and separating with the labeled substance.
  • the temperature t4 reaches the first nucleic acid to be detected (or the first associated product to be detected) and the second nucleic acid to be detected (or the second associated product to be detected) product) and the labeling substance separation/melting temperature, but before reaching the temperature that can separate/melt the third nucleic acid to be detected (or the third associated product to be detected) from the labeling substance), the first nucleic acid to be detected (or The first test related product) and the second test nucleic acid (or the second test related product) are melted/separated from the marker substance, and at this time, only the third test nucleic acid (or the third test related product) is separated from the labeled substance.
  • the labeling substance remains bound, and in this state, the signal detection unit is controlled to perform signal detection on the reaction solution storage unit to obtain a third signal.
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution container to maintain the temperature at T4 for a certain period of time to incubate the micro-liquid, This time can be 10-1000 seconds.
  • the signal detection part is controlled to perform signal detection on the reaction solution containing part to obtain a third signal.
  • the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part to temperature t4 and maintain it for a certain period of time, as shown in Figure 15D and Figure 16D As shown, the time period is interval four, and in interval four, both the first nucleic acid to be detected (or the first associated product to be detected) and the second nucleic acid to be detected (or the second associated product to be detected) are separated/melted from the marker substance And maintain the separation/melting state, the third nucleic acid to be tested (or the third related product to be tested) is still combined with the marker substance, in this state (section 4), the controller controls the signal detection part to send
  • the difference between this embodiment and embodiment 13 is that: due to the different components contained in the sample, the signal obtained in interval 1 of Figure 15A- Figure 15D is not a blank signal, but a micro liquid containing a certain component to be tested generated signal.
  • the component to be detected is the fourth component to be detected (the fourth nucleic acid to be detected).
  • the sample also includes a fourth component to be tested (the fourth nucleic acid to be tested), the fourth nucleic acid to be tested can be amplified by the amplification substance in the reagent, and the resulting amplification product (the fourth nucleic acid to be tested) The associated product to be detected) can be combined with the labeling substance in the reagent, and the labeling substance combined with the fourth nucleic acid amplification product to be detected can generate a detectable signal. It should be noted that the fourth nucleic acid to be detected can be amplified and combined with the labeling substance within a certain temperature range.
  • nucleic acids to be detected for example, the first nucleic acid to be detected and the second nucleic acid to be detected can be amplified by the amplification substance in the reagent, but their products cannot combine with the labeling substance in the reagent.
  • the fourth associated product to be detected can combine with the labeling substance when the temperature is lower than or equal to the temperature A4, and A4 is higher than the temperature A1 (the first nucleic acid to be detected or its associated product can be combined with the labeled substance when the temperature is lower than or equal to the temperature A1 material combination).
  • the fourth nucleic acid to be detected or its related products can be separated/melted from the marker substance within a certain temperature range.
  • the lowest temperature at which nucleic acids or their associated products are separated from labeled substances For example, the fourth nucleic acid to be detected or its related products can be separated from the marker substance when the temperature is higher than or equal to the temperature B4, and the temperature B4 is lower than the temperature B1.
  • the annealing temperature T1 At the annealing temperature T1, other nucleic acids to be detected in the sample, for example, the first nucleic acid to be detected and the second nucleic acid to be detected are only amplified during this process, but not combined with the labeling substance in the reagent;
  • the test nucleic acid can be amplified and can be combined with the labeling substance.
  • the fourth nucleic acid to be detected or its associated product remains in a state of binding to the labeling substance, and the signal detected by the signal detection unit is no longer a blank signal, but a signal generated by the microfluid containing the fourth nucleic acid to be detected.
  • the temperature t1 may be higher than or lower than or equal to the annealing temperature T1.
  • the control unit controls the temperature adjustment unit to adjust the temperature of the micro liquid in the reaction solution storage unit to the temperature T2, the fourth standby
  • the detection nucleic acid or its related product is combined with the labeling substance, and the first nucleic acid to be tested or its related product is combined with the labeling substance;
  • the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction solution containing part to temperature t2, The fourth nucleic acid to be detected or its associated product is separated/melted from the labeling substance, and the first nucleic acid to be detected or its associated product remains bound to the labeling substance.
  • the controller controls the temperature adjustment unit to adjust the temperature of the reaction solution container in the second temperature adjustment mode
  • the controller controls the temperature adjustment unit to adjust the temperature of the micro liquid in the reaction solution container to temperature T3
  • the fourth standby The nucleic acid to be tested or its associated product is combined with the labeling substance, the first nucleic acid to be tested or its associated product is combined with the labeling substance, and the second nucleic acid to be tested or its associated product is combined with the labeling substance;
  • the temperature of the micro-liquid in the liquid is adjusted to temperature t3
  • the fourth nucleic acid to be detected or its associated product and the first nucleic acid to be detected or its associated product are separated/melted from the labeling substance, and the second nucleic acid to be detected or its associated product is separated from the labeling substance.
  • the product remains bound to the labeled substance.
  • Temperature t1, t2, t3, t4, t5 in above-mentioned embodiment 13 to embodiment 16 can be understood as follows:
  • the fourth nucleic acid to be detected or its associated product can remain in a state of binding to the labeling substance in the reagent; if the sample does not contain the fourth nucleic acid to be detected, then No nucleic acid to be detected or its related products are combined with the labeled substance in the reagent;
  • the fourth reagent, the first reagent, the second reagent, and the third reagent in the above-mentioned embodiment 13 to embodiment 16 can be understood as follows:
  • the fourth reagent includes an amplification substance capable of amplifying the fourth nucleic acid to be detected and a labeling substance capable of labeling the fourth nucleic acid to be detected;
  • the first reagent includes an amplification substance capable of amplifying the first nucleic acid to be detected and a labeling substance capable of labeling the first nucleic acid to be detected;
  • the second reagent includes an amplification substance capable of amplifying the second nucleic acid to be detected and a labeling substance capable of labeling the second nucleic acid to be detected;
  • the third reagent includes an amplification substance capable of amplifying the third nucleic acid to be detected and a labeling substance capable of labeling the third nucleic acid to be detected.
  • the primers in the amplification substances for amplifying the fourth nucleic acid to be tested, the first nucleic acid to be tested, the second nucleic acid to be tested, and the third nucleic acid to be tested are different, and the remaining components may be the same or different; mark the fourth nucleic acid to be tested
  • the labeling substances of the nucleic acid, the first nucleic acid to be detected, the second nucleic acid to be detected and the third nucleic acid to be detected may be the same or different.
  • one micro-liquid contains at most one test nucleic acid (the fourth test nucleic acid or the first test nucleic acid or the second test nucleic acid or the third test nucleic acid) and Reagents in sufficient quantity to amplify and label all nucleic acids to be tested.
  • the processes in the methods of the above embodiments can be realized through computer programs to instruct related hardware, and the programs can be stored in a non-volatile computer-readable storage medium When the program is executed, it may include the processes of the embodiments of the above-mentioned methods.
  • the storage medium may be a magnetic disk, an optical disk, a ROM, or the like.
  • Non-volatile memory may include ROM, Programmable ROM (PROM), Erasable PROM (Erasable PROM, EPROM), Electrically Erasable PROM (Electrically Erasable PROM, EEPROM) or flash memory.
  • Volatile memory can include random access memory (RAM), which acts as external cache memory.
  • RAM can take many forms, such as static RAM (Static RAM, SRAM), dynamic RAM (Dynamic Random Access Memory, DRAM), synchronous DRAM (synchronous DRAM, SDRAM), double data rate SDRAM (Double Data Rate) Data Rate SDRAM, DDR SDRAM), enhanced SDRAM (Enhanced Synchronous DRAM, ESDRAM), synchronous link DRAM (Synchlink DRAM, SLDRAM), memory bus direct RAM (Rambus DRAM, RDRAM) and direct memory bus dynamic RAM (Direct Rambus DRAM) , DRDRAM).
  • Each functional unit in each embodiment of the present application may be integrated into one processing unit, each unit may exist separately physically, or two or more units may be integrated into one unit.
  • the above-mentioned integrated units can be implemented in the form of hardware or in the form of software functional units.
  • the above-mentioned integrated units are realized in the form of software function units and sold or used as independent products, they can be stored in a computer-accessible memory.
  • the technical solution of the present application in essence, or the part that contributes to the prior art, or all or part of the technical solution, can be embodied in the form of a software product, and the computer software product is stored in a memory , including several requests to make a computer device (which may be a personal computer, server, or network device, etc., specifically, a processor in the computer device) execute some or all of the steps of the above-mentioned methods in various embodiments of the present application.

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Abstract

提供了一种对靶标核酸进行检测的方法，特别是提供了一种使用通用探针在数字PCR上实现对单重或多重靶核酸进行检测的方法。采用该检测方法，能够实现单管超多重的检测，具有荧光背景低、熔解温度可调整、包容性好、成本低、操作简便、不容易造成污染、灵敏度高和适用范围广等优势。

Description

对靶标核酸进行检测的方法
本申请要求享有2021年12月27日提交的以下专利申请的优先权：
1.名称为“引物探针组合物、检测方法及检测装置”，申请号为CN202111614026.1的中国专利申请；
本申请还要求享有2022年12月02日提交的以下专利申请的优先权：
2.名称为“引物探针组合物及检测方法”，申请号为CN 202211539752.6的中国专利申请；
本申请还要求享有2022年10月18日提交的以下专利申请的优先权：
3.名称为“多重分时核酸检测装置及检测方法”，申请号为CN 202211271772.X的中国专利申请；
以上内容以与本发明一致的程度通过引用并入本文中。
