WO2023125562A1 - 多种靶核酸检测的方法 - Google Patents

多种靶核酸检测的方法 Download PDF

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WO2023125562A1
WO2023125562A1 PCT/CN2022/142473 CN2022142473W WO2023125562A1 WO 2023125562 A1 WO2023125562 A1 WO 2023125562A1 CN 2022142473 W CN2022142473 W CN 2022142473W WO 2023125562 A1 WO2023125562 A1 WO 2023125562A1
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signal
primer
nucleic acid
probe
target nucleic
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French (fr)
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赵雨航
胡莞尔
王海鑫
黄秋萍
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Maccura Biotechnology Co Ltd
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Maccura Biotechnology Co Ltd
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Priority to US18/724,610 priority Critical patent/US20250101514A1/en
Priority to CN202280006311.1A priority patent/CN116670277A/zh
Priority to EP22914814.3A priority patent/EP4458985A4/en
Priority to JP2024539076A priority patent/JP2025501166A/ja
Publication of WO2023125562A1 publication Critical patent/WO2023125562A1/zh
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    • 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
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    • 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
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    • 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
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • the present invention relates to the field of molecular biology, in particular to a method for the detection of multiple target nucleic acids in a single sample container.
  • 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 present invention provides a method for detecting multiple target nucleic acids, including the following:
  • the primer probe composition includes nucleic acid polymerase and dNTPs;
  • reaction system placing the reaction system under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions to obtain reaction products;
  • each signal collection includes at least one signal channel collection
  • n is an integer ⁇ 2, and n ⁇ the number of types of target nucleic acids.
  • the detection of multiple target nucleic acids can be realized (at least 8 multiplex reactions can be realized on digital PCR).
  • the realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence.
  • the fluorescent recognition of digital PCR has low requirements, simple operation and time saving.
  • one signal acquisition is performed for each signal acquisition temperature; if n different signal acquisition temperatures are used, n times of signal acquisition are performed.
  • n is the number of signal collections; n ⁇ the number of target nucleic acid types, that is, the number of signal collections ⁇ the number of target nucleic acid types.
  • the number of target nucleic acid species contained in the signals acquired by two adjacent signal channels differs by at most one; in some embodiments, under the same signal channel, The number of target nucleic acid species contained in the signals collected by two adjacent signal channels differs by one; in some embodiments, the signal collected by the signal channel with the highest signal collection temperature contains at most one target nucleic acid. nucleic acid. In some embodiments, the "one type of target nucleic acid" also includes Type 1 target nucleic acid that is not classified.
  • signal acquisition includes the following operations: the first signal acquisition is performed on the reaction product at the first temperature, and then the temperature is raised to the second temperature for the second signal acquisition, and then the temperature is continued to the third temperature.
  • the third signal collection is carried out at the temperature, and then the heating and signal collection are continued until all the target nucleic acids in the sample to be tested are detected.
  • It can also include the operation of signal acquisition in continuous cooling, for example, the first signal acquisition is performed on the reaction product at the first temperature, then the temperature is lowered to the second temperature for the second signal acquisition, and then the temperature is continued to be lowered to the third temperature.
  • the signal is collected for the third time at the temperature, and then the cooling and signal collection are continued until all the target nucleic acids in the sample to be tested are detected.
  • each signal acquisition includes only one signal channel acquisition, that is, each signal acquisition is one signal acquisition under a certain signal channel (fluorescence channel).
  • two adjacent signal channel acquisitions are two adjacent signal acquisitions, and the number of target nucleic acid species contained in the signals obtained by adjacent two signal channel acquisitions differs by 1, then the number of times n of signal acquisition is equal to the number of target nucleic acid species number of species.
  • the sample to be tested contains 3 kinds of target nucleic acids.
  • the reaction product is collected at the first temperature for the first signal acquisition (equivalent to signal channel acquisition), and the obtained signal contains 3 kinds of targets. Then the temperature was raised to the second temperature for the second signal collection, and the obtained signal contained 2 kinds of targets, and then the temperature was continued to be raised to the third temperature for the third signal collection, and the obtained signal contained 1 kind of target.