技术领域
本发明涉及核酸序列检测领域，具体涉及一种对靶标核酸进行检测的方法，更具体地,本发明涉及一种使用通用探针在数字PCR上实现对单重或多重靶核酸进行检测的方法。
背景技术
聚合酶链式反应(PCR)是不采用活的生物体而对DNA进行酶复制的分子生物学技术。PCR通常用于医学和生物研究实验室以承担多种任务，例如感染性疾病的诊断、基因克隆、实验动物表型鉴定、转录组研究、遗传疾病的检测、基因指纹的鉴定、亲子鉴定等。由于其无可比拟的复制和精确能力，PCR被分子生物学家认为是核酸检测的首选方法。上世纪90年代后期，美国ABI公司推出的实时荧光定量PCR(Real Time Quantitative PCR，qPCR)技术及相关产品更是将PCR发展成为一种高灵敏、高特异性和精确定量的核酸序列分析技术。
数字PCR(digital PCR,dPCR)技术是一种核酸分子绝对定量技术，其利用极限稀释的原理将一个荧光定量PCR反应体系分配到成千上万份单独的纳升级微反应器中，使得每个微反应器中包含或不包含1个或多个拷贝的目标核酸分子(DNA靶标)，再同时进行单分子模板PCR扩增。不同于荧光定量PCR在每个扩增循环进行时采集荧光的方法，数字PCR在扩增结束后对每个反应单元的荧光信号进行独立采集，最后通过泊松分布的原理及阳性/阴性的反应单元的比例得出目标分子的原始拷贝数或浓度。
相较于荧光定量PCR，数字PCR不依靠Ct值和标准曲线就可以进行精确的绝对定量检测，具有灵敏度高以及精确度高的优势。由于数字PCR在进行结果判读时仅判断“有/无“两种扩增状态，因此也不需要检测荧光信号与设定阈值线的交点，完全不依赖于Ct值的鉴定，所以数字PCR反应和结果判读受扩增效率的影响大大降低，对PCR反应抑制物的耐受能力大大提高。此外，数字PCR实验中对反应体系进行分配的过程可以极大程度上在局部降低与靶标序列有竞争作用的背景序列浓度，因此，数字PCR特别适 合在复杂背景中检测稀有突变，目前多应用在液体活检中，实现在肿瘤患者外周血中检测稀有突变标志物。
传统试样检测装置在进行多重核酸检测的过程中，先对包含待测靶序列的核酸进行扩增，然后在不同温度下进行信号采集，不同温度下获得的信号存在信号重叠的情况(即不同靶序列同时产生信号)，传统计算方法是通过图像处理或者其他信号计算方法将重叠的信号分开，再分别计算每种靶序列产生的信号值，此种方法计算复杂，且容易带来计算误差。
发明内容
为解决上述问题，本发明提供了一种对靶标核酸进行检测的方法，可以进行1种靶标或2种以上靶标核酸的检测，该方法先对包含待测靶序列的核酸进行扩增，然后进行信号采集，通过设置扩增时退火温度以及信号采集时的采集温度，能够实现在不同温度下获得单一信号(单次信号采集时最多只有一种靶序列产生信号)。当存在2种以上靶标时，本发明要解决的问题还包括如何在单管反应中检测并区分多种不同靶标，并且在存在多组引物的情况下避免因为引物二聚体而出现假阳性信号。
为此，第一方面，本发明提供了一种对靶标核酸进行检测的方法，包括如下步骤：
S100，将引物探针组合物、待测样本和扩增试剂混合，获得反应体系；所述扩增试剂包括DNA聚合酶和dNTPs；
S200，对所述反应体系进行第1次PCR扩增，扩增时采用的退火温度为T1；
S300，进行以下任一操作：
操作1，包括：
S310，在第1信号采集温度t1下进行第1次信号采集；
S320，进行第2次PCR扩增，扩增时采用的退火温度为T2；
操作2，包括：
S310’，在第1信号采集温度t1下进行第1次信号采集；
操作3，包括：
S310”，进行第2次PCR扩增，扩增时采用的退火温度为T2；
S320”，在第1信号采集温度t1下进行第1次信号采集；
以上操作满足：T1＞T2，T2＜t1；
S400，在第2信号采集温度t2下进行第2次信号采集，所述t2＞t1，且t2＞T1；
其中，待测样本中的待检测靶标的种类为1种；
所述第1次信号采集、第2次信号采集中最多只有1次信号采集中含有靶标的信号。
在某些实施例中，第1次PCR扩增之后或第1和2次PCR扩增之后进行一次信号采集，信号采集温度为依次升高(t2＞t1)。某些实施例中，连续进行2次PCR，或只进行1次PCR，均能生成与探针结合的双链产物，当采用较低的温度(t1)进行信号采集时，由于双链产物的形成，产生可检测的信号变化； 然后采用更高温度(t2)进行信号采集时，由于t2高于所述双链产物的熔解温度，则双链产物呈单链状态，t2时采集的信号与t1时采集的信号不同，即，两次采集的信号不同，能更准确地证明靶核酸的存在。
某些实施例中，进行2次PCR和2次信号采集，且PCR和信号采集交替进行：PCR扩增所采用的退火温度为依次降低(T2＜T1)但信号采集温度为依次升高(t2＞t1)。即，当采用某一温度作为PCR的退火温度进行PCR循环扩增时，靶核酸所形成的单链预产物的退火温度比该温度低，不能与探针结合形成双链产物，不能引起检测信号变化；此次PCR扩增之后，即在另一温度下进行信号采集，靶核酸所形成的单链预产物与探针的双链产物呈双链状态，并产生可检测的靶核酸信号。两次采集的信号不同，更准确地证明靶核酸的存在。
因此，采用上述的PCR检测方法，能更准确地证明靶核酸的存在或不存在：当存在靶核酸时，两次采集的信号不同，只有1次信号采集中含有最多1种靶核酸的信号；若不存在该靶核酸，则两次信号采集都无任何靶核酸的信号。
T2＜T1，是指第2次PCR的退火温度，与第1次PCR的退火温度存在大小差异，两者的退火温度差异，使得第2次PCR扩增后、与某种探针形成的双链产物的种类数，相比第1次PCR扩增后、与该种探针形成的双链产物的种类数，最多增加1种(不增加，是指待测产物中没有相应的靶核酸)。某些实施例中，T2与T1的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
t2＞t1，是指第2次信号采集的温度，与第1次信号采集的温度存在大小差异，两种的温度差异，使得第2次信号采集的信号，与第1次信号采集的信号不同。某些实施例中，t2与t1温度相差6℃以上；例如，相差6-46℃，又例如，相差6-40℃、6-35℃、6-30℃、6-25℃、6-20℃、6-18℃、6-15℃、6-12℃、6-10℃、6-8℃或6-7℃等。
T2＜t1，是指第2次PCR的退火温度，与第1次信号采集的温度存在大小差异。某些实施例中，T2与t1的温度相差5℃以上，例如，相差5-50℃，又例如，相差5-40℃、5-30℃、5-20℃、5-18℃、5-15℃、5-12℃、5-10℃、5-8℃、5-7℃或5-6℃等。
t2＞T1，是指第1次PCR的退火温度，与第2次信号采集的温度存在大小差异。某些实施例中，t2与T1的温度相差7℃以上，例如，相差7-50℃，又例如，相差7-40℃、7-30℃、7-20℃、7-18℃、7-15℃、7-12℃、7-10℃或7-8℃等。
在本发明的一些实施方式中，上述的检测方法，所述第1次PCR扩增时采用的退火温度为T1，所述第1次信号采集时采用的采集温度为t1；所述T1≤t1。
某些实施例中，第1次PCR扩增时(其退火温度为T1)，引物组与探针产生的单链预产物中，有1种单链预产物与探针特异性结合并延伸0个碱基后形成双链产物，该单链预产物的退火温度略小于其熔解温度(通常情况下，寡核苷酸的退火温度通常比该寡核苷酸的熔解温度(Tm)低)。在t1(T1≤t1＜其熔解温度)下进行信号采集，此时该单链预产物与探针呈双链状态，使探针产生可检测的信号变化。某些实 施例中，也可以在t1(t1≤T1)下进行信号采集，此时该单链预产物与探针也呈双链状态，使探针产生可检测的信号变化。本领域技术人员可根据实际需求(例如，信号采集时，单链预产物与探针呈双链状态，采集的信号强度大等实际需求)，对信号采集温度t1的温度进行具体选择。
根据本发明的优选实施方式，退火温度范围为40℃-75℃，即40℃≤T2＜T1≤75℃。
根据本发明的优选实施方式，信号采集温度为0-95℃，即0℃≤t1＜t2≤90℃；通常信号采集温度的选择，本领域技术人员可根据设计的引物探针组合物的实际情况进行确定。例如，通过引物探针组合物的设计，本领域技术人员可以预期代表靶核酸的反应产物的熔解温度为T，若想检测到该靶核酸的信号，则信号采集温度≤T；若不想检测到靶核酸的信号，则信号采集温度＞T。
根据本发明的优选实施方式，步骤S200中所述第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；
和/或，步骤S300中所述第2次PCR扩增，包括如下反应条件：约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育约10-约1800秒。
根据本发明的优选实施方式，步骤S200中所述第1次PCR扩增的反应条件包括：约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；进一步优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；
和/或，步骤S300中所述第2次PCR扩增，包括如下反应条件：约85℃-约105℃变性约2-约60秒，约20℃-约75℃退火及延伸约10-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育约10-约1800秒。
根据本发明的优选实施方式，对所述反应体系进行第1次PCR扩增之前，还包括：将所述反应体系分配到500个以上的反应单元中，每个反应单元含有1个待测样本的靶标核酸或不含有待测样本的靶标核酸；进一步优选地，所述信号采集为通过相机对荧光信号进行采集；所述信号通道采集为在荧光信号通道下通过相机对荧光信号进行采集。
根据本发明的优选实施方式，所述引物探针组合物包括：探针(P)和引物；所述引物与所述待测样本中靶标特异性结合后产生预产物，所述预产物中含有一条可与探针特异性结合的单链预产物，该单链预产物与探针特异性结合并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起探针信号变化；进一步优选地，所述延伸为延伸0-100个碱基，更进一步优选地，所述延伸为延伸1-100个碱基，延伸的碱基个数的上限就是探针的整个长度；和/或，所述引物的浓度为约30nM-约1000nM，所述探针(P)的浓度为约100nM-约1200nM。在发明中，浓度单位nM指的是nmol/L。
具体的，延伸0个碱基指的是该单链预产物与探针(P)特异性结合(杂交)后不发生延伸。本领域技术人员可根据实际需求，通过对引物组中引物序列和/或探针的序列进行设计，实现上述“特异性结合但不发生延伸”的效果，例如，设计使其产生的第一单链预产物结合在探针的5’末端，或者，设计使其产生的第一单链预产物的3’末端含有不与探针互补配对的区域。
根据本发明的优选实施方式，所述探针(P)为一段不与任何靶标序列配对结合的自由设计序列，包括探针信号检测区(H)，探针上修饰有检测标记；所述引物包括正向引物(F)和/或反向引物(R)；
所述正向引物(F)包含与靶标序列特异性配对结合的靶标序列结合区；
所述反向引物(R)包含引物信号检测区(h)和靶标序列结合区，且引物信号检测区(h)位于靶标序列结合区的5’端，或者所述反向引物(R)从5’端至3’端依次包含延伸阻滞区(M)、引物信号检测区(h)和靶序列结合区；
其中，所述反向引物(R)的靶标序列结合区为与靶标序列特异性配对结合的序列；所述反向引物(R)的引物信号检测区(h)均为一段不与任何靶标序列配对结合的序列，所述引物信号检测区(h)的互补序列(h’)与探针(P)的探针信号检测区(H)特异性配对结合；所述反向引物(R)的延伸阻滞区(M)为不与探针(P)任一部分的序列或任何靶标序列相同或互补的自由设计序列；
进一步优选地，所述反向引物(R)中引物信号检测区(h)和靶标序列结合区之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。某些实施例中，反向引物(R)中延伸阻滞区(M)和引物信号检测区(h)之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
本发明中，“特异性配对结合”，或“特异性结合”，是指具有互补序列的两条单链核酸分子在一定的条件下(适宜的温度及离子强度等)碱基互补配对结合。本领域技术人员熟知，两条单链核酸分子并非完全互补配对才能特异性结合，存在少数碱基错配的情况下，也能特异性结合。
根据本发明的优选实施方式，所述正向引物(F)还包括探针锚定区(A)，所述正向引物(F)从5’端至3’端依次包含探针锚定区(A)和靶标序列结合区；其中，所述探针锚定区(A)为一段不与任何靶标序列配对结合的序列；进一步优选地，所述探针锚定区(A)和靶标序列结合区之间有0-20个碱基的间隔；其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围；
所述探针(P)还包括引物锚定区(A’)，所述引物锚定区(A’)为与所述正向引物(F)的探针锚定区(A)互补的序列；进一步优选地，所述探针(P)中的所述引物锚定区(A’)和所述探针信号检测区(H)之间有0-20个碱基的间隔。
根据本发明的优选实施方式，所述正向引物和所述反向引物的结构可以互换；例如，“反向引物(R)包含引物信号检测区(h)和靶标序列结合区，正向引物(F)包含靶标序列结合区”，引物结构可以互换 成“正向引物(F)包含引物信号检测区(h)和靶标序列结合区，反向引物(R)包含靶标序列结合区”；或者，“反向引物(R)包含引物信号检测区(h)和靶标序列结合区，正向引物(F)包含探针锚定区(A)和靶标序列结合区”引物结构可以互换成“正向引物(F)包含引物信号检测区(h)和靶标序列结合区，反向引物(R)包含探针锚定区(A)和靶标序列结合区”。
本领域技术人员熟知，为避免引物二聚体，正向引物(F)中的靶标序列结合区与反向引物(R)中的靶标序列结合区不同，也不能特异性结合。
根据本发明的优选实施方式，所述引物中，有至少1种所述正向引物(F)包含第一正向引物和第二正向引物，所述第一正向引物从5’端至3’端依次包含正向扩增产物捕获区(B)和靶序列结合区；以及，所述第二正向引物包含正向扩增产物捕获区(B)，所述正向扩增产物捕获区(B)不与靶序列或其任意的片段配对结合；进一步优选地，所述第一正向引物中所述正向扩增产物捕获区(B)与所述靶序列结合区之间有0-20个碱基的间隔。
根据本发明的优选实施方式，所述第二正向引物还包括探针锚定区(A)，进一步优选地，所述第二正向引物中所述探针锚定区(A)和所述正向扩增产物捕获区(B)之间有0-20个碱基的间隔。
根据本发明的优选实施方式，所述引物中，有至少1种所述反向引物(R)包含第一反向引物和第二反向引物，所述第一反向引物从5’端至3’端依次包含反向扩增产物捕获区(C)和靶序列结合区；以及，所述第二反向引物从5’端至3’端依次包含引物信号检测区(h)和反向扩增产物捕获区(C)，所述反向扩增产物捕获区(C)不与靶序列或其任意的片段配对结合；
进一步优选地，所述第一反向引物中所述反向扩增产物捕获区(C)和所述靶序列结合区之间有0-20个碱基的间隔；和/或，所述第二反向引物中所述引物信号检测区(h)和所述反向扩增产物捕获区(C)之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
在本发明的一些实施方式中，上述对靶标核酸进行检测的方法，为数字PCR检测方法；在本发明的一些实施方式中，上述对靶标核酸进行检测的方法，对所述反应体系进行第1次PCR扩增之前，还包括：将所述反应体系分配到500个以上的反应单元中，每个反应单元含有1个待测样本的靶标核酸或不含有待测样本的靶标核酸；优选地，所述信号采集为通过相机对荧光信号进行采集；所述信号通道采集为在荧光信号通道下通过相机对荧光信号进行采集。在上述对靶标核酸进行检测的方法中，引物探针组合物可以为本领域常规的引物探针组合物，例如，含有分子信标的引物探针组合物，也可以是下述第三方面提供的用于进行核酸序列检测的引物探针组合物。
为此，第二方面，本发明提供了一种对2种以上靶标核酸进行检测的方法，包括如下步骤：
将引物探针组合物、待测样本和扩增试剂混合，获得反应体系；所述引物探针组合物包括至少1种探针(P)、至少2种针对不同靶标的引物；所述扩增试剂包括DNA聚合酶和dNTPs；
对所述反应体系进行n次PCR扩增和n次信号采集；每次PCR扩增后进行一次信号采集，每次信号采集包括至少1次信号通道采集；每次信号通道采集中最多含有1种靶标的信号；
所述第n次PCR扩增时采用的退火温度为Tn，所述第n次信号采集时采用的采集温度为tn，Tn＜tn；Tn＜T(n-1)，且Tn＜t(n-1)；tn＞t(n-1)，且tn＞T(n-1)；
所述n为≥2的整数，所述n≤待测样本中待测的靶标种类数。
在某些实施例中，每次PCR扩增之后进行一次信号采集，PCR扩增所采用的退火温度为依次降低(Tn＜T(n-1))，但信号采集温度为依次升高(tn＞t(n-1))。即，当采用某一温度作为PCR的退火温度进行PCR循环扩增时，退火温度比该温度高的单链预产物，均能与探针结合形成双链产物而引起检测信号变化，退火温度比该温低的单链预产物，仍处于单链状态；此次PCR扩增之后，即在另一温度下进行信号采集，仅有1种单链预产物与探针的双链产物呈双链状态，并产生可检测的靶核酸信号。(因为，其他种类的单链预产物：一部分由于退火温度更低，在PCR循环扩增阶段不能与探针退火形成双链产物；另一部分由于退火温度高，在PCR循环扩增阶段能与探针退火形成双链产物，但由于这些双链产物的熔解温度更低，低于信号采集的温度，生成的双链产物也在该信号采集温度下呈单链状态。)因此，采用上述的PCR检测方法，能够实现在每次信号通道采集的信号，最多只有1种靶核酸的信号，若不存在该靶核酸，则无任何靶核酸的信号。
Tn＜T(n-1)，是指第n次PCR的退火温度，与第n-1次PCR的退火温度存在大小差异，两者的退火温度差异，使得第n次PCR扩增后、与某种探针形成的双链产物的种类数，相比第n-1次PCR扩增后、与该种探针形成的双链产物的种类数，最多增加1种(不增加，是指待测产物中没有相应的靶核酸)。某些实施例中，Tn与T(n-1)的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
tn＞t(n-1)，是指第n次信号采集的温度，与第n-1次信号采集的温度存在大小差异，两种的温度差异，使得第n次信号采集时的双链产物，与第n-1次信号采集时的双链产物不同。例如，在第n-1次信号采集时的双链产物为甲，第n次信号采集时的双链产物为乙(在第n次信号采集时，由于双链产物甲的熔解温度低于第n次信号采集时的温度，此时，双链产物甲呈单链状态)。某些实施例中，tn与t(n-1)温度相差6℃以上；例如，相差6-46℃，又例如，相差6-40℃、6-35℃、6-30℃、6-25℃、6-20℃、6-18℃、6-15℃、6-12℃、6-10℃、6-8℃或6-7℃等。
Tn＜tn，是指第n次PCR的退火温度，与第n次信号采集的温度存在大小差异，两种温度的差异，使得第n次PCR扩增后可以获得特定的双链产物，在第n次信号采集时可以获得该双链产物的信号。为了获得该双链产物的信号，在第n次信号采集时(信号采集温度tn的条件下)，该双链产物不呈现单链状态。某些实施例中，Tn与tn相差1℃以上，例如，相差1-50℃、1-40℃、1-30℃、1-25℃、2-20℃、2-15℃、2-12℃、2-10℃、2-7℃、2-6℃、2-5℃、3-5℃或4-5℃等。某些实施例中，采用的信号采集温度tn，小于等于该双链产物的熔解温度。
Tn＜t(n-1)，是指第n次PCR的退火温度，与第n-1次信号采集的温度存在大小差异，两种温度的差异，使得第n-1次信号采集时的双链产物，在第n次PCR时，该双链产物依然存在，同时，还产生了与该双链产物不同的另一种双链产物。某些实施例中，Tn与t(n-1)的温度相差5℃以上，例如，相差5-50℃，又例如，相差5-40℃、5-30℃、5-20℃、5-18℃、5-15℃、5-12℃、5-10℃、5-8℃、5-7℃或5-6℃等。
tn＞T(n-1)，是指第n-1次PCR的退火温度，与第n次信号采集的温度存在大小差异。某些实施例中，tn与T(n-1)的温度相差7℃以上，例如，相差7-50℃，又例如，相差7-40℃、7-30℃、7-20℃、7-18℃、7-15℃、7-12℃、7-10℃或7-8℃等。
本发明中，探针的种类数根据其检测标记的种类数进行分类。不同种类的探针，其修饰的检测标记不同(某些实施例中，检测标记即为检测基团)，可以实现在不同的信号通道(或荧光通道)下进行信号采集。例如，本发明的引物探针组合物中含有2种探针，1种探针修饰的检测基团是VIC和BHQ1，与该种探针特异性结合形成的双链产物，在VIC通道进行信号采集；另1种探针修饰的检测基团是FAM和BHQ1，与该种探针特异性结合形成的双链产物，在FAM通道进行信号采集。
某些实施例中，每次PCR扩增后进行一次信号采集，每次信号采集包括m次信号通道采集，m为≥1的整数，m为所述引物探针组合物中探针的检测基团的种类数。如前所述，每次PCR扩增后，相比于上一次PCR扩增，与同种探针形成的双链产物的种类数最多增加1种。如果引物探针组合物中，探针的种类数为m，则每次PCR扩增后，相比于上一次PCR扩增，与m种探针形成的双链产物的种类数最多增加m种(与每1种探针形成的双链产物的种类数最多增加1种)。通过m次不同的信号通道采集，可以实现对增加的m种双链产物所对应的靶核酸信号进行区别，且互不交叉，从而实现多重检测。
根据本发明的优选实施方式，所述至少1次信号采集包括m次信号通道采集，所述m为所述引物探针组合物中探针的检测标记的种类数，并且，n与m的乘积≥待测样本中待测的靶标种类数。为了检测仪器的程序设计方便，某些实施例中，如果引物探针组合物中有2种探针，则每次信号采集都是在2个信号通道下进行2次信号通道采集。某些实施例中，待测样本中有5种靶标，针对这5种靶标的引物探针组合物中，含有2种探针，第一探针所检测的靶标种类是3种，第二探针所检测的靶标种类是2种，则对反应产物只需要进行3次信号采集，每次信号采集都是在2个信号通道下进行的2次信号通道采集。如此，存在某一信号通道下，进行了3次信号采集，但所对应的靶标只有2种，则出现某次信号通道采集的信号无靶标，或者相邻两次信号通道采集的信号相同的情况。在这些实施例中，以检测靶标数量最多的检测通道为准，设置信号采集次数，实现信号采集的次数＜靶核酸的种类数，减少了信号采集的次数。虽然没有完全满足其他信号通道的信号采集次数最少的情况，但对仪器程序的设置更为简便，本领域技术人员可根据实际需求进行选择。某些实施例中，本领域技术人员根据引物探针组合物的设计情况，选择在没有预期靶标的通道不进行信号采集，从而减少信号采集的次数，简化操作步骤。
在本发明的一些实施方式中，上述的检测方法，所述第1次PCR扩增时采用的退火温度为T1，所述第1次信号采集时采用的采集温度为t1；所述T1≤t1。
某些实施例中，第1次PCR扩增时(其退火温度为T1)，引物组与探针产生的单链预产物中，有1种单链预产物与探针特异性结合并延伸0个碱基后形成双链产物，该单链预产物的退火温度略小于其熔解温度(通常情况下，寡核苷酸的退火温度通常比该寡核苷酸的熔解温度(Tm)低)。在t1(T1≤t1＜其熔解温度)下进行信号采集，此时该单链预产物与探针呈双链状态，使探针产生可检测的信号变化。某些实施例中，也可以在t1(t1≤T1)下进行信号采集，此时该单链预产物与探针也呈双链状态，使探针产生可检测的信号变化。本领域技术人员可根据实际需求(例如，信号采集时，单链预产物与探针呈双链状态，采集的信号强度大等实际需求)，对信号采集温度t1的温度进行具体选择。
某些实施例中，退火温度为：40℃≤Tn＜T(n-1)≤75℃(n为≥2的整数)。
某些实施例中，采集温度为：0℃≤t(n-1)＜tn≤90℃(n为≥2的整数)。
根据本发明的优选实施方式，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环。。
根据本发明的优选实施方式，对所述反应体系进行第1次PCR扩增的反应条件包括：约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环。
根据本发明的优选实施方式，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，约20-约60个循环；约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约20℃-约75℃恒温孵育约10-约1800秒；
优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约20℃-约75℃恒温孵育约10-约1800秒。
根据本发明的优选实施方式，对所述反应体系进行第1次PCR扩增的反应条件包括：约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，约35-约50个循环；约90℃-约95℃变性约10-约60秒，约20℃-约75℃退火及延伸约30-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；约20℃-约75℃恒温孵育约10-约1000秒；
优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；约90℃-约95℃变性约10-约60秒，约20℃-约75℃退火及延伸约30-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约90℃-约96℃预变性约5-约15分钟；约90℃-约95℃变性约10-约60秒，约50℃-约75℃退火及延伸约30-约90秒，35-50个循环；约20℃-约75℃恒温孵育约10-约1000秒。
根据本发明的优选实施方式，所述第1次PCR扩增之后的其他PCR扩增，包括如下反应条件：约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育10-1800秒。
根据本发明的优选实施方式，所述第1次PCR扩增之后的其他PCR扩增，包括如下反应条件：约90℃-约95℃变性约10-约60秒，约20℃-约75℃退火及延伸约30-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育10-1000秒。
根据本发明的优选实施方式，至少1种所述引物与靶标特异性结合后产生预产物，所述预产物中含有一条可与探针(P)特异性结合的单链预产物，该单链预产物与探针(P)特异性结合并延伸后形成双链产物，所述双链产物的形成引起探针信号变化；优选地，所述延伸为延伸0-100个碱基，更优选地延伸1-100个碱基，延伸的碱基个数的上限就是探针的整个长度；和/或，所述引物的浓度为约30nM-约1000nM，所述探针(P)的浓度为约100nM-约1200nM。如果是多种靶标共用1种探针时，探针的用量会更多，例如，1300nM、1400nM、1500nM、1600nM、1700nM、1800nM、1900nM、2000nM或更多。
具体的，延伸0个碱基指的是该单链预产物与探针(P)特异性结合(杂交)后不发生延伸。本领域技术人员可根据实际需求，通过对引物组中引物序列和/或探针的序列进行设计，实现上述“特异性结合但不发生延伸”的效果，例如，设计使其产生的第一单链预产物结合在探针的5’末端，或者，设计使其产生的第一单链预产物的3’末端含有不与探针互补配对的区域。
根据本发明的优选实施方式，所述至少2种针对不同靶标的引物包括以下任意一种：