  • the presence of the target nucleic acid can be determined by comparing the signal conditions of two adjacent signal collections. For example, compared to the first signal acquisition, some signals disappear during the second signal acquisition, and the disappeared signal is the first target; compared with the second signal acquisition, another part of the signal disappears during the third signal acquisition.
  • the signal that disappears another part of the signal that disappears is the second target, and the signal that still exists in the third signal acquisition is the third target. In this way, the existence of all three targets can be determined. In addition, the amount of each target can be determined according to the amount of signal disappearance/presence.
  • there are 2 kinds of probes in the primer-probe composition (2 kinds of probes, referring to the modified detection labels or fluorescent groups are different, and 2 types of probes with different base sequences), then there are at least 1 signal acquisition includes 2 signal channel acquisitions. Among them, two signal channel acquisitions, that is, one signal acquisition performed under two signal channels (fluorescent channels) respectively.
  • two signal channel acquisitions that is, one signal acquisition performed under two signal channels (fluorescent channels) respectively.
  • those skilled in the art can select an appropriate signal acquisition temperature and perform signal acquisition in an appropriate signal channel (fluorescent channel) according to the design of the primer-probe composition.
  • the two-dimensional analysis of fluorescence channel and melting temperature can be performed simultaneously in a single-tube reaction, that is, different targets can be detected by using the same fluorescence channel and different signal acquisition temperatures; or using different In the fluorescent channel, the target type detection can be realized by the product of the number of fluorescent channels and the signal acquisition temperature characteristics.
  • the amplification reagents may also include reagents that promote PCR reactions, such as KCl, MgCl2, Tris-HCl, dithiothreitol (DTT), (NH4)2SO4, and the like.
  • the amplification reagent also includes some other enzymes that act on nucleic acid, for example, exonuclease, endonuclease and the like.
  • two adjacent signal channel acquisitions mean that the signal acquisition temperatures used for the two signal channel acquisitions are similar.
  • the reaction product is placed at the first temperature for the first signal channel acquisition, then heated up to the second temperature for the second signal channel acquisition, and then continue to heat up
  • the third signal channel acquisition is carried out at the third temperature
  • the first signal channel acquisition and the second signal channel acquisition are two adjacent signal channel acquisitions
  • the second signal channel acquisition and the third signal channel acquisition It is collected for two adjacent signal channels, but the first signal channel acquisition and the third signal channel acquisition are not collected for two adjacent signal channels.
  • the difference between the signal acquisition temperatures used in two adjacent signal channel acquisitions is more than 4°C, so as to achieve a difference of one target nucleic acid species contained in the signals obtained by two adjacent signal channel acquisitions under the same signal channel. , to avoid the situation that there is no difference in the target nucleic acid signals obtained by two adjacent signal channel acquisitions, thereby reducing the number of signal channel acquisitions and simplifying the operation steps.
  • the signal collection temperature is 0-95°C.
  • the selection of signal collection temperature can be determined by those skilled in the art according to the actual conditions of the designed primer-probe composition. 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.
  • each signal collection includes m signal channel collections; said m is the number of detection labels of probes in the primer-probe composition.
  • 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. If there are 2 types of targets to be detected, only 3 signal acquisitions are required for the reaction product, and each signal acquisition is 2 signal channel acquisitions under 2 signal channels.
  • 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 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 above-mentioned method for detecting multiple target nucleic acids is a digital PCR detection method for multiple target nucleic acids.
  • the reaction system Before placing the reaction system under conditions that allow the nucleic acid polymerase to perform hybridization and extension reactions, it also includes: distributing the reaction system to more than 500 reaction units, each reaction unit containing the target nucleic acid of a sample to be tested Or do not contain the target nucleic acid of the sample to be tested.
  • the signal collection is collecting fluorescence signals by a camera.
  • the acquisition of the signal channel is to collect the fluorescence signal through a camera under the fluorescence signal channel.
  • the primer probe composition, the sample to be tested (containing 2 targets) and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is distributed to more than 500 reaction units to form countless small Liquid droplets, each small droplet contains at most one target nucleic acid of the sample to be tested, but contains components suitable for nucleic acid amplification, such as amplification reagents such as primer-probe composition and nucleic acid polymerase. All the droplets are then placed under conditions that allow nucleic acid polymerases to perform hybridization and extension reactions (in some embodiments, that is, PCR amplification conditions), and the resulting reaction products (amplification products) are subjected to a second temperature at a first temperature.
  • amplification reagents such as primer-probe composition and nucleic acid polymerase.
  • the specific types of the two targets can be analyzed, and by analyzing the number of luminescent droplets in the second photograph, the number of the first target can be obtained, and the first photograph can be analyzed The difference between the number of luminous droplets and the number of luminescent droplets in the second photoshoot can be used to obtain the number of second targets.
  • the above-mentioned conditions for allowing nucleic acid polymerase to perform hybridization and extension reactions include: pre-denaturation at about 85°C-about 105°C for 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 transcriptase, for the
  • 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 0-about 15 minutes; about 85°C-about Denaturation at 105°C for about 1 to about 60 seconds, annealing at about 40°C to about 75°C and extension for about 3 to about 90 seconds, 20-60 cycles.
  • the above-mentioned conditions allowing nucleic acid polymerases to perform hybridization and extension reactions include: 85°C-105°C pre-denaturation for 0-15 minutes; 85°C-105°C denaturation for 2-60 seconds, 40°C-75°C annealing and Extend for 10-90 seconds, 20-60 cycles.
  • the amplification reagent when the target nucleic acid in the sample to be tested contains RNA, the amplification reagent further includes reverse transcriptase, and the conditions allowing the nucleic acid polymerase to perform hybridization and extension reactions include: 30-65°C reverse transcription 2 -30 minutes; pre-denaturation at 85°C-105°C for 0-15 minutes; denaturation at 85°C-105°C for 2-60 seconds, annealing and extension at 40°C-75°C for 10-90 seconds, 20-60 cycles.
  • the above-mentioned primer probe composition includes a first probe and a first primer mixture; the first primer mixture includes at least two primer sets, different primers The groups specifically bind to different kinds of target nucleic acids, respectively.
  • 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.
  • the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe.
  • the double-stranded products have different melting temperatures. In some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.
  • 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 primer set for a certain target nucleic acid does not extend after the first single-stranded pre-product produced by it specifically binds to the probe (ie, extends by 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.
  • the melting temperature of oligonucleotides is related to factors such as the length and composition of oligonucleotides. Those skilled in the art can adjust the melting temperature of the oligonucleotide according to actual needs, for example, increasing the GC content of the oligonucleotide and making the length of the oligonucleotide longer can obtain a higher melting temperature. Therefore, those skilled in the art can adjust the sequence composition of primers and/or probes according to the actual situation, so that the single-stranded pre-products formed by different primers are different, and the positions where different single-stranded pre-products specifically bind to the first probe Different, so that the melting temperature of the different double-stranded products formed is different.
  • those skilled in the art can make different double-stranded products representing different targets according to the actual situation, and different double-stranded products have different melting temperatures, so that signals of different target nucleic acids can be obtained at different signal collection temperatures.
  • the design of the primers and probes can make the melting temperatures of different double-stranded products representing different targets differ by more than 4°C.
  • the primer probe composition also includes a second probe and a second primer mixture; the base sequence of the second probe is different from that of the first probe, and the modified detection label are also different; the second primer mixture includes at least one primer set, and different primer sets specifically bind to different target nucleic acids.
  • 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 increase in the number of types of probes can realize the signal detection of different target nucleic acids in different channels, and can realize the detection of more multiple target nucleic acids.
  • 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 set in the second primer mixture specifically binds to its corresponding target nucleic acid to generate a pre-product
  • the pre-product contains a single-stranded pre-product that specifically binds to the second probe, so The single-stranded pre-product specifically combines with the second probe 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 second primer mixture produce different single-stranded pre-products from their corresponding target nucleic acids
  • different single-stranded pre-products are different from double-stranded products formed by the second probe
  • different double-stranded pre-products are different from the double-stranded products formed by the second probe.