(1)至少两种正向引物(F)和至少一种反向引物(R)；
(2)至少一种正向引物(F)和至少两种反向引物(R)；
(3)至少两种正向引物(F)和至少两种反向引物(R)；
不同的所述正向引物(F)各自独立地包含与不同靶标序列特异性配对结合的靶标序列结合区；
和/或，所述探针(P)为一段不与任何靶序列配对结合的自由设计序列，包括探针信号检测区(H)，所述探针上修饰有检测标记；
和/或，所述反向引物(R)包含引物信号检测区(h)和靶标序列结合区，且所述引物信号检测区(h)位于所述靶标序列结合区的5’端；其中，不同的所述反向引物(R)的靶序列结合区为与不同靶标序列特异性配对结合的序列；不同的所述反向引物(R)的引物信号检测区(h)均为一段不与任何靶标序列配对结合、但与探针(P)的探针信号检测区(H)具有部分或全部相同的序列；优选地，不同反向引物(R)的引物信号检测区(h)的序列彼此不同。
本发明中，反向引物(R)的引物信号检测区(h)为与探针(P)的探针信号检测区(H)具有部分或全部相同的序列，是指，反向引物(R)的引物信号检测区(h)的互补序列与探针(P)的探针信号检测区(H)特异性配对结合。
根据本发明的优选实施方式，所述引物包括至少两种所述引物时，由于正向引物和反向引物的结构可以互换，因此对正向引物和反向引物的比例不限制。在某些实施例中，不含引物信号检测区h的引物的含量多于其他含有引物信号检测区h的引物，优选不含引物信号检测区h的引物的含量为其他含有引物信号检测区h的引物的含量的1.2-20倍，更优选不含引物信号检测区h的引物的含量为其他含有引物信号检测区h的引物的含量的2倍、2.5倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍或10倍。
根据本发明的优选实施方式，所述引物中，有至少1种所述反向引物(R)从5’端至3’端依次包含延伸阻滞区(M)、引物信号检测区(h)和靶标序列结合区；所述延伸阻滞区(M)为不与探针(P)任一部分的序列或任何靶序列相同或互补的自由设计序列；若还有其他种反向引物，所述其他种反向引物(R)的5’端不含有延伸阻滞区(M)。
某些实施例中，若还有其他种反向引物，所述其他种反向引物(R)包括引物信号检测区(h)和靶标序列结合区，且所述引物信号检测区(h)位于靶标序列结合区的3’端；某些实施例中，若还有其他种反向引物，所述其他种反向引物(R)从5’端至3’端依次包含引物信号检测区(h)和靶标序列结合区。
根据本发明的优选实施方式，所述引物中，有至少1种正向引物(F)还包括探针锚定区(A)，所述正向引物(F)中的探针锚定区(A)位于所述靶标序列结合区的5’端；其中，所述探针锚定区(A)为一段不与任何靶标序列配对结合的序列；不同正向引物(F)的靶标序列结合区的序列不同；进一步优选地，正向引物(F)中探针锚定区(A)和靶标序列结合区之间有0-20个碱基的间隔；其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围；
所述探针(P)为一段不与任何靶标序列配对结合的序列，其包括引物锚定区(A’)和探针信号检测区(H)；所述引物锚定区(A’)为与正向引物(F)的探针锚定区(A)互补的序列；进一步优选地，所 述探针(P)中所述引物锚定区(A’)和所述探针信号检测区(H)之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
根据本发明的优选实施方式，所述引物中，有至少1种正向引物(F)从5’端至3’端依次包含探针锚定区(A)和靶标序列结合区；其中，所述探针锚定区(A)为一段不与任何靶标序列配对结合的序列；不同正向引物(F)的靶标序列结合区的序列不同；优选地，正向引物(F)中探针锚定区(A)和靶标序列结合区之间有0-20个碱基的间隔；其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围；
所述探针(P)为一段不与任何靶标序列配对结合的序列，其包括引物锚定区(A’)和探针信号检测区(H)；所述引物锚定区(A’)为与正向引物(F)的探针锚定区(A)反向互补的序列；优选地，所述探针(P)中所述引物锚定区(A’)和所述探针信号检测区(H)之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
根据本发明的优选实施方式，所述引物中，有至少1种所述正向引物(F)包括第一正向引物和第二正向引物，所述第一正向引物从5’端至3’端依次包含正向扩增产物捕获区(B)和靶序列结合区；以及，所述第二正向引物包含正向扩增产物捕获区(B)，所述正向扩增产物捕获区(B)不与靶序列或其任意的片段配对结合，进一步优选地，所述第一正向引物中所述正向扩增产物捕获区(B)与所述靶序列结合区之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
根据本发明的优选实施方式，所述第二正向引物还包括探针锚定区(A)，优选地，所述第二正向引物中探针锚定区(A)和正向扩增产物捕获区(B)之间有0-20个碱基的间隔。
根据本发明的优选实施方式，所述引物中，有至少1种所述反向引物(R)包含第一反向引物和第二反向引物，所述第一反向引物从5’端至3’端依次包含反向扩增产物捕获区(C)和靶序列结合区；以及，所述第二反向引物从5’端至3’端依次包含引物信号检测区(h)和反向扩增产物捕获区(C)，所述反向扩增产物捕获区(C)不与靶序列或其任意的片段配对结合；
优选地，所述第一反向引物中反向扩增产物捕获区(C)和靶序列结合区之间有0-20个碱基的间隔；和/或，所述第二反向引物中引物信号检测区(h)和反向扩增产物捕获区(C)之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
在本发明的一些实施方式中，上述的对2种以上靶标核酸进行检测的方法，为数字PCR检测方法；在本发明的一些实施方式中，对所述反应体系进行第1次PCR扩增之前，还包括：将所述反应体系分配到500个以上的反应单元中，每个反应单元含有1个待测样本的靶标核酸或不含有待测样本的靶标核酸； 优选地，所述信号采集为通过相机对荧光信号进行采集；所述信号通道采集为在荧光信号通道下通过相机对荧光信号进行采集。
在上述对靶标核酸进行检测的方法中，引物探针组合物可以为本领域常规的引物探针组合物，例如，含有分子信标的引物探针组合物，也可以是下述第三方面提供的用于进行核酸序列检测的引物探针组合物。
为此，第三方面，本发明提供了一种用于进行核酸序列检测的引物探针组合物，包括第一探针和第一引物混合物；
所述第一引物混合物包括至少2种引物组，不同种引物组分别与不同种靶核酸特异性结合；
所述第一引物混合物中的引物组与其对应的靶核酸特异性结合后产生预产物，所述预产物中含有与第一探针特异性结合的单链预产物，所述单链预产物与第一探针特异性结合并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起可检测的信号变化；
所述第一引物混合物中不同种的引物组与其对应的靶核酸产生的单链预产物不同；当第一单链预产物与第一探针的退火温度，高于第二单链预产物与第一探针的退火温度时，所述第一单链预产物与第一探针产生的双链产物的熔解温度，低于所述第二单链预产物与第一探针产生的双链产物的熔解温度。
所述第一探针，是指一类修饰的检测标记相同，其碱基序列可以相同、也可以不同的1类探针。即，探针的种类数根据其检测标记的种类数进行分类。某些实施例中，1种探针产生的信号可以在1个检测通道(或荧光通道)中进行信号采集；2种探针产生的信号需要在2个不同检测通道中进行信号采集。某些实施例中，检测标记相同，并不意味着修饰的检测基团完全相同，例如，检测基团包括荧光基团和猝灭基团，只要荧光基团相同即可，也可以实现探针产生的信号可以在1个检测通道(或荧光通道)中进行信号采集。
“至少2种引物组”，是指，引物组的种类至少为2种，每种引物组所针对的靶核酸种类不一样，即，每种引物组与其对应的靶核酸能够实现特异性结合。某些实施例中，1种引物组对1类不分型别的靶核酸都能够实现特异性结合，则这1类不分型的靶核酸，也可以认为是1种靶核酸。
某些实施例中，引物组为双引物情况，即，包括一对针对同一靶核酸的上游引物和下游引物。某些实施例中，引物组为单引物情况，即，仅含有一种针对某靶核酸的引物，该引物能与其对应的靶核酸特异性结合而不能与其他种类的靶核酸特异性结合。
某些实施例中，不同种类的引物组中可能存在共用引物的情况，例如，两种引物组中，均包括同样一种引物。同样一种引物，是指引物的序列完全相同。
通常情况下，寡核苷酸的退火温度通常比该寡核苷酸的熔解温度(Tm)低约5℃，尽管它可以被确定为比所述寡核苷酸的Tm低约4.9、4.8、4.7、4.6、4.5、4.4、4.3、4.2、4.1、3.9、3.8、3.7、3.6、3.5、3.4、3.3、3.2、3.1、3.0℃或更低，或其间的任何范围(包括端值)。寡核苷酸的退火温度、熔解温度，与寡核苷酸的长度、组成等因素有关。本领域技术人员可以根据实际需求，对寡核苷酸的退火温度和熔解 温度进行调节，例如，提高寡核苷酸的GC含量、使寡核苷酸的长度更长，可获得更高的熔解温度。
某些实施例中，某种引物组与其靶核酸产生的单链预产物与探针特异性结合所需的退火温度越低，该单链预产物与探针特异性结合并延伸≥0个碱基形成的双链产物的熔解温度就越高。例如，通过调整引物组的长度和/或序列，使引物组与靶核酸特异性结合后得到的单链预产物不同。不同的单链预产物与相同探针结合的位置不同，从而实现第一单链预产物与探针的退火温度(此处简称“第一退火温度”)、高于第二单链预产物与探针的退火温度(此处简称“第二退火温度”)，即，第一退火温度高于第二退火温度；并且，由于不同的单链预产物与相同探针结合的位置不同，还可以实现对不同单链预产物与探针结合并延伸的长度进行调整，从而实现第一单链预产物与探针产生的双链产物的熔解温度(此处简称“第一熔解温度”)、低于所述第二单链预产物与探针产生的双链产物的熔解温度(此处简称“第二熔解温度”)，即，第一熔解温度低于第二熔解温度。
本发明中，表述“高于”、“低于”用于描述两个温度之间存在差异，且该差异的存在，可以使不同种的单链预产物在某一特定温度进行信号采集时，实现单链或双链的不同状态，例如，某一特定信号温度下，第一单链预产物呈单链状态，而第二单链预产物与探针结合呈现双链状态。某些实施例中，“高于”用于描述两个退火温度之间相差4℃以上；例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。某些实施例中，“低于”用于描述两个熔解温度之间相差6℃以上；例如，相差6-46℃，又例如，相差6-40℃、6-35℃、6-30℃、6-25℃、6-20℃、6-18℃、6-15℃、6-12℃、6-10℃、6-8℃或6-7℃等。
采用上述的引物探针组合物，通过对退火温度和熔解温度的高、低差异的设置，可实现：在某个温度下进行信号采集时，最多只有1种单链预产物与探针处于双链状态，因此，最多产生1种靶核酸的信号(所述“最多”，是指，若待测样本中无该靶核酸，则不产生信号；若有，则只有该种靶核酸的信号，不会同时出现其他靶核酸信号的情况)。因此，实现了同一温度下采集的信号不重叠。
某些实施例中，引物探针组合物中，有1种探针(第一探针)和2种引物组，2种引物组分别为第一引物组和第二引物组；第一引物组和第二引物组分别与第一靶核酸和第二靶核酸特异性结合后，分别产生第一单链预产物和第二单链预产物，第一单链预产物与探针的退火温度为第一退火温度，第二单链预产物与探针的退火温度为第二退火温度；第一单链预产物与探针产生的双链产物为第一双链产物，其熔解温度为第一熔解温度，第二单链预产物与探针产生的双链产物为第二双链产物，其熔解温度为第二熔解温度。在采用T1(第一退火温度≥T1＞第二退火温度)的退火温度进行PCR循环后，仅产生第一双链产物；然后在t1的温度下进行第一次信号采集(t1＜第一熔解温度)，此时，仅第一双链产物处于双链状态，产生的信号为第一靶核酸的信号；然后在采用T2(第二退火温度≥T2)的退火温度进行PCR循环后，产生第二双链产物，且反应体系中还含有第一双链产物；然后在t2的温度下进行第二次信号采集(第一熔解温度＜t2＜第二熔解温度)，此时，仅第二双链产物处于双链状态，产生的信号为第二靶核酸的信号(由于t2＞第一熔解温度，所以第一双链产物为解链状态，无信号产生)。
类似地，当含有3种以上引物组时，当采用某一温度作为PCR的退火温度进行PCR循环扩增时，退火温度比该温度高的单链预产物，均能与探针结合形成双链产物，退火温度比该温低的单链预产物，仍处于单链状态；当采用某一温度进行信号采集时，仅有1种单链预产物与探针的双链产物呈双链状态，并产生可检测的靶核酸信号。其他种类的单链预产物，一部分由于退火温度低，在PCR循环扩增阶段不能与探针退火形成双链产物；另一部分由于退火温度高，在PCR循环扩增阶段能与探针退火形成双链产物，但由于这些双链产物的熔解温度比信号采集的温度低，生成的双链产物也在该信号采集温度下呈单链状态。
本发明中，表述“第一”、“第二”、“第三”、“第四”和“第五”等仅用于描述目的，用以区分所限定的对象，而不以任何方式限定次序或主次。
某些实施例中，上述用于靶核酸检测的引物探针组合物，所述引物探针组合物中包括针对第一靶核酸的第一引物组，所述第一引物组与第一靶核酸特异性结合后产生第一预产物，所述第一预产物中含有与探针特异性结合的第一单链预产物，所述第一单链预产物与探针特异性结合并延伸0个碱基后形成第一双链产物，所述第一双链产物的形成引起可检测的信号变化；所述第一单链预产物与探针的退火温度，高于其他单链预产物与相同探针的退火温度；所述第一单链预产物与探针产生的第一双链产物的熔解温度，低于其他单链预产物与相同探针产生的双链产物的熔解温度。
在该实施例中，针对某一靶核酸的引物组(此处描述为“第一引物组”)，其产生的第一单链预产物与探针特异性结合后不发生延伸。本领域技术人员可根据实际需求，通过对引物组中引物序列和/或探针的序列进行设计，实现上述“特异性结合但不发生延伸”的效果，例如，设计使其产生的第一单链预产物结合在探针的5’末端，或者，设计使其产生的第一单链预产物的3’末端含有不与探针互补配对的区域。
上述“第一”仅用于描述、实现区分的目的，用以区分所限定的对象，而不以任何方式限定次序或主次，不用于限定靶核酸、引物组、预产物、单链预产物、或双链产物的种类数量。在此，仅为了描述在上述的引物探针组合物中，有1种引物组产生的单链预产物与探针特异性结合后，不发生延伸的情况，且该单链预产物与探针的退火温度最高(高于其他任何种类的单链预产物与相同探针的退火温度)、但所形成的双链产物的熔解温度最低(低于其他任何种类的双链产物的熔解温度)。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述引物探针组合物中还包括第二探针和第二引物混合物；所述第二探针与所述第一探针的碱基序列不同，且修饰的检测标记也不相同；所述第二引物混合物包括至少1种引物组，不同种引物组分别与不同种靶核酸特异性结合。
该实施方式中，引物探针组合物中包括另外一种探针(第二探针)。探针的种类数根据其检测标记的种类数进行分类。不同种类的探针由于检测标记不同，需要在不同的检测通道下进行信号采集。某些实施例中，检测标记不同，并不意味着修饰的检测基团完全不相同，例如，检测基团包括荧光基团和猝灭基团，只要荧光基团不相同(猝灭基团可以相同，也可以不同)，也可以实现2种探针产生的信号在2个检测通道(或荧光通道)中进行信号采集。该实施例中，探针的种类数增加，可实现对不同的靶核酸在不同 通道下进行信号检测，可实现更多重的靶核酸检测。
本实施例中，出现“第一探针”、“第二探针”，并不用于限定作用，也不用于限定本实施例中仅有2种探针，此处仅用于区分“第一探针”和“第二探针”属于不同种类的探针，两种探针所修饰的检测标记不同、且碱基序列也不相同。某些实施例中，为检测更多种类的靶核酸，在用于靶核酸检测的引物探针组合物还包括第三探针和第三引物混合物、第四探针和第四引物混合物、第五探针和第五引物混合物、第六探针和第六引物混合物、或者更多探针和引物混合物。不同探针所修饰的检测标记不同、且碱基序列也不相同；不同引物混合物中的引物组也不相同。也就是说，为实现更多重的靶核酸检测，本领域技术人员可以根据需要，设计更多种与靶核酸对应的引物组、以及更多种与引物组对应的探针。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述第二引物混合物中的引物组与其对应的靶核酸特异性结合后产生预产物，所述预产物中含有与第二探针特异性结合的单链预产物，所述单链预产物与第二探针特异性结合并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起可检测的信号变化；
优选地，第二引物混合物中不同种的引物组与其对应的靶核酸产生的单链预产物不同；当第三单链预产物与第二探针的退火温度，高于第四单链预产物与第二探针的退火温度时，所述第三单链预产物与第二探针产生的双链产物的熔解温度，低于所述第四单链预产物与第二探针产生的双链产物的熔解温度。
上述退火温度和熔解温度的高低关系设置，能够实现在某个检测通道的某一检测温度下，采集的信号最多为1种靶核酸的信号(所述“最多”，是指，若待测样本中无该靶核酸，则不产生信号；若有，则只有该种靶核酸的信号，不会同时出现其他靶核酸信号的情况)。
本发明中，“第三”、“第四”等仅用于描述目的，用以区分所限定的对象，而不以任何方式限定次序或主次，此实施例中的“第三”仅用于与“第四”进行区分，即“第三单链预产物”和“第四单链预产物”属于不同的种类，其对应的靶核酸种类不同。
本发明中，“第三”、“第四”等仅用于描述目的，用以区分所限定的对象，也不用于限定单链预产物的种类数量。例如，本实施例中出现“第四单链预产物”，并不用于限定本实施例中仅有4种靶核酸。某些实施例中，还包括4种以上靶核酸，产生4种及以上的单链预产物。某些实施例中，第二引物混合物中包括3种及以上的引物组，这些引物组与其对应的靶核酸产生的单链预产物的退火温度、熔解温度，与第三单链预产物、第四单链预产物类似，即，某种引物组与其靶核酸产生的单链预产物与探针特异性结合所需的退火温度越低，该单链预产物与探针特异性结合并延伸≥0个碱基形成的双链产物的熔解温度就越高。以实现以下效果：当采用某一温度作为PCR的退火温度进行PCR循环扩增时，退火温度比该温度高的单链预产物，均能与第二探针结合形成双链产物，退火温度比该温低的单链预产物，仍处于单链状态；当采用某一温度进行信号采集时，仅有1种单链预产物与第二探针的双链产物呈双链状态，并产生可检测的靶核酸信号。其他种类的单链预产物，一部分由于退火温度低，在PCR循环扩增阶段不能与第二探针退火形成双链产物；另一部分由于退火温度高，在PCR循环扩增阶段能与第二探针退火形成双链产物，但由于 这些双链产物的熔解温度比信号采集的温度低，生成的双链产物也在该信号采集温度下呈单链状态。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述第一单链预产物与第一探针产生的双链产物的熔解温度，等于或相近于所述第三单链预产物与第二探针产生的双链产物的熔解温度；优选地，所述第一单链预产物与第一探针的退火温度，等于或相近于所述第三单链预产物与第二探针的退火温度。
该实施方式中，引物探针组合物中包括2种以上的探针。探针的种类数根据其检测标记的种类数进行分类。不同种类的探针由于检测标记不同，需要在不同的检测通道下进行信号采集。
本发明中，表述“等于”、“相近于”用于描述两个温度之间不存在差异，或者存在的差异较小。并且，由于差异较小，或者不存在差异，导致不同种的单链预产物在某一特定温度进行信号采集时，实现单链或双链的相同状态。例如，某一特定温度下，第一单链预产物与第三单链预产物均处于单链状态，或者第一单链预产物与第三单链预产物均处于双链状态。值得注意的是，虽然他们在某个特定温度进行信号采集时，可以均保持双链状态，但他们呈现双链状态时所结合的探针种类不同。某些实施例中，“等于”、“相近于”用于描述两个退火温度/两个熔解温度之间相差4℃以内；例如，相差0-4℃，优选地，相差002℃，更优选地，相差001℃。
该实施方式中，“第一”、“第二”、“第三”等仅用于描述目的，用以区分所限定的对象，而不以任何方式限定次序或主次，也不用于限定单链预产物或探针的种类数量。例如，本实施例中出现“第三单链预产物”，并不用于限定本实施例中仅有3种靶核酸、有3种单链预产物。此实施例中的“第三”仅用于与“第一”进行区分，即“第一单链预产物”和“第三单链预产物”属于不同的种类，其对应的靶核酸种类不同。又例如，本实施例中出现“第一探针”、“第二探针”，并不用于限定本实施例中仅有2种探针，此处仅用于区分“第一探针”和“第二探针”属于不同种类的探针，两种探针所修饰的检测基团不同、且碱基序列也不相同。该实施例还包括3种、或3种以上探针的情况。为实现更多重的靶核酸检测，本领域技术人员可以根据需要，设计更多种探针。
通过上述熔解温度相同或相近的设置，可实现：在某个温度下进行信号采集时，可以有2种以上的单链预产物与探针处于双链状态。但是，由于该温度下，呈现双链状态的不同单链预产物所结合的探针的种类是不一样的，可以通过采用相同温度、但在不同的检测通道下进行信号采集，以实现对不同的单链预产物所对应的靶核酸进行识别。如此，通过增加检测通道(荧光通道)的方式，可实现更多重的核酸检测。此外，还可以减少信号采集温度的变化次数，即，在某个信号采集温度下，只需转换检测通道即可实现对不同靶标进行检测；也可以减少PCR循环的次数，即，在某一次PCR循环后，相比于上一次PCR扩增，与每1种探针形成的双链产物的种类数最多增加1种(如果有2种探针，则增加2种双链产物)。
但是，值得注意的是，并不是只能通过上述熔解温度相同或相近的设置，才能实现在某个温度下进行信号采集时，可以有2种以上的单链预产物与探针处于双链状态(呈现双链状态的不同单链预产物所结合的探针的种类不同)。当熔解温度不相同或不相近的情况下(例如，两者相差6℃以上)，通过调整信 号采集的温度，使其低于不同单链预产物与不同种类探针所产生的不同双链产物的熔解温度，也可实现上述效果。例如，第一单链预产物与第一探针产生的双链产物的熔解温度Tm1，不等于或不相近于所述第三单链预产物与第二探针产生的双链产物的熔解温度Tm2，通过调整信号采集的温度，使其低于Tm1和Tm2，则在该信号采集温度下，两种双链产物依然呈双链状态。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述第一探针或第二探针为一段不与任何靶核酸特异性结合的序列，其包括探针信号检测区(H)，不同探针的探针信号检测区(H)的序列彼此不同；
所述引物组包括第一引物和第二引物，所述第一引物包含靶标序列结合区1；所述第二引物包含引物信号检测区(h)和靶标序列结合区2，且引物信号检测区(h)位于靶标序列结合区2的5’端；所述引物信号检测区(h)为一段不与任何靶核酸特异性结合、且与其对应探针的探针信号检测区(H)具有部分或全部相同的序列；不同引物组中第二引物的引物信号检测区(h)的序列彼此不同。
在某些实施例中，所述靶标序列结合区1和靶标序列结合区2分别与靶核酸的不同位置特异性结合。
某些实施例中，引物信号检测区(h)与探针的探针信号检测区(H)具有部分或全部相同的序列，是指，引物信号检测区(h)的反向互补序列(h’)，能够与探针的探针信号检测区(H)的部分序列或全部序列特异性结合。
在某些实施例中，上述第一引物和第二引物与靶核酸特异性结合后产生预产物，所述预产物中含有一条具有引物信号检测区(h)的反向互补序列(h’)的单链预产物，该单链预产物的反向互补序列(h’)与探针的探针信号检测区(H)特异性结合后并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起可检测的信号变化。
某些实施例中，不同引物组中的第二引物的引物信号检测区(h)的序列彼此不同，即，不同引物组中的第二引物的引物信号检测区(h)的序列的碱基不同和/或长度不同，使得不同单链预产物的反向互补序列(h’)的序列不同，不同单链预产物与探针特异性结合并延伸0个、或＞0个碱基后形成不同的双链产物，不同的双链产物的熔解温度能够彼此分开；或者，不同单链预产物分别与不同种类的探针特异性结合并延伸≥0个碱基后，形成不同的双链产物，不同的双链产物的熔解温度不能彼此分开，但由于不同双链产物的探针种类不同，可通过不同检测通道将其区分开。即，可通过熔解温度和/或检测通道，将不同种类的靶核酸进行区分，实现多重检测。
某些实施例中，第二引物的引物信号检测区(h)和靶标序列结合区2之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述第一探针或第二探针还包含引物锚定区(A’)；所述第一引物混合物或第二引物混合物中的至少1种引物组的第一引物还包含探针锚定区(A)，所述探针锚定区(A)位于靶标序列结合区1的5’端；所述探针锚定区(A)不与任何 靶核酸特异性结合、但与所述引物锚定区(A’)特异性结合。
上述引物组中的第一引物和第二引物与靶核酸特异性结合后产生预产物，该预产物中含有一条具有探针锚定区(A)、引物信号检测区(h)的反向互补序列(h’)的单链预产物，该单链预产物的探针锚定区(A)和反向互补序列(h’)，分别与探针的引物锚定区(A’)、探针信号检测区(H)特异性结合后并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起探针产生可检测的信号变化。
通过调整不同引物组中第一引物的探针锚定区(A)，和/或，不同引物组中第二引物的引物信号检测区(h)的序列组成和/或长度，使得不同引物组与其靶核酸所产生的单链预产物不同，不同的单链预产物与探针特异性结合所产生的退火温度不同、特异性结合并延伸≥0个碱基后形成的双链产物的熔解温度也不相同，以实现某种引物组与其靶核酸产生的单链预产物与探针特异性结合所需的退火温度越低，该单链预产物与探针特异性结合并延伸≥0个碱基形成的双链产物的熔解温度就越高。
某些实施例中，不同引物组中第一引物的靶标序列结合区1的序列不同；不同引物组中第一引物的探针锚定区(A)的序列可以相同、也可以不同；优选地，第一引物的探针锚定区(A)和靶标序列结合区1之间有0-20个碱基的间隔。其中，间隔的碱基数量可以为0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或其组成的范围。
在本发明的一些实施方式中，上述用于靶核酸检测的引物探针组合物，所述第一引物混合物或第二引物混合中，最多有1种引物组中的第二引物还包括延伸阻滞区(M)，所述延伸阻滞区(M)位于引物信号检测区(h)的5’端，所述延伸阻滞区(M)及其互补序列，均不与任何探针或任何靶核酸特异性结合。
上述第一引物和含有延伸阻滞区(M)的第二引物分别与靶核酸特异性结合后产生预产物，所述预产物中含有一条具有引物信号检测区(h)的反向互补序列(h’)的单链预产物，所述单链预产物的反向互补序列(h’)与探针特异性结合并延伸0个碱基后形成双链产物(由于延伸阻滞区的存在，单链预产物与探针特异性结合后不产生延伸)，从而引起探针产生可检测的信号变化。
在本发明的一些实施例中，引物与靶核酸特异性结合后，产生的单链预产物与探针特异性结合后不发生延伸(即，延伸0个碱基)。本领域技术人员可根据实际需求，通过对引物组中引物序列和/或探针的序列进行设计，实现上述“特异性结合但不发生延伸”的效果，例如，设计使其产生的第一单链预产物结合在探针的5’末端，或者，设计使其产生的第一单链预产物的3’末端含有不与探针互补配对的区域。
“第一引物混合物”、“第二引物混合物”，等仅用于描述目的，用以区分所限定的对象。两者均表示一组与探针特异性结合的引物混合物，该引物混合物中包括多种引物组，这些引物组中的第二引物含有的引物信号检测区(h)的反向互补序列(h’)，均可以与同一种探针特异性结合。但是，“第一引物混合物”和“第二引物混合物”中，其引物组所特异性结合的探针的种类不同。
某些实施例中，一组与同一种探针特异性结合的引物混合物中，最多有1种引物组中的第二引物含有延伸阻滞区(M)，该种引物组与靶核酸特异性结合后产生的单链预产物，与探针特异性结合所需的退 火温度最高、与探针特异性结合后不延伸产生的双链产物的熔解温度最低。
本发明中，“第一引物”、“第二引物”仅用于描述目的，用以区分所限定的对象，而不以任何方式限定次序或主次。例如，上述引物探针组合物中，引物组的第一引物和第二引物的结构可以互换，例如，第一引物包含引物信号检测区(h)和靶标序列结合区，第二引物包含靶标序列结合区；又例如，第一引物包含引物信号检测区(h)和靶标序列结合区，第二引物包含探针锚定区(A)和靶标序列结合区。某些实施例中，“第一引物”又名“正向引物”，“第二引物”又名“反向引物”。
本发明中，探针为一段不与任何靶核酸配对结合的自由设计序列，探针上修饰有检测标记。优选地，所述检测标记包括第一检测基团和第二检测基团，且所述第一检测基团和第二检测基团通过距离的变化产生信号的变化；优选地，所述第一检测基团与所述第二检测基团之间间隔3-250埃；优选地，间隔3-201埃；更优选地，间隔3-140埃；和/或，所述第一检测基团为荧光报告基团，所述第二检测基团为淬灭基团或其它能够与所述第一检测基团通过荧光共振能量转移产生信号变化的修饰基团。
本发明中，所述第一检测基团和第二检测基团在探针上的位置，使得单链预产物的反向互补序列(h’)与探针(P)特异性结合并延伸≥0个碱基后形成双链产物，只要该双链产物的形成，能够导致所述第一检测基团与第二检测基团的位置发生变化，即可引起探针产生可检测的信号变化。所述第一检测基团和所述第二检测基团的位置可以互换。
某些实施例中，在没有待测靶核酸存在时，无单链预产物与探针特异性结合，探针呈单链状态或其他二级结构，此时第一检测基团和第二检测基团之间的距离较近，荧光共振能量转移的效率较高；若第一引物中含有探针锚定区(A)，在没有待测靶核酸存在时，即使探针的引物锚定区(A’)与第一引物中的探针锚定区(A)互补，但探针的其他部分呈单链状态或其他二级结构，此时第一检测基团和第二检测基团之间的距离依然较近，荧光共振能量转移的效率较高。而当待测靶核酸存在时，第一引物与第二引物分别与靶序列特异性结合并延伸产生双链扩增产物，其中一条单链扩增产物与探针特异性结合形成双链产物，此时第一检测基团与第二检测基团之间的距离变长，荧光共振能量转移效率降低，使得荧光信号发生变化，可被仪器检测到。
为此，第四方面，本发明提供一种PCR检测试剂盒，包括第三方面的用于靶核酸检测的引物探针组合物。本领域技术人员知道，为实现PCR检测，该试剂盒还包括用于扩增的扩增试剂，例如，DNA聚合酶和dNTPs。当所述核酸模板为RNA时，所述扩增试剂还包括逆转录酶。扩增试剂还包括一些促进PCR反应的试剂，例如KCl、MgCl 2、Tris-HCl、二硫苏糖醇(DTT)、(NH 4) 2SO 4等。某些优选实施例中，本发明提供的PCR检测试剂盒为数字PCR检测试剂盒，其包括第三方面的用于靶核酸检测的引物探针组合物。
为此，第五方面，本发明提供一种多重分时核酸检测装置，包括
反应液容纳部，用于容纳数个包含试样和试剂的微液体；
温度调节部，用于调节所述反应液容纳部中微液体的温度；
信号检测部，用于检测所述反应液容纳部中的微液体产生的信号；
控制部，
所述控制部控制所述温度调节部以第一温度调节模式调节所述反应液容纳部的温度，使数个微液体中仅第一部分微液体产生有效信号；控制所述信号检测部对所述反应液容纳部进行信号检测以获得第一信号；
所述控制部控制所述温度调节部以第二温度调节模式调节所述反应液容纳部的温度，使同一个所述反应液容纳部容纳中的数个微液体，仅不同于所述第一部分微液体的第二部分微液体产生有效信号；控制所述信号检测部对所述反应液容纳部进行信号检测以获得第二信号。
所述微液体为包含试样和试剂的混合物。
上述第一温度调节模式和第二温度调节模式不同。某些实施例中，所述不同，是指两种温度调节模式所采用的最低温度不同。某些实施例中，第一温度调节模式采用的最低温度高于第二温度调节模式采用的最低温度。
在其中一个实施例中，所述控制部控制所述温度调节部将所述反应液容纳部中微液体的温度调整至温度t2的状态下，控制所述信号检测部对所述反应液容纳部进行信号检测以获得第一信号，
所述控制部控制所述温度调节部将所述反应液容纳部中微液体的温度调整至温度t3的状态下，控制所述信号检测部对所述反应液容纳部进行信号检测以获得第二信号，
其中，t2＜t3。
某些实施例中，t2与t3温度相差6℃以上；例如，相差6-46℃，又例如，相差6-40℃、6-35℃、6-30℃、6-25℃、6-20℃、6-18℃、6-15℃、6-12℃、6-10℃、6-8℃或6-7℃等。
在其中一个实施例中，所述试剂包括第一试剂和第二试剂；
所述反应液容纳部中微液体的温度为t2时，第一部分微液体中的第一试剂与试样结合而产生信号，
所述反应液容纳部中微液体的温度为t3时，第二部分微液体中的第二试剂与试样结合而产生信号。
在其他一个实施例中，所述第一温度调节模式包括孵育阶段和信号采集阶段，
在孵育阶段，所述温度调节部周期性地改变所述反应液容纳部中微液体的温度以使所述反应液容纳部反复从高温温度降至低温温度T2，或所述温度调节部使所述反应液容纳部保持恒温温度T2，
在信号采集阶段，所述信号检测部对所述反应液容纳部进行信号检测以获得第一信号。
在其中一个实施例中，所述第二温度调节模式包括孵育阶段和信号采集阶段，
在孵育阶段，所述温度调节部周期性地改变所述反应液容纳部中微液体的温度以使所述反应液容纳部反复从高温温度降至低温温度T3，或所述温度调节部使所述反应液容纳部保持恒温温度T3，
在信号采集阶段，所述信号检测部对所述反应液容纳部进行信号检测以获得第二信号，
其中，T2＞T3，t2＞T3。
某些实施例中，T2与T3的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、 4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
某些实施例中，t2与T3的温度相差5℃以上，例如，相差5-50℃，又例如，相差5-40℃、5-30℃、5-20℃、5-18℃、5-15℃、5-12℃、5-10℃、5-8℃、5-7℃或5-6℃等。
在其中一个实施例中，所述反应液容纳部中微液体的温度为T2时，第一部分微液体中的第一试剂与试样结合产生信号；
所述反应液容纳部中微液体的温度为T3时，第一部分微液体中的第一试剂与试样结合产生信号，第二部分微液体中的第二试剂与试样结合产生信号。
在其中一个实施例中，所述控制部控制所述温度调节部以第一温度调节模式和第二调节模式调节所述反应液容纳部中微液体的温度之前：
所述控制部控制所述温度调节部周期性地改变所述反应液容纳部中微液体的温度以使所述反应液容纳部反复从高温温度降至低温温度T1以使数个微液体中仅第四部分微液体产生有效信号，第四部分微液体与第一部分微液体和第二部分微液体均不同，其中，T1＞T2。
某些实施例中，T1与T2的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
在其中一个实施例中，所述试剂还包括第四试剂；
所述反应液容纳部中微液体的温度为T1时，第四部分微液体中的第四试剂与试样结合产生信号；
所述反应液容纳部中微液体的温度为T2时，第四部分微液体中的第四试剂与试样结合产生信号，第一部分微液体中的第一试剂与试样结合产生信号；
所述反应液容纳部中微液体的温度为T3时，第四部分微液体中的第四试剂与试样结合产生信号，第一部分微液体中的第一试剂与试样结合产生信号，第二部分微液体中的第二试剂与试样结合产生信号。
在其中一个实施例中，控制所述信号检测部对所述反应液容纳部进行信号检测以获得第四信号。
在其中一个实施例中，所述信号检测部对所述反应液容纳部进行信号检测以获得第四信号的过程中，所述控制部控制所述温度调节部将所述反应液容纳部中微液体的温度调整至t1或保持在T1，
所述反应液容纳部中微液体的温度为T1或t1的状态下，第四部分微液体中的第四试剂与试样保持结合而继续产生信号。
在其中一个实施例中，初始状态下，至多一个单位的试样被分配至所述微液体。
在其中一个实施例中，所述信号为荧光信号，所述信号检测部为相机。
在其中一个实施例中，所述控制部控制所述信号检测部在所述反应液容纳部平铺微液体的位置，对所述反应液容纳部进行信号检测。
在其中一个实施例中，所述控制部控制所述温度调节部在所述反应液容纳部平铺微液体的位置，对所述反应液容纳部进行温度调节。
为此，第六方面，本发明提供一种检测方法，用于对数个包含试样和试剂的微液体进行检测，包括 步骤：
以第一温度调节模式调节数个微液体的温度以使数个微液体中仅第一部分微液体产生有效信号；对数个微液体进行信号检测以获得第一信号；
以第二温度调节模式调节所述微液体的温度以使数个微液体中仅第二部分微液体产生有效信号，第二部分微液体与第一部分微液体不同；对数个微液体进行信号检测以获得第二信号。
在其中一个实施例中，将数个微液体的温度调整至t2的状态下，对数个微液体进行信号检测以获得第一信号，
将数个微液体的温度调整至t3的状态下，对数个微液体进行信号检测以获得第二信号，
其中，t2＜t3。
某些实施例中，t2与t3温度相差6℃以上；例如，相差6-46℃，又例如，相差6-40℃、6-35℃、6-30℃、6-25℃、6-20℃、6-18℃、6-15℃、6-12℃、6-10℃、6-8℃或6-7℃等。
在其中一个实施例中，所述试剂包括第一试剂和第二试剂；
所述反应液容纳部中微液体的温度为t2时，第一部分微液体中的第一试剂与试样结合而产生信号，
所述反应液容纳部中微液体的温度为t3时，第二部分微液体中的第二试剂与试样结合而产生信号。
在其中一个实施例中，所述第一温度调节模式包括孵育阶段和信号采集阶段，
在孵育阶段，调节数个微液体的温度以使数个微液体反复从高温温度降至低温温度T2，或调节数个微液体的温度以使数个微液体保持恒温温度T2，
在信号采集阶段，对数个微液体进行信号检测以获得第一信号。
在其中一个实施例中，所述第二温度调节模式包括孵育阶段和信号采集阶段，
在孵育阶段，调节数个微液体的温度以使数个微液体反复从高温温度降至低温温度T3，或调节数个微液体的温度以使数个微液体保持恒温温度T3，
在信号采集阶段，对数个微液体进行信号检测以获得第二信号，
其中，T2＞T3，t2＞T3。
某些实施例中，T2与T3的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
某些实施例中，t2与T3的温度相差5℃以上，例如，相差5-50℃，又例如，相差5-40℃、5-30℃、5-20℃、5-18℃、5-15℃、5-12℃、5-10℃、5-8℃、5-7℃或5-6℃等。
在其中一个实施例中，数个微液体的温度为T2时，第一部分微液体中的第一试剂与试样结合产生信号；
数个微液体的温度为T3时，第一部分微液体中的第一试剂与试样结合产生信号，第二部分微液体中的第二试剂与试样结合产生信号。
在其中一个实施例中，在以第一温度调节模式和第二调节模式调节数个微液体的温度之前：
周期性地改变数个微液体的温度以使数个微液体反复从高温温度降至低温温度T1以使数个微液体中仅第四部分微液体产生有效信号，第四部分微液体与第一部分微液体和第二部分微液体均不同，其中，T1＞T2。
某些实施例中，T1与T2的温度相差4℃以上，例如，相差4-40℃，又例如，相差4-30℃、4-20℃、4-18℃、4-15℃、4-12℃、4-10℃、4-8℃、4-6℃或4-5℃等。
在其中一个实施例中，所述试剂还包括第四试剂；
数个微液体的温度为T1时，第四部分微液体中的第四试剂与试样结合产生信号；
数个微液体的温度为T2时，第四部分微液体中的第四试剂与试样结合产生信号，第一部分微液体中的第一试剂与试样结合产生信号；
数个微液体的温度为T3时，第四部分微液体中的第四试剂与试样结合产生信号，第一部分微液体中的第一试剂与试样结合产生信号，第二部分微液体中的第二试剂与试样结合产生信号。
在其中一个实施例中，对数个微液体进行信号检测以获得第四信号。
在其中一个实施例中，对数个微液体进行信号检测以获得第四信号的过程中，将数个微液体的温度调整至t1或保持在T1，
数个微液体的温度为T1或t1的状态下，第四部分微液体中的第四试剂与试样保持结合而继续产生信号。
在其中一个实施例中，初始状态下，至多一个单位的试样被分配至所述微液体。
在其中一个实施例中，所述信号为荧光信号，通过相机对数个微液体进行信号检测。
本发明的检测方法及引物探针组合物与现有技术相比，其优点在于：
(1)检测信号互不交叉：本发明提供的检测方法及引物探针组合物，对每个荧光通道的每次信号采集，最多只有1种靶核酸的检测信号，检测结果更直观、准确，避免2种以上靶核酸的荧光信号重叠、产生检测误差的情况；
(2)多重检测：本发明提供的检测方法及引物探针组合物，可在数字PCR单管反应中同时进行荧光通道与熔解温度两个维度的分析，即使用同一荧光通道，通过熔解温度特征，可以检测不同的靶标(靶核酸)；或使用不同的荧光通道中，即可实现荧光通道数量与熔解温度特征之乘积的靶标种类检测；
(3)荧光背景低：每个荧光通道可仅使用1种探针，即可通过不同单链预产物与探针的不同熔解温度进行区分，大大降低了数字PCR反应中的荧光背景，提高了反应灵敏度；同时在试剂中减少了荧光探针的数量，大大降低了试剂成本，利于大规模推广应用；
(4)熔解温度可调整：本发明提供的方法是利用二次扩增产物(靶核酸与引物组扩增产生单链预产物，此为“一次扩增产物”；单链预产物与探针特异性结合后形成双链产物，此为“二次扩增产物”)的不同熔解温度进行区分，因此通过调整引物信号检测区(h)的长度或序列组成，或调整引物信号检测区(h)与探针(P)的全信号检测区(H)反向互补的位置，进而增高或降低与探针(P)形成的双链产物的熔解 温度，从而调整不同的信号采集温度；
(5)包容性好：本发明所述引物探针设计方法，与靶序列(靶核酸)有两部分(第一引物、第二引物)互补配对(其探针不与靶核酸互补配对)，相比Taqman水解探针法需要三部分(第一引物、第二引物和Taqman探针)与靶序列配对，在靶序列存在较多高变异区时，如病毒或细菌基因组，本发明所述引物探针设计方法的包容性更好，设计难度更低；
(6)样本消耗少：本发明提供的引物探针组合物及检测方法，对样本的消耗少，特别适合用于稀有样本的检测，可以大大提高加入检测反应的样本浓度，提高检测灵敏度，同时单管反应为后续进行免提取一管法检测提供了可能性；
(7)灵敏度高：本发明所述方法对靶序列的长度要求非常低，当靶标类型是短片段核酸时，例如游离核酸时，更短的靶序列长度在检测中有更高的灵敏度；
(8)适用范围广：本发明所述方法可适用于多种样本类型的核酸检测，包括血清样本、血浆样本、全血样本、痰液样本、拭子样本、灌洗液样本、新鲜组织样本、福尔马林固定石蜡包埋组织(FFPE)等。
本发明的多重分时核酸检测装置及检测方法与现有技术相比，其优点在于：可以实现不同待测核酸在不同时间产生有效信号，即，对不同的待测核酸的信号进行隔离，避免信号重叠、产生检测误差的情况，实现多重核酸的分时、高精度检测。
附图说明
附图说明是用来对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用来解释本发明，但不构成对本发明的限制。在附图中：
图1为本发明中一种引物探针设计原理图(针对3种靶标)；
当样本中不存在靶序列或扩增尚未开始时，探针(P)只有引物锚定区(A’)与正向引物(F1/2/3)的探针锚定区(A)反向互补，探针其余部分不与任何序列相互作用，此时探针(P)上修饰的第一检测基团与第二检测基团之间的距离为d1；在PCR扩增时，在较高的第一退火温度(T1)下退火，正向引物(F1)与反向引物(R1)分别与靶序列1特异性结合并延伸产生双链预产物，其中一条单链预产物(S1)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h1’)，延伸阻滞区的反向互补序列(M’)；正向引物(F2)与反向引物(R2)分别与靶序列2特异性结合并延伸产生双链预产物，其中一条单链预产物(S2)的5’端到3’端依次是探针锚定区(A2)，靶序列，引物信号检测区的反向互补序列(h2’)；任选的，如果存在正向引物(F3)和反向引物(R3)，正向引物(F3)与反向引物(R3)分别与靶序列3特异性结合并延伸产生双链预产物，其中一条单链预产物(S3)的5’端到3’端依次是探针锚定区(A3)，靶序列，引物信号检测区的反向互补序列(h3’)。待PCR扩增结束后，在较低的第一信号采集温度(t1)进行第一次信号采集。此时探针(P)的探针信号检测区(H)与单链预产物(S1)的引物信号检测区的反向互补序列(h1’)反向互补配对，探针(P)的引物锚定区(A1’)与单链预产物 (S1)5’端的探针锚定区(A1)反向互补配对，得到与探针(P)形成的双链产物(D1)，而单链预产物(S1)3’端的延伸阻滞区的反向互补序列(M’)不与探针(P)或靶序列的任意部分互补；此时探针(P)的全部或部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号，可被仪器检测到；而此时由于单链预产物(S2)3’端的引物信号检测区的反向互补序列(h2’)和单链预产物(S3)3’端的引物信号检测区的反向互补序列(h3’)的退火温度均小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，进行单链预产物(S2)的二次扩增循环，循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))，此时探针(P)的探针信号检测区(H)与单链预产物(S2)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A2’)与单链预产物(S2)5’端的探针锚定区(A2)反向互补配对，单链预产物(S2)的3’端可继续延伸到探针(P)的5’端，得到探针(P)的部分或全部反向互补序列，即完成以单链预产物(S2)为引物，以探针(P)为模板的二次扩增，得到与探针(P)形成的扩增双链产物(D2)，此时探针(P)的全部或部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号；在第二退火温度(T2)循环扩增结束后，再将温度升高至较高的第二信号采集温度(t2)进行第二次信号采集(t2>t1，且t2>T1)，此时单链预产物(S1)与探针(P)形成的杂交双链产物(D1)由于第二信号采集温度(t2)高于杂交双链产物(D1)的熔解温度而无法形成双链结构；且由于单链预产物(S3)3’端的引物信号检测区的反向互补序列(h3’)的退火温度小于第二退火温度(T2)，因此不与探针(P)的探针信号检测区(H)反向互补发生延伸；而单链预产物(S2)与探针(P)形成的扩增双链产物(D2)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到；在第二信号采集温度(t2)下完成第二次信号采集后，进行单链预产物(S3)的二次扩增循环，循环条件为高温解链，并在更低的第三退火温度(T3)下退火(T3<T2，且T3<t2)，此时探针(P)的探针信号检测区(H)与单链预产物(S3)3’端的引物信号检测区的反向互补序列(h3’)互补配对，探针的引物锚定区(A3’)与单链预产物(S3)5’端的探针锚定区(A3)反向互补配对，单链预产物(S3)的3’端可继续延伸到探针(P)的5’端，得到探针(P)的部分或全部反向互补序列，即完成以单链预产物(S3)为引物，以探针(P)为模板的二次扩增，得到与探针(P)形成的扩增双链产物(D3)，此时探针(P)的全部或部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号；在第三退火温度(T3)循环扩增结束后，再将温度升高至更高的第三信号采集温度(t3)进行第二次信号采集(t3>t2，且t3>T2)，此时单链预产物(S1)与探针(P)形成的杂交双链产物(D1)由于第三信号采集温度(t3)高于杂交双链产物(D1)的熔解温度而无法形成双链结构；单链预产物(S2)与探针(P)形成的扩增双链产物(D2)由于第三信号采集温度(t3)高于扩增双链产物(D2)的熔解温度而无法形成双链结构；而单链预产物(S3)与探针(P)形成的扩增双链产物(D3)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到。
图2A为实施例1的数字PCR检测反应在第一信号采集温度(60℃)的信号图；图2B为实施例1的数字PCR检测反应在第二信号采集温度(78℃)的信号图。
图3A为实施例2的数字PCR检测反应在第一信号采集温度(60℃)的信号图；图3B为实施例2的数字PCR检测反应在第二信号采集温度(78℃)的信号图。
图4A为实施例3的数字PCR检测反应在第一信号采集温度(60℃)的信号图；图4B为实施例3的数字PCR检测反应在第二信号采集温度(78℃)的信号图。
图5A为实施例4的数字PCR检测反应在第一信号采集温度(56℃)的信号图；图5B为实施例4的数字PCR检测反应在第二信号采集温度(72℃)的信号图。
图6A为实施例5的数字PCR检测反应在第一信号采集温度(55℃)的信号图；图6B为实施例5的数字PCR检测反应在第二信号采集温度(65℃)的信号图；图6C为实施例5的数字PCR检测反应在第三信号采集温度(73℃)的信号图。
图7A为实施例6的数字PCR检测反应在第一信号采集温度(72℃)时的信号图，由图可以看到有区别于背景的阳性液滴；图7B为实施例6的数字PCR检测反应在第二信号采集温度(82℃)时的信号图，由图可以看到均为阴性液滴。
图8A为本实施例7在第一信号采集温度(60℃)、VIC荧光通道采集的信号图，可以看到有区别于背景的阳性液滴；图8B为本实施例7中在第二信号采集温度(68℃)、VIC荧光通道采集的信号图，可以看到有区别于背景的阳性液滴，且该温度和通道下出现的阳性液滴与第一信号采集温度(60℃)VIC通道出现的阳性液滴位置不同；图8C为实施例7中在第二信号采集温度(68℃)、FAM荧光通道采集的信号图，可以看到有区别于背景的阳性液滴，且该温度和通道下出现的阳性液滴与第一信号采集温度(60℃)VIC通道和第二信号采集温度(68℃)VIC通道出现的阳性液滴的位置不同。
图9A为实施例8在第一信号采集温度(60℃)的信号图。图9B为实施例8在第二信号采集温度(78℃)的信号图。
图10A为实施例9在第一信号采集温度(60℃)的信号图。图10B为实施例9在第二信号采集温度(78℃)的信号图。
图11A为实施例10在第一信号采集温度(60℃)的信号图。图11B为实施例10在第二信号采集温度(78℃)的信号图。
图12A为实施例11在第一信号采集温度(60℃)的信号图。图12B为实施例11的在第二信号采集温度(78℃)的信号图。
图13A为实施例1在第一信号采集温度(61℃)的信号图。图13B为实施例12在第二信号采集温度(68℃)的信号图。图13C为实施例12在第三信号采集温度(76℃)的信号图。
图14A是本申请一实施例公开的多重分时核酸检测装置的示意图；图14B是本申请又一实施例公开的多重分时核酸检测装置的示意图；图14C是本申请又一实施例公开的由多个反应液容纳部组成的孔板结 构图。
图15A是本申请一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图15B是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图15C是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图15D是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图。
图16A是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图16B是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图16C是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图；图16D是本申请又一实施例公开的控制器控制温度调节部调节反应液容纳部温度的温度变化曲线图。
具体实施方式
为使本发明更加容易理解，下面将结合实施例来详细说明本发明，这些实施例仅起说明性作用，并不局限于本发明的应用范围。
第一实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
以下实施例中，均采用全自动数字PCR分析系统及试剂耗材进行检测和数据分析(其中，实施例1-7采用的是迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材；实施例8-12采用的是北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材)，该数字PCR分析系统是将含有样本、引物探针组合物和扩增试剂的反应体系分散到4个反应孔中进行扩增和检测(每个反应孔中含有多个小液滴，每个小液滴即为1个反应单元)，为避免累赘，以下实施例的附图均提供其中1个反应孔的液滴结果图。
实施例1
(1)用于检测非小细胞肺癌EGFR 21号外显子L858R突变的单重检测引物探针体系如表1所示：
表1实施例1引物探针的碱基序列
Figure PCTCN2022142406-appb-000001
上述引物探针中：
正向引物F1(SEQ ID NO:3)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，F1全长32bp，其3’端第1至19个碱基为靶序列结合区，其5’端的第1至10个碱基为探针锚定区(A1)，与探针P1(SEQ ID NO:1)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R1(SEQ ID NO:2)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，R1全长50bp，其3’端第1至20个碱基为靶序列结合区，其5’端的第1至5个碱基为延伸阻滞区(M)，其5’端的第6至27个碱基为引物信号检测区(h3)，与探针P1(SEQ ID NO:1)的5’端第13至34个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表2所示。
表2实施例1的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F1) 500nM
反向引物(R1) 100nM
探针(P1) 400nM
核酸模板 150拷贝
超纯水 加至20μL
注表2中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4；mM指的是mmol/L(下同)。