  • the chain products have different melting temperatures. In some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.
  • 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, and the first primer comprises a target sequence binding region 1;
  • the second primer comprises a primer signal The detection region (h) and the 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 segment that does not specifically bind to any target nucleic acid , and the probe signal detection region (H) of the corresponding probe has part or all of the same sequence;
  • the sequences of the primer signal detection region (h) of the second primer 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 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.
  • 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 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.
  • 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 primer probe composition, the sample to be tested, and the amplification reagent are mixed to obtain a reaction system, and then the reaction system is placed under conditions that allow nucleic acid polymerase to perform hybridization and extension reactions to obtain reaction products, Specifically include the following:
  • the forward primer (F1) and reverse primer (R1) specifically bind to target sequence 1 and extend to generate a double-stranded pre-product.
  • Amplified product, the 5' end to the 3' end of a single-stranded pre-amplified product (S1) is the probe anchor region (A1), target sequence, and the reverse complementary sequence of the primer signal detection region (h1') , the reverse complementary sequence (M') of the extension block region; the forward primer (F2) and the reverse primer (R2) respectively specifically bind to the target sequence 2 and extend to generate double-stranded pre-amplified products, one of which is single-stranded
  • the 5' end to the 3' end of the pre-amplification product (S2) is followed by the probe anchor region (A2), the target sequence, and the reverse complementary sequence (h2') of the primer signal detection region; optional, if there is a positive
  • the forward primer (F3) and reverse primer (R3), the forward primer (F3) and the reverse primer (R3) specifically bind to the target sequence 3 and extend to generate double-stranded pre-amplified products, one of which is single-stranded pre-amplified
  • the probe signal of the probe (P) The detection region (H) is reverse complementary to the reverse complementary sequence (h1') of the primer signal detection region of the single-stranded preamplification product (S1), and the primer anchor region (A1') of the probe (P) is paired with the single The probe anchor region (A1) at the 5' end of the strand preamplification product (S1) is reverse-complementary paired to obtain a hybrid double-stranded product (D1) formed with the probe (P), whose melting temperature is T1, while the single-stranded
  • the reverse complementary sequence (M') of the extension block region at the 3' end of the preamplified product (S1) is not complementary to any part of the probe (P) or target sequence; at this time, the probe signal of the probe (P)
  • 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 (P) to obtain a probe Part or all of the reverse complementary sequence of the needle (P), that is, to complete the secondary amplification with the single-stranded pre-amplification product (S3) as the primer and the probe (P) as the template, and obtain the formation of the probe (P)
  • the amplified double-stranded product (D3) has a melting temperature of T3, and T3>T2.
  • the reaction product is placed at n different signal acquisition temperatures for n times of signal acquisition, and then analyzed under the same signal channel, whether the signals obtained by two signal acquisitions with adjacent signal acquisition temperatures exist or not The difference, determine the presence or absence of the target nucleic acid in the sample to be tested, specifically include the following:
  • the first signal acquisition is performed at the first signal acquisition temperature (t1, t1 ⁇ T1), since the single-stranded amplification products S1, S2 and S3 respectively form a full or partial double-stranded structure D1, D2 with the probe (P) , D3, compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes farther, and the efficiency of fluorescence resonance energy transfer is lower, so that the fluorescence signal changes, which can be detected by detected by the instrument; then the temperature is raised to the second signal acquisition temperature (t2, T1 ⁇ t2 ⁇ T2) for the second signal acquisition, the amplification formed by the single-stranded pre-amplification product (S1) and the probe (P)
  • the double-stranded product (D1) cannot form a double-stranded structure because the second signal collection temperature (t2) is higher than the melting temperature of the amplified double-stranded product (D1), and no fluorescent signal is generated, while the single-stranded pre-amplified
  • the reaction product is placed at n different signal acquisition temperatures for n times of signal acquisition, and then analyzed under the same signal channel, whether the signals obtained by two signal acquisitions with adjacent signal acquisition temperatures exist or not The difference, determine the presence or absence of the target nucleic acid in the sample to be tested, specifically include the following:
  • the first signal acquisition is performed at the first signal acquisition temperature (t3, T2 ⁇ t3 ⁇ T3), and the single-stranded pre-amplification product ( S1)
  • the amplified double-stranded product (D1) formed with the probe (P) and the amplified double-stranded product (D2) formed by the single-stranded pre-amplified product (S2) and the probe (P) are due to the first signal acquisition temperature (t3) is higher than the melting temperature of the amplified double-stranded products (D1 and D2) and cannot form a double-stranded structure, while the amplified double-stranded product (D3) formed by the single-stranded pre-amplified product (S3) and the probe (P) )
  • the distance between the first detection group and the second detection group becomes farther, and the efficiency of fluorescence resonance energy transfer is lower, so that the fluorescence signal changes
  • the amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) is still double-stranded and can be detected by the instrument.