样本制备：使用非小细胞肺癌EGFR 21号外显子L858R突变DNA作为阳性样本,用带有信号检测区域序列的反向引物(R1)对非小细胞肺癌EGFR 21号外显子L858R突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表2中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；78℃进行第二次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图2A为反向引物(R1)扩增出的单重PCR非小细胞肺癌EGFR 21号外显子L858R突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到有区别于背景的阳性液滴。
图2B为反向引物(R1)扩增出的单重PCR非小细胞肺癌EGFR 21号外显子L858R突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到均为阴性液滴。
由图2A和2B可以看出：采用本发明的检测方法，可以在不同信号采集温度下获得不同的检测信号结果，对靶标信号进行“开”和“关”，实现对靶标更准确的检测。
实施例2
(1)用于检测非小细胞肺癌EGFR 21号外显子L858R突变的单重检测引物探针体系如表3所示：
表3实施例2引物探针的序列
Figure PCTCN2022142406-appb-000002
上述引物探针中：
正向引物F1(SEQ ID NO:3)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，F1全长32bp，其3’端第1至19个碱基为靶序列结合区，其5’端的第1至10个碱基为探针锚定区(A1)，与探针P1(SEQ ID NO:1)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R2(SEQ ID NO:4)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，R2全长33bp，其3’端第1至20个碱基为靶序列结合区，其5’端的第1至10个碱基为引物信号检测区(h2)，与探针P1(SEQ ID NO:1)的3’端第1至10个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表4所示。
表4实施例2的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F1) 500nM
反向引物(R2) 100nM
探针(P1) 400nM
核酸模板 130拷贝
超纯水 加至20μL
注表4中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 21号外显子L858R突变DNA作为阳性样本,用带有信号检测区域序列的反向引物(R2)对非小细胞肺癌EGFR 21号外显子L858R突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表4中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；78℃进行第二次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图3A为反向引物(R2)扩增出的单重PCR非小细胞肺癌EGFR21号外显子L858R突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到均为阴性液滴。
图3B为反向引物(R2)扩增出的单重PCR非小细胞肺癌EGFR 21号外显子L858R突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到有区别于背景的阳性液滴。
由图3A和3B可以看出：采用本发明的引物探针组合物、以及数字PCR检测方法，可以在不同信号采集温度下获得不同的检测信号结果，对靶标信号进行“关”和“开”，实现对靶标更准确的检测。
实施例3
(1)用于检测非小细胞肺癌EGFR 21号外显子L858R突变的单重检测引物探针体系如表5所示：
表5实施例3引物探针的碱基序列
Figure PCTCN2022142406-appb-000003
Figure PCTCN2022142406-appb-000004
上述引物探针中：
正向引物F1(SEQ ID NO:3)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，F1全长32bp，其3’端第1至19个碱基为靶序列结合区，其5’端的第1至10个碱基为探针锚定区(A1)，与探针P1(SEQ ID NO:1)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R2(SEQ ID NO:4)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，R2全长33bp，其3’端第1至20个碱基为靶序列结合区，其5’端的第1至10个碱基为引物信号检测区(h2)，与探针P1(SEQ ID NO:1)的3’端第1至10个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表6所示。
表6实施例3的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F1) 500nM
反向引物(R2) 100nM
探针(P1) 400nM
核酸模板 160拷贝
超纯水 加至20μL
注表6中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 21号外显子L858R突变DNA作为阳性样本，用带有信号检测区域序列的反向引物(R2)对非小细胞肺癌EGFR 21号外显子L858R突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表6中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；40℃恒温孵育5分钟；78℃进行第二次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图4A为反向引物(R2)扩增出的单重PCR非小细胞肺癌EGFR 21号外显子L858R突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到均为阴性液滴(处于边缘和/或看起来比较大的液滴，为实验操作问题，并非阳性液滴)。
图4B为反向引物(R2)扩增出的单重PCR非小细胞肺癌EGFR 21号外显子L858R突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到有区别于背景的阳性液滴。
由图4A和4B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，可以在不同信号采集温度下(60℃、78℃)获得不同的检测信号结果，对靶标信号进行“关”和“开”，实现对靶标更准确的检测。
实施例4
(1)用于检测耐碳青霉烯类抗生素的基因VIM突变和耐碳青霉烯类抗生素的基因OXA-48突变的双重检测引物探针体系如表7所示：
表7实施例4引物探针的碱基序列
Figure PCTCN2022142406-appb-000005
上述引物探针中：
正向引物F1-4(SEQ ID NO:6)，为针对耐碳青霉烯类抗生素的基因VIM突变靶序列设计的特异性引物，F1-4全长37bp，其3’端第1至23个碱基为靶序列结合区，其5’端的第1至11个碱基为探针锚定区(A)，与探针P2-4(SEQ ID NO:5)的5’端第1至11个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R1-4(SEQ ID NO:7)，为针对耐碳青霉烯类抗生素的基因VIM突变靶序列设计的特异性引物，R1-4全长55bp，其3’端第1至25个碱基为靶序列结合区，其5’端的第1至5个碱基为信号阻滞区(M)，其5’端的第6至27个碱基为引物信号检测区(h1)，与探针P2-4(SEQ ID NO:5)的5’端第14至35个碱基序列相同；
正向引物F2-4(SEQ ID NO:8)，为针对耐碳青霉烯类抗生素的基因OXA-48突变靶序列设计的特异 性引物，F2-4全长33bp，其3’端第1至19个碱基为靶序列结合区，其5’端的第1至11个碱基为探针锚定区(A)，与探针P2-4(SEQ ID NO:5)的5’端第1至11个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R2-4(SEQ ID NO:9)，为针对耐碳青霉烯类抗生素的基因OXA-48突变靶序列设计的特异性引物，R2-4引物全长37bp，其3’端第1至24个碱基为靶序列结合区，其5’端的第1至10个碱基与探针P2-4(SEQ ID NO:5)的3’端的第1至10个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表8所示。
表8实施例4的检测反应体系
Figure PCTCN2022142406-appb-000006
注表8中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用耐碳青霉烯类抗生素的基因VIM突变的核酸和耐碳青霉烯类抗生素的基因OXA-48突变的核酸作为阳性样本，用带有不同信号检测区域序列的反向引物(R1-4和R2-4)对耐碳青霉烯类抗生素的基因VIM突变和耐碳青霉烯类抗生素的基因OXA-48突变靶序列进行检测，以验证双重突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表8中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；56℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；72℃进行第二次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图5A为本实施例在第一信号采集温度(56℃)时的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因VIM突变阳性液滴(处于边缘和/或看起来比较大的液滴，为实验操作问题，并非阳性液滴)。
图5B为本实施例在第二信号采集温度(72℃)时的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因OXA-48突变阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(56℃)出现的阳性液滴位置不同。
由图5A和图5B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，实现在不同的信号检测温度下(56℃、72℃)，分别得到不同靶序列的检测信号，且互不交叉。
实施例5
(1)用于检测耐碳青霉烯类抗生素的基因IMP突变，耐碳青霉烯类抗生素的基因OXA-48突变和耐碳青霉烯类抗生素的基因KPC突变的三重检测引物探针体系，如表9所示：
表9实施例5引物探针的碱基序列
Figure PCTCN2022142406-appb-000007
上述引物探针中：
所述正向引物F1-5(SEQ ID NO:11)，为针对耐碳青霉烯类抗生素的基因IMP突变靶序列设计的特异性引物，F1-5全长42bp，其3’端第1至24个碱基为靶序列结合区，其5’端的第1至15个碱基为探针锚定区(A)，与探针P1-5(SEQ ID NO:10)的5’端第1至15个碱基序列，即引物锚定区(A’)，反向互补；
所述反向引物R1-5(SEQ ID NO:12)，为针对耐碳青霉烯类抗生素的基因IMP突变靶序列设计的特异性引物，R1-5全长56bp，其3’端第1至23个碱基为靶序列结合区，其5’端的第1至5个碱基为信号阻滞区(M)，其5’端的第6至30个碱基为引物信号检测区(h1)，与探针P1-5(SEQ ID NO:10)的5’端第17至41个碱基序列相同；
所述正向引物F2-5(SEQ ID NO:13)，为针对耐碳青霉烯类抗生素的基因OXA-48突变靶序列设计的特异性引物，F2-5全长37bp，其3’端第1至19个碱基为靶序列结合区，其5’端的第1至15个碱基为探针锚定区(A)，与探针P1-5(SEQ ID NO:10)的5’端第1至15个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R2-5(SEQ ID NO:14)，为针对耐碳青霉烯类抗生素的基因OXA-48突变靶序列设计的特异性引物，R2-5引物全长38bp，其3’端第1至24个碱基为靶序列结合区，其5’端的第1至11个碱基与探针P1-5(SEQ ID NO:10)的3’端的第15至25个碱基序列相同。
所述正向引物F3-5(SEQ ID NO:15)，为针对耐碳青霉烯类抗生素的基因KPC突变靶序列设计的特异性引物，F3-5全长39bp，其3’端第1至21个碱基为靶序列结合区，其5’端的第1至15个碱基为探针锚定区(A)，与探针P1-5(SEQ ID NO:10)的5’端第1至15个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R3-5(SEQ ID NO:16)，为针对耐碳青霉烯类抗生素的基因KPC突变靶序列设计的特异性引物，R3-5引物全长33bp，其3’端第1至22个碱基为靶序列结合区，其5’端的第1至8个碱基与探针P1-5(SEQ ID NO:10)的3’端的第1至8个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表10所示。
表10实施例5的检测反应体系
Figure PCTCN2022142406-appb-000008
Figure PCTCN2022142406-appb-000009
注表10中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用耐碳青霉烯类抗生素的基因IMP突变的核酸样本，耐碳青霉烯类抗生素的基因OXA-48突变的核酸样本和耐碳青霉烯类抗生素的基因KPC突变的核酸样本作为阳性样本,用带有不同信号检测区域序列的反向引物(R1-5/R2-5/R3-5)对耐碳青霉烯类抗生素的基因IMP突变，耐碳青霉烯类抗生素的基因OXA-48突变和耐碳青霉烯类抗生素的基因KPC突变靶序列进行检测，以验证三重突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表10中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；55℃进行第一次信号采集；94℃变性15秒，52℃退火延伸20秒，共进行5个循环；65℃进行第二次信号采集。94℃变性15秒，40℃退火延伸20秒，共进行5个循环；73℃进行第三次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图6A为本实施例中耐碳青霉烯类抗生素的基因IMP突变，耐碳青霉烯类抗生素的基因OXA-48突变和耐碳青霉烯类抗生素的基因KPC突变阳性样本在第一信号采集温度(55℃)时的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因IMP突变阳性液滴。
图6B为本实施例中耐碳青霉烯类抗生素的基因IMP突变，耐碳青霉烯类抗生素的基因OXA-48突变和耐碳青霉烯类抗生素的基因KPC突变阳性样本在第二信号采集温度(65℃)时的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因OXA-48突变阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(55℃)出现的阳性液滴位置不同。
图6C为本实施例中耐碳青霉烯类抗生素的基因IMP突变，耐碳青霉烯类抗生素的基因OXA-48突变和耐碳青霉烯类抗生素的基因KPC突变阳性样本在第三信号采集温度(73℃)时的液滴图，为耐碳青霉烯类抗生素的基因KPC突变阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(55℃)和第二信号采集温度(65℃)出现的阳性液滴位置不同。
由图6A-6C可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，实现在不同的信号检测温度下(55℃、65℃、73℃)，分别得到不同靶序列的检测信号，且互不交叉。
实施例6
(1)用于检测非小细胞肺癌EGFR 21号外显子L858R突变和非小细胞肺癌EGFR 21号外显子L858R突变的单重检测引物探针体系如表11所示：
表11实施例6引物探针的碱基序列
Figure PCTCN2022142406-appb-000010
上述引物探针中：
正向引物F2-6(SEQ ID NO:18)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，F2-6全长32bp，其3’端第1至第19个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A2)，与探针P1-6(SEQ ID NO:5)的5’端第1至10个碱基序列，即引物锚定区(A2’)，反向互补；
反向引物R2-6(SEQ ID:17)，为针对非小细胞肺癌EGFR 21号外显子L858R突变靶序列设计的特异性引物，R2-6全长33bp，其3’端第1至第20个碱基为靶序列结合区，其5’端的第1至第10个碱基为引物信号检测区(h2)，与探针P1-6(SEQ ID NO:5)的3’端第1至10个碱基序列相同。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表12所示。
表12实施例6的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F2-6) 500nM
反向引物(R2-6) 100nM
探针(P1-6) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表12中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 21号外显子L858R突变作为阳性样本,使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表12中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；72℃进行第一次信号采集；82℃进行第二次信号采集。
数据分析(采用迈克生物股份有限公司D600全自动数字PCR分析系统进行数据分析)：
通过不同信号采集温度下采集的信号对结果进行判读。
图7A为在第一信号采集温度(72℃)时的液滴图，可以看到有区别于背景的阳性液滴。
图7B为在第二信号采集温度(82℃)时的液滴图，可以看到均为阴性液滴。
由图7A和7B可以看出：采用本发明的检测方法，可以在不同信号采集温度下(72℃、82℃)获得不同的检测信号结果，对靶标信号进行“开”和“关”，实现对靶标更准确的检测。
实施例7
(1)用于检测耐碳青霉烯类抗生素的基因KPC突变，耐碳青霉烯类抗生素的基因IMP突变和耐碳青霉烯类抗生素的基因VIM突变的三重检测引物探针体系，如表13所示：
表13实施例7引物探针的碱基序列
Figure PCTCN2022142406-appb-000011
Figure PCTCN2022142406-appb-000012
上述引物探针中：
正向引物F1-7(SEQ ID NO:20)，为针对耐碳青霉烯类抗生素的基因KPC突变靶序列设计的特异性引物，F1-7全长36bp，其3’端第1至第21个碱基为靶序列结合区，其5’端的第1至第12个碱基为探针锚定区(A)，与探针P1-7(SEQ ID NO:19)的5’端第1至12个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R1-7(SEQ ID：21)，为针对耐碳青霉烯类抗生素的基因KPC突变靶序列设计的特异性引物，R1-7全长52bp，其3’端第1至第22个碱基为靶序列结合区，其5’端的第1至第5个碱基为信号阻滞区(M)，其5’端的第6至第27个碱基为引物信号检测区(h1)，与探针P1-7(SEQ ID NO:19)的5’端第14至35个碱基序列相同；
正向引物F2-7(SEQ ID NO:22)，为针对耐碳青霉烯类抗生素的基因IMP突变靶序列设计的特异性引物，F2-7全长39bp，其3’端第1至第24个碱基为靶序列结合区，其5’端的第1至第12个碱基为探针锚定区(A)，与探针P1-7(SEQ ID NO:19)的5’端第1至12个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R2-7(SEQ ID NO:23)，为针对耐碳青霉烯类抗生素的基因IMP突变靶序列设计的特异性引物，R2-7引物全长37bp，其3’端第1至第23个碱基为靶序列结合区，其5’端的第1至第11个碱基与探针探针P1-7(SEQ ID NO:19)的3’端的第1至第11个碱基序列相同。
正向引物F3-7(SEQ ID NO:24)，为针对耐碳青霉烯类抗生素的基因VIM突变靶序列设计的特异性引物，F3-7全长36bp，其3’端第1至第23个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P2-7(SEQ ID NO:1)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R3-7(SEQ ID NO:19)，为针对耐碳青霉烯类抗生素的基因VIM突变靶序列设计的特异性引物，R3-7引物全长38bp，其3’端第1至第25个碱基为靶序列结合区，其5’端的第1至第10个碱基与探针探针P2-7(SEQ ID NO:1)的3’端的第1至第10个碱基序列相同。
利用上述引物探针进行检测的原理如下：
当对病原靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-7)与反向引物(R1-7)分别与靶序列1(耐碳青霉烯类抗生素的基因KPC突变)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S1)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h1’)，延伸阻滞区的反向互补序列(M’)；正向引物(F2-7)与反向引物(R2-7)分别与靶序列2(耐碳青霉烯类抗生素的基因IMP突变)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S2)的5’端到3’端依次是探针锚定区(A2)，靶序列，引物信号检测区的反向互补序列(h2’)；正向引物(F3-7)与反向引物(R3-7)分别与靶序列3(耐碳青霉烯类抗生素的基因VIM突变)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S3)的5’端到3’端依次是探针锚定区(A3)， 靶序列，引物信号检测区的反向互补序列(h3’)。
待PCR扩增结束后，在较低的第一信号采集温度(t1)进行信号采集。此时探针(P1-7)的探针信号检测区(H)与单链预扩增产物(S1)的引物信号检测区的反向互补序列(h1’)反向互补配对，探针(P1-7)的引物锚定区(A1’)与单链预扩增产物(S1)5’端的探针锚定区(A1)反向互补配对，得到与探针(P1-7)形成的杂交双链产物(D1)，而单链预扩增产物(S1)3’端的延伸阻滞区的反向互补序列(M’)不与探针(P1-7)或靶序列的任意部分互补；此时探针(P1-7)的部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号，可被仪器检测到。而此时由于单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)和单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h3’)的退火温度均小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，进行单链预扩增产物(S2和S3)的二次扩增循环，循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1)，此时探针(P1-7)的探针信号检测区(H)与单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A2’)与单链预扩增产物(S2)5’端的探针锚定区(A2)反向互补配对，单链预扩增产物(S2)的3’端可继续延伸到探针(P)的5’端，得到探针(P1-7)的全部反向互补序列，即完成以单链预扩增产物(S2)为引物，以探针(P1-7)为模板的二次扩增，得到与探针(P1-7)形成的扩增双链产物(D2)，此时探针(P1-7)的部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号；此时探针(P2-7)的探针信号检测区(H)与单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h3’)互补配对，探针的引物锚定区(A3’)与单链预扩增产物(S3)5’端的探针锚定区(A3)反向互补配对，单链预扩增产物(S3)的3’端可继续延伸到探针(P2-7)的5’端，得到探针(P2-7)的全部反向互补序列，即完成以单链预扩增产物(S3)为引物，以探针(P2-7)为模板的二次扩增，得到与探针(P2-7)形成的扩增双链产物(D3)，此时探针(P2-7)的部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表14所示。
表14实施例7的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F1-7) 250nM
反向引物(R1-7) 100nM
正向引物(F2-7) 250nM
反向引物(R2-7) 100nM
正向引物(F3-7) 250nM
反向引物(R3-7) 100nM
探针(P1-7) 400nM
探针(P2-7) 400nM
耐碳青霉烯类抗生素的基因KPC突变核酸模板 150拷贝
耐碳青霉烯类抗生素的基因IMP突变核酸模板 80拷贝
耐碳青霉烯类抗生素的基因VIM突变核酸模板 120拷贝
超纯水 加至20μL
注表14中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用碳青霉烯类抗生素的基因KPC突变、基因IMP突变和基因VIM突变作为阳性样本，将三种样本混合，进行检测，以验证三重靶标的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表14中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用迈克生物股份有限公司D600全自动数字PCR分析系统及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集(VIC通道)；94℃变性15秒，52℃退火延伸20秒，共进行5个循环；68℃进行第二次信号采集(包括两次信号通道采集，分别为VIC通道和FAM通道)。
数据分析：
通过不同信号通道和不同信号采集温度下的阳性液滴对结果进行判读。
图8A为本实施例在第一信号采集温度(60℃)、VIC荧光通道采集的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因KPC突变阳性液滴。
图8B为本实施例中在第二信号采集温度(68℃)、VIC荧光通道采集的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因IMP突变阳性液滴，且该温度和通道下出现的阳性液滴与第一信号采集温度(60℃)VIC通道出现的阳性液滴位置不同。
图8C为本实施例中在第二信号采集温度(68℃)、FAM荧光通道采集的液滴图，可以看到有区别于背景的阳性液滴，为耐碳青霉烯类抗生素的基因VIM突变阳性液滴，且该温度和通道下出现的阳性液滴与第一信号采集温度(60℃)VIC通道和第二信号采集温度(68℃)VIC通道出现的阳性液滴的位置不同。
由图8A至8C可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，通过不同的信号采集温度和不同的信号采集通道，获得不同靶序列的检测信号，且互不交叉，检测结果更直观、清楚。
实施例8
(1)用于检测非小细胞肺癌EGFR 20号外显子H773_V774insH突变的单重检测引物探针体系如表15所示：
表15实施例8引物探针的碱基序列
Figure PCTCN2022142406-appb-000013
上述引物探针中：
正向引物F1-8(SEQ ID NO:27)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，F1-8全长29bp，其3’端第1至第16个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A1)，与探针P1-8(SEQ ID NO:25)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R1-8(SEQ ID NO：26)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，R1-8全长51bp，其3’端第1至第21个碱基为靶序列结合区，其5’端的第1至第5个碱基为延伸阻滞区(M)，其5’端的第6至第27个碱基为引物信号检测区(h3)，与探针P1-8(SEQ ID NO:25)的3’端第26至47个碱基序列相同。
利用上述引物探针进行检测的原理如下：当对靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-8)与反向引物(R1-8)分别与靶序列1特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S1)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h1’)，延伸阻滞区的反向互补序列(M’)；待PCR扩增结束后，在较低的第一信号采集温度(t1)进行信号采集。此时探针(P1-8)的探针信号检测区(H)与单链预扩增产物(S1)的引物信号检测区的反向互补序列(h1’)反向互补配对，探针(P1-8)的引物锚定区(A1’)与单链预扩增产物(S1)5’端的探针锚定区(A1)反向互补配对，得到与探针(P1-8)形成的杂交双链产物(D1)，而单链预扩增产物(S1)3’端的延伸阻滞区的反向互补序列(M’)不与探针(P1-8)或靶序列的任意部分互补；此时探针(P)的全部或部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号，可被仪器检测到。在第一信号采集温度(t1)采集信号完成后，再进入循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))的程序，并在第二信号采集温度(t2)下采集信号(t2>t1，且t2>T1)，此时单链预扩增产物(S1)与探针(P1-8)形成的杂交双链产物(D1)由于第二信号采集温度(t2)高于杂交双链产物(D1)的熔解温度而无法形成双链结构，第一检测基团与第二检测基团之间的距离发生变化，使得荧光信号发生变化，可被仪器检测到。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表16所示。
表16实施例8的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F1-8) 500nM
反向引物(R1-8) 100nM
探针(P1-8) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表16中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 20号外显子H773_V774insH突变DNA作为阳性样本,用带有信号检测区域序列的反向引物(R1)对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表16中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；78℃进行第二次信号采集。
数据分析(采用北京达微生物科技有限公司OS-500数字PCR一体机分析软件进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图9A为反向引物(R1-8)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到有区别于背景的阳性液滴。
图9B为反向引物(R1-8)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到均为阴性液滴。
由图9A和图9B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，可以在不同信号采集温度下获得不同的检测信号结果，对靶标信号进行“开”和“关”，实现对靶标更准确的检测。
实施例9
(1)用于检测非小细胞肺癌EGFR 20号外显子H773_V774insH突变的单重检测引物探针体系如表17所示：
表17实施例9引物探针的碱基序列
Figure PCTCN2022142406-appb-000014
上述引物探针中：
正向引物F1-9(SEQ ID NO:27)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，F1-9全长29bp，其3’端第1至第16个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A1)，与探针P1-9(SEQ ID NO:25)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R2-9(SEQ ID NO:28)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，R2-9全长32bp，其3’端第1至第21个碱基为靶序列结合区，其5’端的第1至第8个碱基为引物信号检测区(h2)，与探针P1-9(SEQ ID NO:25)的3’端第1至8个碱基序列相同。
利用上述引物探针进行检测的原理如下：当对靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-9)与反向引物(R2-9)分别与靶序列特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S2)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h2’)；在较低的第一信号采集温度(t1)进行第一次信号采集(t1>T1)，由于单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)的退火温度小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P1-9)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，再进入循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))的程序，此时探针(P1-9)的探针信号检测区(H)与单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A1’)与单链预扩增产物(S2)5’端的探针锚定区(A1)反向互补配对，单链预扩增产物(S2)的3’端可继续延伸到探针(P1-9)的5’端，得到探针(P1-9)的反向互补序列，即完成以单链预扩增产物(S2)为引物，以探针(P1-9)为模板的二次扩增，得到与探针(P1-9)形成的扩增双链产物(D2)，此时将温度升高至较高的第二信号采集温度(t2)采集信号(t2>t1，且t2>T1)，单链预扩增产物(S2)与探针(P1-9)形成的扩增双链产物(D2)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表18所示。
表18实施例9的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
反转录酶 5U
正向引物(F1-9) 500nM
反向引物(R2-9) 100nM
探针(P1-9) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表18中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 20号外显子H773_V774insH突变DNA作为阳性样本，用带有信号检测区域序列的反向引物(R2-9)对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表18中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；78℃进行第二次信号采集。
数据分析(采用北京达微生物科技有限公司OS-500数字PCR一体机分析软件进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图10A为反向引物(R2-9)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到均为阴性液滴。
图10B为反向引物(R2-9)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到有区别于背景的阳性液滴。
由图10A和图10B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，可以在不同信号采集温度下获得不同的检测信号结果，对靶标信号进行“关”和“开”，实现对靶标更准确的检测。
实施例10
(1)用于检测非小细胞肺癌EGFR 20号外显子H773_V774insH突变的单重检测引物探针体系如表19所示：
表19实施例10引物探针的碱基序列
Figure PCTCN2022142406-appb-000015
上述引物探针中：
正向引物F1-10(SEQ ID NO:27)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，F1-10全长29bp，其3’端第1至第16个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A1)，与探针P1-10(SEQ ID NO:25)的5’端第1至10个碱基序列，即引物锚定区(A1’)，反向互补；
反向引物R2-10(SEQ ID NO:28)，为针对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列设计的特异性引物，R2-10全长32bp，其3’端第1至第21个碱基为靶序列结合区，其5’端的第1至第8个碱基为引物信号检测区(h2)，与探针P1-10(SEQ ID NO:25)的3’端第1至8个碱基序列相同。
利用上述引物探针进行检测的原理如下：当对靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-10)与反向引物(R2-10)分别与靶序列2特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S3)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h2’)；在较低的第一信号采集温度(t1)进行第一次信号采集(t1>T1)，由于单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h2’)的退火温度小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P1-10)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，再进入循环条件为恒温扩增，恒温采用较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))的程序，此时探针(P1-10)的探针信号检测区(H)与单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A1’)与单链预扩增产物(S3)5’端的探针锚定区(A1)反向互补配对，单链预扩增产物(S3)的3’端可继续延伸到探针(P1)的5’端，得到探针(P1-10)的反向互补序列，即完成以单链预扩增产物(S3)为引物，以探针(P1-10)为模板的二次扩增，得到与探针(P1-10)形成的扩增双链产物(D3)，此时将温度升高至较高的第二信号采集温度(t2)采集信号(t2>t1，且t2>T1)，单链预扩增产物(S3)与探针(P)形成的扩增双链产物(D3)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表20所示。
表20实施例10的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
反转录酶 5U
正向引物(F1-10) 500nM
反向引物(R2-10) 100nM
探针(P1-10) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表20中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用非小细胞肺癌EGFR 20号外显子H773_V774insH突变DNA作为阳性样本，用带有信号检测区域序列的反向引物(R2-10)对非小细胞肺癌EGFR 20号外显子H773_V774insH突变靶序列进行检测，以验证单一突变的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表20中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；40℃恒温孵育5分钟；78℃进行第二次信号采集。
数据分析(采用北京达微生物科技有限公司OS-500数字PCR一体机分析软件进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图11A为反向引物(R2-10)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到均为阴性液滴(处于边缘和/或看起来比较大的液滴，为实验操作问题，并非阳性液滴)。
图11B为反向引物(R2-10)扩增出的单重PCR非小细胞肺癌EGFR 20号外显子H773_V774insH突变阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到有区别于背景的阳性液滴。
由图11A和图11B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，可以在不同信号采集温度下获得不同的检测信号结果，对靶标信号进行“关”和“开”，实现对靶标更准确的检测。
实施例11
(1)用于检测甲型流感病毒(Influenza A virus，IAV)和呼吸道合胞病毒(Respiratory Syncytial Virus， RSV)的双重检测引物探针体系如表21所示：
表21实施例11引物探针的碱基序列
Figure PCTCN2022142406-appb-000016
上述引物探针中：
正向引物F1-11(SEQ ID NO:30)，为针对呼吸道合胞病毒靶序列设计的特异性引物，F1-11全长42bp，其3’端第1至第29个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P1-11(SEQ ID NO:25)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R1-11(SEQ ID NO：29)，为针对呼吸道合胞病毒靶序列设计的特异性引物，R1-11全长54bp，其3’端第1至第24个碱基为靶序列结合区，其5’端的第1至第6个碱基为信号阻滞区(M)，其5’端的第7至第27个碱基为引物信号检测区(h1)，与探针P1-11(SEQ ID NO:25)的3’端第26至46个碱基序列相同；
正向引物F2-11(SEQ ID NO:31)，为针对甲型流感病毒靶序列设计的特异性引物，F2-11全长32bp，其3’端第1至第20个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P1-11(SEQ ID NO:25)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R2-11(SEQ ID NO:32)，为针对甲型流感病毒靶序列设计的特异性引物，R2-11引物全长31bp，其3’端第1至第19个碱基为靶序列结合区，其5’端的第1至第8个碱基与探针探针P1-11(SEQ ID NO:25)的3’端的第1至第8个碱基序列相同。
利用上述引物探针进行检测的原理如下：当对靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-11)与反向引物(R1-11)分别与靶序列1(呼吸道合胞病毒)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S1)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h1’)，延伸阻滞区的反向互补序列(M’)；正向引物(F2-11)与反向引物(R2-11)分别与靶序列2(甲型流感病毒)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S2)的5’端到3’端依次是探针锚定区(A2)，靶序列，引物信号检测区的反向互补序列(h2’)。待PCR扩增结束后，在较低的第一信号采集温度(t1)进行信号采集。此时探针(P1-11)的探针信号检 测区(H)与单链预扩增产物(S1)的引物信号检测区的反向互补序列(h1’)反向互补配对，探针(P1-11)的引物锚定区(A1’)与单链预扩增产物(S1)5’端的探针锚定区(A1)反向互补配对，得到与探针(P1-11)形成的杂交双链产物(D1)，而单链预扩增产物(S1)3’端的延伸阻滞区的反向互补序列(M’)不与探针(P1-11)或靶序列的任意部分互补；此时探针(P1-11)的部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号，可被仪器检测到。而此时由于单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)的退火温度小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P1-11)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，进行单链预扩增产物(S2)的二次扩增循环，循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))，此时探针(P1-11)的探针信号检测区(H)与单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A2’)与单链预扩增产物(S2)5’端的探针锚定区(A2)反向互补配对，单链预扩增产物(S2)的3’端可继续延伸到探针(P1-11)的5’端，得到探针(P1-11)的全部反向互补序列，即完成以单链预扩增产物(S2)为引物，以探针(P1-11)为模板的二次扩增，得到与探针(P1-11)形成的扩增双链产物(D2)，此时将温度升高至较高的第二信号采集温度(t2)采集信号(t2>t1，且t2>T1)，单链预扩增产物(S2)与探针(P1-11)形成的扩增双链产物(D2)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到；而此时单链预扩增产物(S1)与探针(P1-11)形成的杂交双链产物(D1)由于第二信号采集温度(t2)高于杂交双链产物(D1)的熔解温度而无法形成双链结构。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表22所示。
表22实施例11的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
反转录酶 5U
正向引物(F1-11) 500nM
反向引物(R1-11) 100nM
正向引物(F2-11) 500nM
反向引物(R2-11) 100nM
探针(P1-11) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表22中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用甲型流感病毒(Influenza A virus，IAV)和呼吸道合胞病毒(Respiratory Syncytial Virus， RSV)作为阳性样本，用带有不同信号检测区域序列的反向引物(R1-11和R2-11)对甲型流感病毒(Influenza A virus，IAV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)靶序列进行检测，以验证双重病毒感染的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表22中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：55℃逆转录15分钟；95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；60℃进行第一次信号采集；94℃变性15秒，40℃退火延伸20秒，共进行10个循环；78℃进行第二次信号采集。
数据分析(采用北京达微生物科技有限公司OS-500数字PCR一体机分析软件进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图12A为本实施例中甲型流感病毒(Influenza A virus，IAV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)阳性样本在第一信号采集温度(60℃)时的液滴图，可以看到有区别于背景的阳性液滴，为甲型流感病毒阳性液滴(处于边缘和/或看起来比较大的液滴，为实验操作问题，并非阳性液滴)。
图12B为本实施例中甲型流感病毒(Influenza A virus，IAV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)阳性样本在第二信号采集温度(78℃)时的液滴图，可以看到有区别于背景的阳性液滴，为呼吸道合胞病毒阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(60℃)出现的阳性液滴位置不同。
由图12A和图12B可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，实现在不同的信号检测温度下(t1、t2)，分别得到不同靶序列的检测信号，且互不交叉。
实施例12
(1)用于检测甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)的三重检测引物探针体系，如表23所示：
表23实施例12引物探针的碱基序列
Figure PCTCN2022142406-appb-000017
Figure PCTCN2022142406-appb-000018
上述引物探针中：
正向引物F1-12(SEQ ID NO:35)，为针对甲型流感病毒(Influenza A virus，IAV)靶序列设计的特异性引物，F1-12全长33bp，其3’端第1至第20个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P1-12(SEQ ID NO:33)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R1-12(SEQ ID NO:34)，为针对甲型流感病毒(Influenza A virus，IAV)靶序列设计的特异性引物，R1-12全长47bp，其3’端第1至第20个碱基为靶序列结合区，其5’端的第1至第5个碱基为信号阻滞区(M)，其5’端的第6至第24个碱基为引物信号检测区(h1)，与探针P1-12(SEQ ID NO:33)的3’端第30至48个碱基序列相同；
正向引物F2-12(SEQ ID NO:37)，为针对乙型流感病毒(Influenza B virus，IBV)靶序列设计的特异性引物，F2-12全长33bp，其3’端第1至第20个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P1-12(SEQ ID NO:33)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R2-12(SEQ ID NO:36)，为针对乙型流感病毒(Influenza B virus，IBV)靶序列设计的特异性引物，R2-12引物全长35bp，其3’端第1至第21个碱基为靶序列结合区，其5’端的第1至第11个碱基与探针探针P1-12(SEQ ID NO:33)的3’端的第17至第27个碱基序列相同。