  • the instrument detects the fluorescent signal; then lower the temperature to the third signal acquisition temperature (t1, t1 ⁇ T1) for the third signal acquisition, the amplification formed by the single-stranded pre-amplification product (S1) and the probe (P)
  • the double-stranded product (D1) compared with the state of the probe (P) before PCR, the distance between the first detection group and the second detection group becomes longer, and the fluorescence resonance energy transfer efficiency is lower, so that the fluorescence signal occurs
  • the change can be detected by the instrument, the amplified double-stranded product (D3) formed by the single-stranded pre-amplification product (S3) and the probe (P) and the double-stranded product (D3) formed by the single-stranded pre-amplified product (S2) and the probe (P)
  • a second aspect of the present invention provides a device for detection of various target nucleic acids, comprising:
  • the reaction liquid containing part is used to accommodate several micro-liquids, each of which contains a reaction reagent, and some of the micro-liquids also contain one of the first analyte or the second analyte;
  • 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
  • control unit controls the temperature adjustment unit to adjust the temperature of the micro-liquids in the reaction liquid container, so that several micro-liquids containing the first analyte or the second analyte generate signals at the same time;
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t1, and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte generate signals, forming first mixed signal;
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t2, and only a few micro-liquids containing the second analyte generate signals to form a second signal;
  • control part controls the signal detection part to collect the signal generated by the micro-liquid at the temperature t1 and t2, and output the first mixed signal and the second signal;
  • a signal analysis unit calculates the first signal according to the first mixed signal and the second signal collected by the signal detection unit.
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquid in the reaction liquid containing part to t3, and several micro-liquids containing the first analyte, containing the first Several microfluids containing the second analyte and several microfluids containing the third analyte generate signals to form a second mixed signal, wherein t3 ⁇ t1, and the difference between t3 and t1 is more than 4°C.
  • the difference between t1 and t2 is more than 4°C.
  • the difference between t1 and t2 is 4°C.
  • the signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal.
  • the first signal is the fluorescence signal generated by the microfluid containing the first analyte
  • the second signal is the fluorescence generated by the microfluid containing the second analyte signal
  • the first mixed signal is a fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte.
  • the signal analysis part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the the first signal.
  • the signal analysis unit subtracts the second mixed signal generated by the micro-liquid from the micro-liquid containing the first analyte and the micro-liquid containing the second analyte at the same position.
  • the first mixed signal generated by the micro-fluid is used to obtain a third signal
  • the third signal is a fluorescence signal generated by the micro-fluid containing the third analyte.
  • each of the micro-liquids contains a reaction reagent
  • the reaction 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
  • labeled substances can combine with nucleic acids at a certain temperature to produce detectable signals.
  • the reaction reagent includes a primer probe composition and an amplification reagent;
  • the amplification reagent includes nucleic acid polymerase and dNTPs;
  • the primer probe composition includes 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;
  • the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe.
  • the melting temperatures of the double-stranded products are different; in some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.