正向引物F3-12(SEQ ID NO:39)，为针对呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)靶序列设计的特异性引物，F3-12全长35bp，其3’端第1至第22个碱基为靶序列结合区，其5’端的第1至第10个碱基为探针锚定区(A)，与探针P1-12(SEQ ID NO:33)的5’端第1至10个碱基序列，即引物锚定区(A’)，反向互补；
反向引物R3-12(SEQ ID NO:38)，为针对呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)靶序列设计的特异性引物，R3-12引物全长29bp，其3’端第1至第19个碱基为靶序列结合区，其5’端的第1至第7个碱基与探针探针P1-12(SEQ ID NO:33)的3’端的第1至第7个碱基序列相同。
利用上述引物探针进行检测的原理如下：当对靶标进行特异性扩增时，在较高的第一温度(T1)下退火，正向引物(F1-12)与反向引物(R1-12)分别与靶序列1(呼吸道合胞病毒)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S1)的5’端到3’端依次是探针锚定区(A1)，靶序列，引物信号检测区的反向互补序列(h1’)，延伸阻滞区的反向互补序列(M’)；正向引物(F2-12)与反向引 物(R2-12)分别与靶序列2(甲型流感病毒)特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S2)的5’端到3’端依次是探针锚定区(A2)，靶序列，引物信号检测区的反向互补序列(h2’)；正向引物(F3-12)与反向引物(R3-12)分别与靶序列3特异性结合并延伸产生双链预扩增产物，其中一条单链预扩增产物(S3)的5’端到3’端依次是探针锚定区(A3)，靶序列，引物信号检测区的反向互补序列(h3’)。待PCR扩增结束后，在较低的第一信号采集温度(t1)进行信号采集。此时探针(P1-12)的探针信号检测区(H)与单链预扩增产物(S1)的引物信号检测区的反向互补序列(h1’)反向互补配对，探针(P1-12)的引物锚定区(A1’)与单链预扩增产物(S1)5’端的探针锚定区(A1)反向互补配对，得到与探针(P1-12)形成的杂交双链产物(D1)，而单链预扩增产物(S1)3’端的延伸阻滞区的反向互补序列(M’)不与探针(P1-12)或靶序列的任意部分互补；此时探针(P1-12)的部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号，可被仪器检测到。而此时由于单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)和单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h3’)的退火温度均小于第一退火温度(T1)以及第一信号采集温度(t1)，因此不与探针(P1-12)的探针信号检测区(H)反向互补发生延伸；在第一信号采集温度(t1)下完成第一次信号采集后，进行单链预扩增产物(S2)的二次扩增循环，循环条件为高温解链，并在较低的第二退火温度(T2)下退火(T2<T1，且T2<t1))，此时探针(P1-12)的探针信号检测区(H)与单链预扩增产物(S2)3’端的引物信号检测区的反向互补序列(h2’)互补配对，探针的引物锚定区(A2’)与单链预扩增产物(S2)5’端的探针锚定区(A2)反向互补配对，单链预扩增产物(S2)的3’端可继续延伸到探针(P1-12)的5’端，得到探针(P1-12)的全部反向互补序列，即完成以单链预扩增产物(S2)为引物，以探针(P1-12)为模板的二次扩增，得到与探针(P1-12)形成的扩增双链产物(D2)，此时探针(P1-12)的全部序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号；而此时单链预扩增产物(S1)与探针(P1-12)形成的杂交双链产物(D1)由于第二信号采集温度(t2)高于杂交双链产物(D1)的熔解温度而无法形成双链结构；且由于单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h3’)的退火温度小于第二退火温度(T2)，因此不与探针(P1-12)的探针信号检测区(H)反向互补发生延伸；在第二信号采集温度(t2)下完成第二次信号采集后，进行单链预扩增产物(S3)的二次扩增循环，循环条件为高温解链，并在更低的第三退火温度(T3)下退火(T3<T2，且T3<t2)，此时探针(P1-12)的探针信号检测区(H)与单链预扩增产物(S3)3’端的引物信号检测区的反向互补序列(h3’)互补配对，探针的引物锚定区(A3’)与单链预扩增产物(S3)5’端的探针锚定区(A3)反向互补配对，单链预扩增产物(S3)的3’端可继续延伸到探针(P1-12)的5’端，得到探针(P1-12)的部分或全部反向互补序列，即完成以单链预扩增产物(S3)为引物，以探针(P1-12)为模板的二次扩增，得到与探针(P1-12)形成的扩增双链产物(D3)，此时探针(P1-12)的全部或部分序列由单链变为双链状态，第一检测基团与第二检测基团之间的距离发生变化，产生信号；在第三退火温度(T3)循环扩增结束后，再将温度升高至更高的第三信号采集温度(t3)进行第二次信号采集(t3>t2，且t3>T2)，此时单链预扩增产物(S1)与探针(P1-12)形成的杂交双链产物(D1)由于第三信号采集温度(t3)高于杂交双链产物(D1)的熔解温度而无法形成双链结构；单链预扩增产物(S2)与探针(P1-12)形成的扩增双链产物(D2)由于第三信号采集温度(t3)高于扩增双链产物(D2)的熔解温度而无法形成双链结构；而单 链预扩增产物(S3)与探针(P1-12)形成的扩增双链产物(D3)形成了双链状态，第一检测基团与第二检测基团之间的距离变远，荧光共振能量转移效率较低，使得荧光信号发生变化，可被仪器检测到。
(2)利用上述引物探针体系进行PCR检测，检测时采用的反应体系如表24所示。
表24实施例12的检测反应体系
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
反转录酶 5U
正向引物(F1-12) 250nM
反向引物(R2-12) 100nM
正向引物(F2-12) 250nM
反向引物(R2-12) 100nM
正向引物(F3-12) 250nM
反向引物(R3-12) 100nM
探针(P1-12) 400nM
核酸模板 30拷贝
超纯水 加至20μL
注表24中：2X PCR Reaction Buffer包括：3mM MgCl 2、pH为8.3的30mM Tris-HCl、0.5mM dNTP和70mM(NH 4) 2SO 4。
样本制备：使用甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)作为阳性样本，用带有不同信号检测区域序列的反向引物(R1-12/R2-12/R3-12)对甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)靶序列进行检测，以验证三重病毒感染的检测能力。使用纯水作为无模板对照(NTC)。
反应配制：
样本准备完成后，按照表24中所述配比进行反应体系的配置。
数字PCR扩增及信号采集(采用北京达微生物科技有限公司OS-500数字PCR一体机及试剂耗材进行检测)：
将数字PCR管盖封好后轻轻混匀样本，然后短暂离心后置于室温中静置5min。再次将数字PCR管置于掌上离心机中，短暂离心后转移到数字PCR仪的样品架上。所使用扩增及采光程序为：55℃逆转录15分钟；95℃预变性2分钟；94℃变性10秒，56℃退火延伸30秒，共进行45个循环；61℃进行第一次信号采集；94℃变性15秒，52℃退火延伸20秒，共进行5个循环；68℃进行第二次信号采集。94℃变性15秒，40℃退火延伸20秒，共进行5个循环；76℃进行第三次信号采集。
数据分析(采用北京达微生物科技有限公司OS-500数字PCR一体机分析软件进行数据分析)：
通过不同信号采集温度下的阳性液滴对结果进行判读。
图13A为本实施例中甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)阳性样本在第一信号采集温度(61℃)时的液滴图，可以看到有区别于背景的阳性液滴，为甲型流感病毒阳性液滴。
图13B为本实施例中甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)阳性样本在第二信号采集温度(68℃)时的液滴图，可以看到有区别于背景的阳性液滴，为乙型流感病毒阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(61℃)出现的阳性液滴位置不同。
图13C为本实施例中甲型流感病毒(Influenza A virus，IAV)，乙型流感病毒(Influenza B virus，IBV)和呼吸道合胞病毒(Respiratory Syncytial Virus，RSV)阳性样本在第三信号采集温度(76℃)时的液滴图，为呼吸道合胞病毒阳性液滴，且该温度下出现的阳性液滴与第一信号采集温度(61℃)和第二信号采集温度(68℃)出现的阳性液滴位置不同。
由图13A至图13C可看出：采用本发明的引物探针组合物、以及数字PCR检测方法，实现在不同的信号检测温度下(t1、t2、t3)，分别得到不同靶序列的检测信号，且互不交叉。
以上所述的仅是本发明的优选实例。应当指出对于本领域的普通技术人员来说，在本发明所提供的技术启示下，作为本领域的公知常识，还可以做出其它等同变型和改进，也应视为本发明的保护范围。
第二实施方式
需要说明的是，本申请实施例及附图中的术语“包括”和“具有”以及它们任何变形，意图在于覆盖不排他的包含。例如包含了一系列步骤或单元的过程、方法、系统、产品或设备没有限定于已列出的步骤或单元，而是可选地还包括没有列出的步骤或单元，或可选地还包括对于这些过程、方法、产品或设备固有的其它步骤或单元。
可以理解，本申请所使用的术语“第一”、“第二”等可在本文中用于描述各种元件，但这些元件不受这些术语限制。这些术语仅用于将第一个元件与另一个元件区分。举例来说，在不脱离本申请的范围的情况下，可以将第一移动信息称为第二移动信息，且类似地，可将第二移动信息称为第一移动信息。第一移动信息和第二移动信息两者都是移动信息，但其不是同一个移动信息。另外，需要说明的是，本申请实施例中所使用的术语“多个”指的是两个或两个以上。
下面以实施例的方式，对本申请技术方案做进一步的说明。
本实施方式的多重分时核酸检测装置用于对待测样品进行检测，待测样品中可以包括一种待测成分，也可以包括两种、三种、四种、五种甚至更多种的待测成分，通过本实施方式的多重分时核酸检测装置可以实现对待测成分进行多重检测。
请参阅图14A和图14B本实施方式的多重分时核酸检测装置包括反应液容纳部1、温度调节部2、信号检测部3、控制部4。其中，反应液容纳部1用于容纳包含待测成分的试样和能够与待测成分反应的试 剂，为待测成分与试剂发生各种反应提供场所。其中，温度调节部2用于调节反应液容纳部1中的混合液的温度，以使待测成分与试剂在不同的温度下发生不同的反应。其中，信号检测部3用于检测混合液中经过特定反应后的特定物质产生的信号，该信号可以是光学信号，也可以是电信号。其中，控制器4用于控制温度调节部2以不同的温度模式调节反应液容纳部1中的混合液的温度，并控制信号检测部3检测混合液中的特定物质产生的信号。
Ⅰ、试样
试样为来自人体或者动物体的体液，试样中包括不同的待测成分，待测成分可以为核酸或者蛋白质。本实施方式以待测成分为核酸来对技术方案进行说明。对于直接采自人体或动物体的试样，可以执行核酸提取纯化操作，以将核酸提取出来，这一操作可以人工执行，也可以用专业化的核酸提取仪执行。对于直接采自人体或动物体的试样，也可以不执行核酸提取纯化操作。试样中包含不同的待测核酸分子，例如，第一待测成分(第一待测核酸)、第二待测成分(第二待测核酸)、第三待测成分(第三待测核酸)、第四待测成分(第四待测核酸)、第五待测成分(第五待测核酸)等。
Ⅱ、试剂
试剂中包括用于扩增核酸的扩增物质和用于与标记核酸的标记物质，扩增物质可以扩增不同的核酸分子，标记物质在一定温度下能够与核酸结合从而产生可检测的信号。不同核酸与标记物质结合的温度范围不同，结合后与标记物质分离的温度范围也不同。扩增不同待测核酸的扩增物质中的引物不同，标记不同待测核酸的标记物质可相同，也可以不同，本申请采用相同的标记物质标记不同的待测核酸。
Ⅲ、微液体
微液体可以为形成于乳液的微小液滴，也可以为容器上的微孔内容纳的微量液体。在每个微液体中包含至多一个单位的核酸分子和足以扩增多种靶核酸和标记多种靶核酸的试剂。例如，若待测样本中有两种待测核酸，即第一待测核酸、第二待测核酸，经过微分区处理后，每个第一待测核酸、每个第二待测核酸被分配至不同的微液体中，在一个微液体中，有足量的试剂，既可以满足扩增和标记微液体中第一待测核酸的需求，也可以满足扩增和标记微液体中第二待测核酸的需求。
制作微液体的过程可以是高频振动形成微液体的方式：通过微管道吸取试样和足量的试剂，将微管道伸入油性液体下方高频振动，在振动的过程中将微管道中的试样和试剂的微液体以一定速度排出，从而通过振动甩出微滴，即微液体，微液体与油性液体不相容、不反应。通过设置合理的振动频率、振动幅度、微液体排出速度，可以保证形成的每个微液体中至多只有1个单位的核酸分子。
也可以是在容器上设置数个微分区，通过将试样和试剂的微液体加入数个微分区中，使每个微分区中至多有1个单位的核酸分子。
Ⅳ、反应液容纳部
如图14A所示，反应液容纳部为开口向上的盒状容器，通过液体加注机构将待测核酸与试剂加入该盒状容器(即如上所述的高频振动形成微液体的方式)，也可以由实验室工作人员将待检测核酸和试剂加入 该盒装容器。
如图14B所示，反应液容纳部为与微流道连通的腔室，通过微流体驱动结构驱动待测核酸与试剂进入该腔室。
为了实现对待测核酸的绝对定量检测，包含待测核酸和试剂的微液体被包裹于20000个左右的微液体中，每个微液体中含有一个或不含待检核酸分子，无论待测核酸中有几种待测成分，其均被分配至不同的微液体中(这是理想状态，可能有少量微液体中包含一个以上待测核酸分子)。
本实施例中，以反应液容纳部为盒装容器进行说明，向反应液容纳部中注入油性液体，该油性液体不与试样和试剂相容，也不与试样和试剂反应，采用高频振动方式在油性液体内形成微液体，并使微液体在自身重力作用下平铺在反应液容纳部的底部。
如图14C所示，孔板100是由多个反应液容纳部1组成，反应液容纳部1底部为平板形状，可使得多个微液体平铺在反应液容纳部1的底部。
Ⅴ、温度调节部
如图14A和图14B所示，温度调节部2设置在反应液容纳部1的下方、上方或者侧方，可以向反应液容纳部1提供热量，也可以吸收反应液容纳部1的热量，从而实现对反应液容纳部1内的微液体升温和降温的功能。温度调节部2可以使反应液容纳部1内的微液体的温度在4℃0105℃的范围内变化。如图14A所示，当反应液容纳部1为开口向上的盒状容器时，可以在温度调节部上设置专门用于放置反应液容纳部的放置位，需要对反应液容纳部1进行温度调节时，将其放置在放置位上。如图14B所示，当反应液容纳部1为与微流道连通的腔室时，可以结合加热效率等因素，将温度调节部2设置在反应液容纳部1的合理方位。
在本实施例中，温度调节部2设置在由多个反应液容纳部1组成的孔板100的下方，接触对平整的孔板100的进行升温、降温操作，从而使得孔板100中的各个反应液容纳部1中的微液体随孔板100温度的升降而发生温度的改变。
Ⅵ、信号检测部
如图所示，信号检测部3设置在反应液容纳部1的附近，根据反应液容纳部1中的微液体所产生信号的不同，信号检测部可以为检测光信号(例如荧光信号)的器件(相机或者光电器件)或者能够检测电信号的器件。例如，信号检测部为相机时，可以对平铺在反应液容纳部中的信号进行拍照。信号检测部3可以相对于反应液容纳部1可移动，当需要检测信号时，控制器控制驱动机构将信号检测部3移动至靠近反应液容纳部1的位置(例如，如图14A所示的上方)。如图14B所示，信号检测部3也可以相对于反应液容纳部固定设置。
Ⅶ、控制部
如图所示，控制部4与温度调节部2、信号检测部3可通讯地连接。控制部可以包括第一控制部、第二控制部(图中未示出)，第一控制部与温度调节部可通讯地连接，用以控制温度调节部以不同的模式运 行，第二控制部与信号检测部可通讯地连接，用以控制信号检测部采集来自反应液容纳部中的微液体的信号。如图14A和图14B所示，控制部4也可以为单一部件，其可同时控制温度调节部和信号检测部。
实施例13
待测试样中包含第一待测成分(第一待测核酸)和第二待测成分(第二待测核酸)，需要说明的是，由第一待测核酸产生的第一待测关联产物(待测核酸经过聚合酶链式反应(PCR)和/或核酸杂交和/或核酸酶切等操作后，产生与其对应的待测关联产物，通常情况下，待测关联产物为核酸)，可在第一结合温度范围内与试剂中的标记物质结合；由第二待测核酸产生的第二待测关联产物，可在第二结合温度范围内与试剂中的标记物质结合，第一结合温度范围和第二结合温度范围的最高温度不同。本实施例中，第二结合温度范围的最高温度低于第一结合温度范围的最高温度。例如，第一待测关联产物在温度低于或等于温度A1时能够与标记物质结合，第二待测关联产物在温度低于或者等于温度A2时能够与标记物质结合，其中温度A2低于温度A1。第一待测关联产物可在第一分离温度范围内与试剂中的标记物质分离，第二待测关联产物可在第二分离温度范围内与试剂中的标记物质分离，第一分离温度范围和第二分离温度范围的最低温度不同。本实施例中，第二分离温度范围的最低温度高于第一分离温度范围的最低温度。例如，第一待测关联产物在温度高于或等于温度B1时能够与标记物质分离，第二待测关联产物在温度高于或等于温度B2时能够与标记物质分离，其中温度B2高于温度B1。
为了实现对试样中的不同待测核酸进行多重检测，并且防止不同待测核酸之间信号的相互干扰，本实施例中，将试样和试剂(包含扩增物质和标记物质)的混合液加入反应液容纳部形成数个微液体，控制器执行以下控制：
如图15A至图15D所示，横轴代表时间，纵轴代表温度值，控制器控制温度调节部调节反应液容纳部中微液体的温度，使试样中的待测核酸产生与之关联的待测关联产物(待测核酸包含第一待测核酸、第二待测核酸，但是存在于不同的微液体中)；此过程中，微液体会经历多个温度循环，在每个温度循环中，微液体会变化为多个不同的温度，并在该温度下持续一定时间，循环个数可以为多个，例如20-60个，例如，40个左右。每个循环中，微液体会达到高温状态(温度t5)，双链核酸变成单链核酸，然后微液体达到低温状态(温度T1)，单链核酸经历退火及延伸过程(退火、延伸也可以分别在不同温度下进行，延伸温度高于退火温度)。经过多个温度循环后，试样中的不同待测核酸和/或不同待测关联产物的数量均增加(即每个微液体中的待测核酸和/或待测关联产物的量均增加，但是待测核酸的种类不变)，更容易被检测到。通过合理设置扩增过程的退火温度T1，待测核酸和/或待测关联产物的数量增加，但不与试剂中的标记物质结合。退火温度T1(低温温度T1)可在第一范围内取值，但是不能与后续的T2的可取范围(第二范围)重合，并且第一范围内的值均大于第二范围内的值。
如图15A至15D所示，在进行40个温度循环之前，可以先将微液体加热至温度t0，使试样中的待测核酸预变性一段时间(2分钟左右)。预变性一段时间，是为了将核酸从双链状态变成单链状态。
40个循环完成后，控制器可以控制温度调节部将反应液容纳部中微液体的温度调整至温度t1并维持 一定时间，如图15A至图15D所示，此区间段为区间一，控制器可以控制信号检测部执行一次检测动作，此次检测的信号作为空白信号。温度t1可以高于或者低于退火温度T1；循环完成后，温度调节部也可以使微液体的温度保持在温度T1状态，控制器控制信号检测部执行一次检测动作。由于此次检测获得的信号为空白信号，为提高效率，也可以将此次检测动作省略。
接着，控制器控制温度调节部以第一温度调节模式调节反应液容纳部中微液体的温度，经历第一温度调节模式的调节后，微液体中产生可与标记物质结合的第一待测关联产物，结合了第一待测关联产物的标记物质会产生信号，在此状态下，控制器控制信号检测部对反应液容纳部进行信号检测以获得第一信号。由于单个待测核酸分子被分配到一个微液体中，即一个微液体中仅有一个待测核酸分子，这个待测核酸分子可能是第一待测核酸，也可以能是第二待测核酸，经过第一温度调节模式的调节，仅含有第一待测核酸的微液体能够产生信号，若多个微液体发生重叠，则此种微液体发出的信号为无效信号；没有发生重叠的单个微液体发出的信号为有效信号，此类微液体为第一部分微液体，即数个微液体中仅第一部分微液体产生有效信号。微液体中扩增第一待测核酸的扩增物质和标记第一待测关联产物的标记物质为第一试剂。
具体的，第一温度调节模式包括孵育阶段和信号采集阶段，在孵育阶段产生可与标记物质结合的特定种类的待测关联产物，在信号采集阶段实现特定种类中某些种类的待测关联产物与标记物质的不结合，剩下一种待测关联产物保持与标记物质保持结合，并完成信号检测部对反应液容纳部的信号检测。若经过第一温度调节模式的孵育阶段孵育后，仅有一种待测关联产物与标记物质结合，那么在信号采集阶段，就保持该种待测关联产物与标记物质的结合状态。
在孵育阶段，为了实现第一待测关联产物与标记物质结合，温度调节部周期性地改变反应液容纳部中微液体的温度以使反应液容纳部反复从高温温度降至低温温度T2，或温度调节部使反应液容纳部保持恒温温度T2，温度T2低于退火温度T1。反应液容纳部中微液体的温度为T2时，第一部分微液体中的第一待测关联产物与第一试剂结合产生信号。温度T2可在第二范围内取值，但是不能与后续的T3的可取范围(第三范围)重合，并且第二范围内的值均大于第三范围内的值，第二范围内的值均小于第一范围内的值。
为了提高待测核酸与标记物质的结合率，如图15A和图15C所示，在第一温度调节模式的孵育阶段，控制部控制温度调节部周期性地改变反应液容纳部中的微液体的温度，在每个温度循环中，由第一待测核酸产生的第一待测关联产物在T2下与标记物质结合，在高温温度t5与标记物质解链，反复经历过多个循环后，与标记物质结合的第一待测关联产物的数量达到理想最大值，该循环次数为15个左右，例如，10个循环。最后一个循环中，在第一待测关联产物与标记物质结合的状态下，控制器控制信号检测部检对微液体进行信号检测以获取第一信号。当然，如图15A和图15C所示，也可以在最后一个循环结束后(即在信号采集阶段)，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t2(温度t2未达到能使第一待测关联产物与标记物质分离/解链的温度)，使第一待测关联产物与标记物质保持结合状态，并维持一定时间，该时间段为区间二，在区间二控制器控制信号检测部对反应液容纳部进行信号检测以获得第一信号。第一信号包含第一部分微液体产生的有效信号，若存在重叠的微液体，还包括重叠微液 体产生的无效信号，在后续分析过程中，此种无效信号将被排除。
也可以如图15B和图15D所示，在第一温度调节模式的孵育阶段，控制部控制温度调节部调节反应液容纳部中的微液体的温度保持在温度T2，持续一定时间以对微液体进行孵育，该时间可以为10-1800秒。孵育时间结束后，控制信号检测部对反应液容纳部进行信号检测以获得第一信号。当然，如图16B和图16D所示，恒温孵育结束后(信号采集阶段)，控制部也可以控制温度调节部将反应液容纳部中的微液体的温度调整至温度t2，使第一待测核酸与标记物质保持结合状态，并维持一定时间，该时间段为区间二，在区间二控控制器控制信号检测部对反应液容纳部进行信号检测以获得第一信号。
接着，控制器控制温度调节部以第二温度调节模式调节反应液容纳部中微液体的温度，经历第二温度调节模式的调节后，预扩增产物中仅有第二待测关联产物与试剂中的标记物质结合上，结合了第二待测关联产物的标记物质会产生信号，在此状态下，控制器控制信号检测部对反应液容纳部进行信号检测以获得第二信号。同样，由于单个核酸分子被分配到一个微液体中，即一个微液体中仅有一个核酸分子，这个核酸分子可能是第一待测核酸，也可以能是第二待测核酸，经过第二温度调节模式的调节，仅第二待测核酸所在的微液体能够产生信号，若多个微液体发生重叠，则此种微液体发出的信号为无效信号；没有发生重叠的单个微液体发出的信号为有效信号，此类微液体为第二部分微液体，即数个微液体中仅第二部分微液体产生有效信号。微液体中扩增第二待测核酸的扩增物质和标记第二待测核酸的标记物质为第二试剂。
具体的，第二温度调节模式包括孵育阶段和信号采集阶段，在孵育阶段实现特定种类待测关联产物与标记物质的结合，在信号采集阶段实现特定种类中某些种类的待测关联产物与标记物质的分离，剩下一种待测关联产物保持与标记物质保持结合状态，并完成信号检测部对反应液容纳部的信号检测。
在孵育阶段，为了实现第二待测关联产物与标记物质结合，温度调节部周期性地改变反应液容纳部中微液体的温度以使反应液容纳部反复从高温温度降至低温温度T3，或温度调节部使反应液容纳部保持恒温温度T3，温度T3低于温度T2。反应液容纳部中微液体的温度为T3时，第二部分微液体中的第二待测关联产物与第二试剂结合产生信号，同时，含有第一待测关联产物的微液体中的第一待测关联产物也能与第一试剂结合产生信号。温度T3可在第三范围内取值，但是不能与后续的T4的可取范围(第四范围)重合，并且第三范围内的值均大于第四范围内的值，第三范围内的值均小于第二范围内的值。
为了提高待测关联产物与标记物质的结合率，如图15A和图15C所示，在第二温度调节模式的孵育阶段，控制部控制温度调节部周期性地改变反应液容纳部中的微液体的温度，在每个温度循环中，由第二待测核酸产生的第二待测关联产物在T3下与标记物质结合，在高温温度t5与标记物质解链，反复经历过多个循环后，与标记物质结合的第二待测关联产物数量达到理想最大值，该循环次数为15个左右，例如，10个循环。在最后一个循环结束后(即在信号采集阶段)，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t3(温度t3达到使第一待测关联产物与标记物质分离/解链的温度，未达到能使第二待测关联产物与标记物质分离/解链的温度)，使第一待测关联产物与标记物质分离/解链(第一部分微液体中的第一待测关联产物与标记物质分离/解链，因结合产生的信号消失)，使第二待测关联产物与 标记物质保持结合状态，并维持一定时间，该时间段为区间三，在区间三控制器控制信号检测部对反应液容纳部进行信号检测以获得第二信号。第二信号包含第二部分微液体产生的有效信号，若存在重叠的微液体，还包括重叠微液体产生的无效信号，在后续分析过程中，此种无效信号将被排除。
由于第三范围内的值均小于第二范围内的值，在温度达到第三温度范围时，第一待测关联产物也会与标记物质结合。因此，在每个温度循环中第一待测关联产物和第二待测关联产物均会经历与标记物质结合和分离的过程。在最后一个循环后，当温度达到t3或者超过t3还未达到t4(见下文描述，温度t4达到能使第一待测关联产物和第二待测关联产物与标记物质分离/解链的温度，但是未达到能使第三待测关联产物与标记物质分离/解链的温度)之前，第一待测关联产物与标记物质解链，此时，只有第二待测关联产物与标记物质结合，在此状态下，控制信号检测部对反应液容纳部进行信号检测以获得第二信号。
也可以如图15B和图15D所示，在第二温度调节模式的孵育阶段，控制部控制温度调节部调节反应液容纳部中的微液体的温度保持在温度T3，持续一定时间以对微液体进行孵育，该时间可以为10-1800秒。孵育时间结束后，控制信号检测部对反应液容纳部进行信号检测以获得第二信号。在温度T3下，第一待测关联产物和第二待测关联产物均会与标记物质结合，在结束孵育后(即信号检测阶段)，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t3并维持一定时间，在温度t3下，第一待测关联产物与标记物质解链/分离；如图15B和图15D所示，该时间段为区间三，在区间三，第一待测关联产物与标记物质分离/解链并保持分离/解链状态，第二待测关联产物依然与标记物质结合，在此状态下(区间三)，控制器控制信号检测部对反应液容纳部进行信号检测以获得第二信号。
通过控制器的上述控制，可以实现信号检测部检测微液体中产生的信号时，只有一种待检测核酸或其关联产物与标记物质结合，从而将两种待测核酸的微液体产生的信号完全隔离开，实现高精度的两重核酸检测。
实施例14
本实施例与实施例13的不同之处在于，在试样中的核酸在扩增过程中，同时实现与试剂中的标记物质的结合，可以提升检测效率，提高检测通量。
具体如下：
如图16A至图16D所示，控制器控制温度调节部以第一温度调节模式调节反应液容纳部中微液体的温度，使微液体中的核酸被试剂中的扩增物质扩增，实现数量增加的同时，并且其产物可与试剂中的标记物质结合。此过程中，微液体会经历多个温度循环，在每个温度循环中，微液体会变化为多个不同的温度，并在该温度下持续一定时间，循环次数为40个左右。扩增过程的退火温度为温度T2，由于退火温度达到了第一待测关联产物(例如，第一待测核酸的扩增产物)能够与标记物质结合的温度，因此，可以在此第一温度调节模式的调节下同时实现待测核酸的扩增和其扩增产物与标记物质的结合，完成扩增后，含有第一待测核酸的第一部分微液体会产生信号，此时控制器控制信号检测部对反应液容纳部进行信号检测以获 得第一信号，具体方式可以为，在第一温度调节模式下历经40个循环后，控制器控制温度调节部将反应液容纳部中微液体的温度调整至温度t2，使第一待测关联产物与标记物质保持结合状态，并维持一定时间，该时间段为区间二，在区间二控制器控制信号检测部对反应液容纳部进行信号检测以获得第一信号。当然，也可以在循环完成后，将反应液容纳部中微液体的温度保持在T2，控制器控制控制器控制信号检测部对反应液容纳部进行信号检测以获得第一信号。
接着，控制器控制温度调节部以第二温度调节模式调节反应液容纳部中微液体的温度，该控制与实施例13相同，不再赘述。
实施例15
本实施例与实施例13或实施例14的不同之处在于：
第一，试样中还包括第三待测成分(第三待测核酸)，需要说明的是，第三待测核酸或第三待测关联产物(第三待测核酸经过聚合酶链式反应(PCR)和/或核酸杂交和/或核酸酶切等操作后，产生与其对应的第三待测关联产物，通常情况下，第三待测关联产物为核酸)可在一定温度范围内与试剂中的标记物质结合，本实施例中，第三待测核酸或第三待测关联产物与标记物质结合的最高温度，低于第二待测核酸或第二待测关联产物与标记物质结合的最高温度。例如，第三待测核酸或第三待测关联产物在温度低于或者等于温度A3时能够与标记物质结合，温度A3低于温度A2(第二待测核酸或第二待测关联产物在温度低于或者等于温度A2时能够与标记物质结合)。第三待测核酸或第三待测关联产物可在一定温度范围内与试剂中的标记物质分离，本实施例中，第三待测核酸或第三待测关联产物与标记物质分离的最低温度高于第二待测核酸或第二待测关联产物与标记物质分离的最低温度。例如，第三待测核酸或第三待测关联产物在温度高于或等于温度B3时能够与标记物质分离，温度B3高于温度B2(第二待测核酸或第二待测关联产物在温度高于或等于温度B2时能够与标记物质分离)。
第二，在控制器控制温度调节部以第二温度调节模式调节反应液容纳部中微液体的温度，并控制器控制信号检测部获得第二信号后，控制器还进行如下控制：
控制器控制温度调节部以第三温度调节模式调节反应液容纳部中微液体的温度，经历第三温度调节模式的调节后，产物中仅有第三待测核酸或第三待测关联产物与试剂中的标记物质结合，并因为结合而导致标记物质产生信号，在此状态下，控制器控制信号检测部对反应液容纳部进行信号检测以获得第三信号。同样，由于单个核酸分子被分配到一个微液体中，即一个微液体中仅有一个核酸分子，这个核酸分子可能是第一待测核酸或第二待测核酸或第三待测核酸，经过第三温度调节模式的调节，仅第三待测核酸所在的微液体能够产生信号，若多个微液体发生重叠，则此种微液体发出的信号为无效信号，没有发生重叠的单个微液体发出的信号为有效信号，此类微液体为第三部分微液体，即数个微液体中仅第三部分微液体产生有效信号。微液体中扩增第三待测核酸的扩增物质和标记第三待测核酸的标记物质为第三试剂。
具体的，第三温度调节模式包括孵育阶段和信号采集阶段，在孵育阶段实现特定种类待测核酸或其对 应的待测关联产物与标记物质的结合，在信号采集阶段实现特定种类中某些种类的待测核酸或其对应的待测关联产物与标记物质的分离，剩下一种待测核酸或其对应的待测关联产物保持与标记物质保持结合，并完成信号检测部对反应液容纳部的信号检测。
在孵育阶段，为了实现第三待测核酸或其对应的第三待测关联产物与标记物质结合，温度调节部周期性地改变反应液容纳部中微液体的温度以使反应液容纳部反复从高温温度降至低温温度T4，或温度调节部使反应液容纳部保持恒温温度T4，温度T4低于温度T3。反应液容纳部中微液体的温度为T4时，含有第三待测核酸的微液体中的第三待测核酸或第三待测关联产物与第三试剂结合产生信号。温度T4可在第四范围内取值，但是不能与后续的T5(如果有第四温度调节模式)的可取范围(第五范围)重合，并且第四范围内的值均大于第五范围内的值，第四范围内的值均小于第三范围内的值。
为了提高待测核酸与标记物质的结合率，如图15C和图16C所示，在第三温度调节模式的孵育阶段，控制部控制温度调节部周期性地改变反应液容纳部中的微液体的温度，在每个温度循环中，第三待测核酸或第三待测关联产物在低温温度T4下与标记物质结合，在高温温度t5与标记物质解链/分离，反复经历过多个循环后，与标记物质结合的第三待测核酸或第三待测关联产物的数量达到理想最大值，该循环次数为15个左右(例如，9个循环，10个循环等)。在最后一个循环结束后(即在信号采集阶段)，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t4(温度t4达到使第一待测核酸(或第一待测关联产物)和第二待测核酸(或第二待测关联产物)与标记物质分离/解链的温度，未达到能使第三待测核酸/第三待测关联产物与标记物质分离/解链的温度)，使第一待测核酸(或第一待测关联产物)与标记物质分离/解链(含有第一待测核酸的微液体中，第一待测核酸/第一待测关联产物与标记物质分离/解链，因结合产生的信号消失)，使第二待测核酸/第二待测关联产物与标记物质分离/解链(含有第二待测核酸的微液体中,第二待测核酸/第二待测关联产物与标记物质分离/解链，因结合产生的信号消失)，使第三待测核酸/第三待测关联产物与标记物质保持结合状态，并维持一定时间，该时间段为区间四，在区间四控制器控制信号检测部对反应液容纳部进行信号检测以获得第三信号。第三信号包含第三部分微液体产生的有效信号，若存在重叠的微液体，还包括重叠微液体产生的无效信号，在后续分析过程中，此种无效信号将被排除。
由于第四范围内的值均小于第三范围内的值，在温度达到第四范围时，第一待测核酸(或第一待测关联产物)和第二待测核酸(或第二待测关联产物)也会与标记物质结合。因此，在每个温度循环中第一待测核酸(或第一待测关联产物)、第二待测核酸(或第二待测关联产物)和第三待测核酸(或第三待测关联产物)均会经历与标记物质结合和分离的过程。在最后一个循环后，当温度达到t4或者超过t4还未达到t5(温度t4达到能使第一待测核酸(或第一待测关联产物)和第二待测核酸(或第二待测关联产物)与标记物质分离/解链的温度，但是未达到能使第三待测核酸(或第三待测关联产物)与标记物质分离/解链的温度)之前，第一待测核酸(或第一待测关联产物)和第二待测核酸(或第二待测关联产物)均与标记物质解链/分离，此时，只有第三待测核酸(或第三待测关联产物)与标记物质保持结合，在此状态下， 控制信号检测部对反应液容纳部进行信号检测以获得第三信号。
也可以如图15D所示，在第二温度调节模式的孵育阶段，控制部控制温度调节部调节反应液容纳部中的微液体的温度保持在温度T4，持续一定时间以对微液体进行孵育，该时间可以为10-1000秒。孵育时间结束后，控制信号检测部对反应液容纳部进行信号检测以获得第三信号。在温度T4下，第一待测核酸(或第一待测关联产物)、第二待测核酸(或第二待测关联产物)和第三待测核酸(或第三待测关联产物)均会与标记物质结合，在结束孵育后(即信号检测阶段)，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t4并维持一定时间，如图15D和图16D所示，该时间段为区间四，在区间四，第一待测核酸(或第一待测关联产物)和第二待测核酸(或第二待测关联产物)均与标记物质分离/解链并保持分离/解链状态，第三待测核酸(或第三待测关联产物)依然与标记物质结合，在此状态下(区间四)，控制器控制信号检测部对反应液容纳部进行信号检测以获得第三信号。
通过控制器的上述控制，可以实现信号检测部检测微液体中产生的信号时，只有一种待检测核酸或其关联产物与标记物质结合，从而将三种待测核酸的微液体产生的信号完全隔离开，实现高精度的三重核酸检测。
实施例16
本实施例与实施例13的不同之处在于：由于试样中所含的成分不同，导致图15A-图15D的区间一获得的信号不是空白信号，而是含有某个待测成分的微液体产生的信号。本实施例中，该待测成分为第四待测成分(第四待测核酸)。
在实施例13的基础上，试样中还包括第四待测成分(第四待测核酸)，第四待测核酸能够被试剂中的扩增物质扩增，产生的扩增产物(第四待测关联产物)能与试剂中的标记物质结合，结合了第四待测核酸扩增产物的标记物质可以产生可检测的信号。需要说明的是，第四待测核酸被扩增和与标记物质结合可在一定温度范围内实现。在该温度范围内，其他待测核酸，例如，第一待测核酸和第二待测核酸能够被试剂中的扩增物质扩增，但是其产物不能与试剂中的标记物质结合。例如，第四待测关联产物在温度低于或等于温度A4时能够与标记物质结合，A4高于温度A1(第一待测核酸或其关联产物在温度低于或等于温度A1时能够与标记物质结合)。第四待测核酸或其关联产物在一定温度范围内能够与标记物质分离/解链，本实施例中，第四待测核酸或其关联产物与标记物质分离的最低温度低于第一待测核酸或其关联产物与标记物质分离的最低温度。例如，第四待测核酸或其关联产物在温度高于或等于温度B4时能够与标记物质分离，温度B4低于温度B1。
在退火温度T1下，试样中的其他待测核酸，例如，第一待测核酸和第二待测核酸在此过程中只被扩增，而不与试剂中的标记物质结合；第四待测核酸能够被扩增并且能够与标记物质结合。在温度t1下，第四待测核酸或其关联产物保持与标记物质结合的状态，信号检测部检测的信号不再是空白信号，而是包含第四待测核酸的微液体产生的信号。温度t1可以高于或者低于或者等于退火温度T1。
此后的控制过程与实施例13不相同的部分在于：
控制器控制温度调节部以第一温度调节模式调节反应液容纳部温度的过程中，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度T2的情况下，第四待测核酸或其关联产物与标记物质结合、第一待测核酸或其关联产物与标记物质结合；控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t2的情况下，第四待测核酸或其关联产物与标记物质分离/解链，第一待测核酸或其关联产物与标记物质保持结合。
控制器控制温度调节部以第二温度调节模式调节反应液容纳部温度的过程中，控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度T3的情况下，第四待测核酸或其关联产物与标记物质结合、第一待测核酸或其关联产物与标记物质结合、第二待测核酸或其关联产物与标记物质结合；控制部控制温度调节部将反应液容纳部中的微液体的温度调整至温度t3的情况下，第四待测核酸或其关联产物和第一待测核酸或其关联产物均与标记物质分离/解链，第二待测核酸或其关联产物与标记物质保持结合状态。
其余的控制过程与实施例13相同的部分，不在此赘述。
上述实施例13至实施例16中的温度t1、t2、t3、t4、t5可以作如下理解：
在温度t1下，如果试样中包含第四待测核酸，则第四待测核酸或其关联产物能够与试剂中的标记物质保持结合状态；如果试样中不包含第四待测核酸，则没有待测核酸或其关联产物与试剂中的标记物质结合；
在温度t2下，仅第一待测核酸或其关联产物能够与试剂中的标记物质保持结合状态；
在温度t3下，仅第二待测核酸或其关联产物能够与试剂中的标记物质保持结合状态；
在温度t4下，仅第三待测核酸或其关联产物能够与试剂中的标记物质保持结合状态；
在温度t5下，所有待测核酸或其关联产物均解链。
上述实施例13至实施例16中的第四试剂、第一试剂、第二试剂、第三试剂可以作如下理解：
第四试剂包括可以扩增第四待测核酸的扩增物质和能够标记第四待测核酸的标记物质；
第一试剂包括可以扩增第一待测核酸的扩增物质和能够标记第一待测核酸的标记物质；
第二试剂包括可以扩增第二待测核酸的扩增物质和能够标记第二待测核酸的标记物质；
第三试剂包括可以扩增第三待测核酸的扩增物质和能够标记第三待测核酸的标记物质。
其中，扩增第四待测核酸、第一待测核酸、第二待测核酸、第三待测核酸的扩增物质中的引物不同，其余成分可以相同，也可以不同；标记第四待测核酸、第一待测核酸、第二待测核酸第三待测核酸的标记物质可以相同，也可以不同。
在初始状态下，即还未孵育微液体之前，一个微液体中，至多包含一个待测核酸(第四待测核酸或第一待测核酸或第二待测核酸或第三待测核酸)和能够扩增和标记所有待测核酸的足量的试剂。
当然，本领域技术人员可以根据上述实施例合理推测出如何利用本申请公开的技术方案实现高精度的四重核酸检测、五重核酸检测、六重核酸检测等。
本领域普通技术人员可以理解实现上述实施例方法中的全部或部分流程，是可以通过计算机程序来指令相关的硬件来完成，所述的程序可存储于一非易失性计算机可读取存储介质中，该程序在执行时，可包括如上述各方法的实施例的流程。其中，该存储介质可为磁碟、光盘、ROM等。
如此处所使用的对存储器、存储、数据库或其它介质的任何引用可包括非易失性和/或易失性存储器。合适的非易失性存储器可包括ROM、可编程ROM(Programmable ROM，PROM)、可擦除PROM(Erasable PROM，EPROM)、电可擦除PROM(Electrically Erasable PROM，EEPROM)或闪存。易失性存储器可包括随机存取存储器(random access memory，RAM)，它用作外部高速缓冲存储器。作为说明而非局限，RAM可为多种形式，诸如静态RAM(Static RAM，SRAM)、动态RAM(Dynamic Random Access Memory，DRAM)、同步DRAM(synchronous DRAM，SDRAM)、双倍数据率SDRAM(Double Data Rate SDRAM，DDR SDRAM)、增强型SDRAM(Enhanced Synchronous DRAM，ESDRAM)、同步链路DRAM(Synchlink DRAM，SLDRAM)、存储器总线直接RAM(Rambus DRAM，RDRAM)及直接存储器总线动态RAM(Direct Rambus DRAM，DRDRAM)。
应理解，说明书通篇中提到的“一个实施例”或“一实施例”意味着与实施例有关的特定特征、结构或特性包括在本申请的至少一个实施例中。因此，在整个说明书各处出现的“在一个实施例中”或“在一实施例中”未必一定指相同的实施例。此外，这些特定特征、结构或特性可以以任意适合的方式结合在一个或多个实施例中。本领域技术人员也应该知悉，说明书中所描述的实施例均属于可选实施例，所涉及的动作和模块并不一定是本申请所必须的。
在本申请的各种实施例中，应理解，上述各过程的序号的大小并不意味着执行顺序的必然先后，各过程的执行顺序应以其功能和内在逻辑确定，而不应对本申请实施例的实施过程构成任何限定。
在本申请各实施例中的各功能单元可以集成在一个处理单元中，也可以是各个单元单独物理存在，也可以两个或两个以上单元集成在一个单元中。上述集成的单元既可以采用硬件的形式实现，也可以采用软件功能单元的形式实现。
上述集成的单元若以软件功能单元的形式实现并作为独立的产品销售或使用时，可以存储在一个计算机可获取的存储器中。基于这样的理解，本申请的技术方案本质上或者说对现有技术做出贡献的部分或者该技术方案的全部或者部分，可以以软件产品的形式体现出来，该计算机软件产品存储在一个存储器中，包括若干请求用以使得一台计算机设备(可以为个人计算机、服务器或者网络设备等，具体可以是计算机设备中的处理器)执行本申请的各个实施例上述方法的部分或全部步骤。
以上对本申请实施例公开的一种多重分时核酸检测装置及检测方法进行了详细介绍，本文中应用了具体个例对本申请的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本申请的方法及其核心思想。同时，对于本领域的一般技术人员，依据本申请的思想，在具体实施方式及应用范围上均会有改 变之处，综上所述，本说明书内容不应理解为对本申请的限制。

Claims (15)

  1. 一种对靶标核酸进行检测的方法，其特征在于，包括如下步骤：
    S100，将引物探针组合物、待测样本和扩增试剂混合，获得反应体系；所述扩增试剂包括DNA聚合酶和dNTPs；
    S200，对所述反应体系进行第1次PCR扩增，扩增时采用的退火温度为T1；
    S300，进行以下任一操作：
    操作1，包括：
    S310，在第1信号采集温度t1下进行第1次信号采集；
    S320，进行第2次PCR扩增，扩增时采用的退火温度为T2；
    操作2，包括：
    S310’，在第1信号采集温度t1下进行第1次信号采集；
    操作3，包括：
    S310”，进行第2次PCR扩增，扩增时采用的退火温度为T2；
    S320”，在第1信号采集温度t1下进行第1次信号采集；
    以上操作满足：T2＜T1，T2＜t1；优选地，40℃≤T2＜T1≤75℃，和/或0℃≤t1＜t2≤90℃；
    S400，在第2信号采集温度t2下进行第2次信号采集，所述t2＞t1，且t2＞T1；
    其中，待测样本中的待检测靶标的种类为1种；
    所述第1次信号采集、第2次信号采集中最多只有1次信号采集中含有靶标的信号。
  2. 根据权利要求1所述的方法，其特征在于，步骤S200中所述第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；
    和/或，步骤S300中所述第2次PCR扩增，包括如下反应条件：约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育约10-约1800秒。
  3. 根据权利要求1或2所述的方法，其特征在于，对所述反应体系进行第1次PCR扩增之前，还包括：将所述反应体系分配到500个以上的反应单元中，每个反应单元含有1个待测样本的靶标核酸或不含有待测样本的靶标核酸；优选地，所述信号采集为通过相机对荧光信号进行采集；所述信号通道采集为在荧光信号通道下通过相机对荧光信号进行采集。
  4. 根据权利要求1或2所述的方法，其特征在于，所述引物探针组合物包括：探针(P)和引物；所述引物与所述待测样本中靶标特异性结合后产生预产物，所述预产物中含有一条可与探针特异性结合的单链预产物，该单链预产物与探针特异性结合并延伸≥0个碱基后形成双链产物，所述双链产物的形成引起探针信号变化；优选地，所述延伸为延伸0-100个碱基，更优选地，延伸1-100个碱基；和/或，所述引物的浓度为约30nM-约1000nM，所述探针(P)的浓度为约100nM-约1200nM。
  5. 根据权利要求4所述的方法，其特征在于，所述探针(P)为一段不与任何靶标序列配对结合的自由设计序列，包括探针信号检测区(H)，探针上修饰有检测标记；所述引物包括正向引物(F)和/或反向引物(R)；
    所述正向引物(F)包含与靶标序列特异性配对结合的靶标序列结合区；
    所述反向引物(R)包含引物信号检测区(h)和靶标序列结合区，且引物信号检测区(h)位于靶标序列结合区的5’端，或者所述反向引物(R)从5’端至3’端依次包含延伸阻滞区(M)、引物信号检测区(h)和靶序列结合区；
    其中，所述反向引物(R)的靶标序列结合区为与靶标序列特异性配对结合的序列；所述反向引物(R)的引物信号检测区(h)均为一段不与任何靶标序列配对结合的序列，所述引物信号检测区(h)的互补序列(h’)与探针(P)的探针信号检测区(H)特异性配对结合；所述反向引物(R)的延伸阻滞区(M)为不与探针(P)任一部分的序列或任何靶标序列相同或互补的自由设计序列；
    优选地，所述反向引物(R)中引物信号检测区(h)和靶标序列结合区之间有0-20个碱基的间隔。
  6. 根据权利要求5所述的方法，其特征在于，所述正向引物(F)还包括探针锚定区(A)，所述正向引物(F)中的探针锚定区(A)位于所述靶标序列结合区的5’端；其中，所述探针锚定区(A)为一段不与任何靶标序列配对结合的序列；优选地，所述正向引物(F)中的探针锚定区(A)和靶标序列结合区之间有0-20个碱基的间隔；
    所述探针(P)还包括引物锚定区(A’)，所述引物锚定区(A’)为与所述正向引物(F)的探针锚定区(A)互补的序列；优选地，所述探针(P)中的所述引物锚定区(A’)和所述探针信号检测区(H)之间有0-20个碱基的间隔。
  7. 一种对2种以上靶标核酸进行检测的方法，其特征在于，包括如下步骤：
    将引物探针组合物、待测样本和扩增试剂混合，获得反应体系；所述引物探针组合物包括至少1种探针(P)、至少2种针对不同靶标的引物；所述扩增试剂包括DNA聚合酶和dNTPs；
    对所述反应体系进行n次PCR扩增和n次信号采集；每次PCR扩增后进行一次信号采集，每次信号采集包括至少1次信号通道采集；每次信号通道采集中最多含有1种靶标的信号；
    所述第n次PCR扩增时采用的退火温度为Tn，所述第n次信号采集时采用的采集温度为tn，Tn＜T(n-1)，且Tn＜t(n-1)；tn＞t(n-1)，且tn＞T(n-1)；优选地，40℃≤Tn＜T(n-1)≤75℃，和/或0℃≤t(n-1)＜tn≤90℃；
    所述n为≥2的整数，所述n≤待测样本中待测的靶标种类数；
    优选地，所述至少1次信号采集包括m次信号通道采集；所述m为所述引物探针组合物中探针的检测标记的种类数，并且，n与m的乘积≥待测样本中待测的靶标种类数。
  8. 根据权利要求7所述的方法，其特征在于，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环。
  9. 根据权利要求7所述的方法，其特征在于，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，约20-约60个循环；约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约20℃-约75℃恒温孵育约10-约1800秒；
    优选地，当待测样本中靶标为RNA时，所述扩增试剂还包括逆转录酶，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，对所述反应体系进行第1次PCR扩增的反应条件包括：约30-约65℃逆转录约2-约30分钟；约85℃-约105℃预变性约0-约15分钟；约85℃-约105℃变性约1-约60秒，约40℃-约75℃退火及延伸约3-约90秒，20-60个循环；约20℃-约75℃恒温孵育约10-约1800秒。
  10. 根据权利要求7-9中任一项所述的方法，其特征在于，所述第1次PCR扩增之后的其他PCR扩增，包括如下反应条件：约85℃-约105℃变性约1-约60秒，约20℃-约75℃退火及延伸约3-约90秒，1-20个循环；或者，约20℃-约75℃恒温孵育10-1800秒。
  11. 根据权利要求7所述的方法，其特征在于，对所述反应体系进行第1次PCR扩增之前，还包括：将所述反应体系分配到500个以上的反应单元中，每个反应单元含有1个待测样本的靶标核酸或不含有待测样本的靶标核酸；优选地，所述信号采集为通过相机对荧光信号进行采集；所述信号通道 采集为在荧光信号通道下通过相机对荧光信号进行采集。
  12. 根据权利要求7-11中任一项所述的方法，其特征在于，至少1种所述引物与靶标特异性结合后产生预产物，所述预产物中含有一条可与探针(P)特异性结合的单链预产物，该单链预产物与探针(P)特异性结合并延伸后形成双链产物，所述双链产物的形成引起探针信号变化；优选地，所述延伸为延伸0-100个碱基；更优选地，所述延伸为延伸1-100个碱基；和/或，所述引物的浓度为约30nM-约1000nM，所述探针(P)的浓度为约100nM-约1200nM。
  13. 根据权利要求7-11中任一项所述的方法，其特征在于，所述至少2种针对不同靶标的引物包括以下任意一种：
    (1)至少两种正向引物(F)和至少一种反向引物(R)；
    (2)至少一种正向引物(F)和至少两种反向引物(R)；
    (3)至少两种正向引物(F)和至少两种反向引物(R)；
    不同的所述正向引物(F)各自独立地包含与不同靶标序列特异性配对结合的靶标序列结合区；
    和/或，所述探针(P)为一段不与任何靶序列配对结合的自由设计序列，包括探针信号检测区(H)，所述探针上修饰有检测标记；
    和/或，所述反向引物(R)包含引物信号检测区(h)和靶标序列结合区，且所述引物信号检测区(h)位于所述靶标序列结合区的5’端；其中，不同的所述反向引物(R)的靶序列结合区为与不同靶标序列特异性配对结合的序列；不同的所述反向引物(R)的引物信号检测区(h)均为一段不与任何靶标序列配对结合的序列，所述引物信号检测区(h)的互补序列与探针(P)的探针信号检测区(H)特异性配对结合；优选地，不同反向引物(R)的引物信号检测区(h)的序列彼此不同。
  14. 根据权利要求7-13中任一项所述的方法，其特征在于，所述引物中，有至少1种所述反向引物(R)从5’端至3’端依次包含延伸阻滞区(M)、引物信号检测区(h)和靶标序列结合区；所述延伸阻滞区(M)为不与探针(P)任一部分的序列或任何靶序列相同或互补的自由设计序列；若还有其他种反向引物，所述其他种反向引物(R)的5’端不含有延伸阻滞区(M)。
  15. 根据权利要求7-14中任一项所述的方法，其特征在于，所述引物中，有至少1种正向引物(F)还包括探针锚定区(A)，所述正向引物(F)中的探针锚定区(A)位于所述靶标序列结合区的5’端；其中，所述探针锚定区(A)为一段不与任何靶标序列配对结合的序列；不同正向引物(F)的靶标序列结合区的序列不同；优选地，正向引物(F)中探针锚定区(A)和靶标序列结合区之间有0-20个碱基的间隔；
    所述探针(P)为一段不与任何靶标序列配对结合的序列，其包括引物锚定区(A’)和探针信号检测区(H)；所述引物锚定区(A’)为与正向引物(F)的探针锚定区(A)互补的序列；优选地，所述探针(P)中所述引物锚定区(A’)和所述探针信号检测区(H)之间有0-20个碱基的间隔。
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