  • the primer probe composition also includes a second probe and a second primer mixture
  • the base sequence of the second probe is different from that of the first probe, and the modified detection label is also different;
  • the second primer mixture includes at least one primer set, and different primer sets are respectively matched with different target Nucleic acid specific binding;
  • the primer set in the second primer mixture specifically binds to its corresponding target nucleic acid to generate a pre-product
  • the pre-product contains a single-stranded pre-product that specifically binds to the second probe, so The single-stranded pre-product specifically binds to the second probe 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 second primer mixture produce different single-stranded pre-products from their corresponding target nucleic acids
  • different single-stranded pre-products are different from double-stranded products formed by the second probe
  • different double-stranded pre-products are different from the double-stranded products formed by the second probe.
  • the melting temperatures of the chain products are different; preferably, the melting temperatures of different double-chain products differ by more than 4°C.
  • 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), and a probe signal detection region of different probes
  • the sequences of (H) 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 identical sequences; the sequences of the primer signal detection regions (h) of the second primers in different primer sets are different from each other;
  • the first probe or the second probe further comprises a primer anchor region (A'); the first primer of at least one primer set in the first primer mixture or the second primer mixture further comprises a probe Needle anchoring region (A), described probe anchoring region (A) is positioned at the 5' end of target sequence binding region 1; Described probe anchoring region (A) is not specifically combined with any target nucleic acid, but with The primer anchor region (A') specifically binds;
  • the second primer in at most one primer set further includes an extension block region (M), and the extension block region (M) is located in the primer signal detection region.
  • the 5' end of region (h), said extension block region (M) and its complement, neither specifically binds to any probe or to any target nucleic acid.
  • the primer probe composition and digital PCR detection method of the present invention have the following advantages:
  • the method is simple: the method of the present invention can realize at least 8 multiple reactions on the digital PCR, and different targets can be distinguished by taking photos for a limited number of times to distinguish the presence or absence of fluorescence, and the requirements for the fluorescence recognition of digital PCR are low , easy to operate and save time;
  • the method of the present invention can simultaneously analyze the two dimensions of fluorescence channel and melting temperature in a single-tube reaction, that is, using the same fluorescence channel, different signals can be collected at different temperatures to detect different targets; or Using different fluorescent channels, the target type detection can be realized by the product of the number of fluorescent channels and the signal acquisition temperature characteristics;
  • each fluorescent channel uses only one probe, which can be distinguished by the different melting temperatures of the amplified products, which greatly reduces the fluorescent background in the PCR reaction and improves the reaction sensitivity;
  • the melting temperature can be adjusted: the method of the present invention utilizes the different melting temperatures of the secondary amplification products to distinguish, so by adjusting the length or sequence of the primer signal detection region (h), or adjusting the primer signal detection region (h) ) the reverse complementary position to the full signal detection region (H) of the probe (P), thereby increasing or decreasing the melting temperature of the secondary amplified double-stranded product formed with the probe (P);
  • the primer probe design method of the present invention has two parts of reverse complementary pairing with the target sequence, i.e. forward primer (F) and reverse primer (R), compared with Taqman hydrolysis probe method needs The three parts are reverse-complementary paired with the template.
  • F forward primer
  • R reverse primer
  • the primer probe design method of the present invention is more tolerant and less difficult to design;
  • Single-tube reaction less consumption of samples, especially suitable for the detection of rare samples, can greatly increase the concentration of samples added to the detection reaction, and improve detection sensitivity. the possibility
  • the method of the present invention has very low requirements on the length of the target sequence.
  • the target type is a short fragment of nucleic acid, such as 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
  • Fig. 1A is a signal diagram collected at 50°C in Example 1
  • Fig. 1B is a signal diagram collected at 68°C in Example 1;
  • Figure 2A is a signal diagram collected at 50°C in Example 2;
  • Figure 2B is a signal diagram collected at 68°C in Example 2;
  • Figure 2C is a signal diagram collected at 77°C in Example 2;
  • Figure 3A is a signal diagram collected at 70°C in Example 3
  • Figure 3B is a signal diagram collected at 50°C in Example 3;
  • Figure 4A is a signal diagram collected at 77°C in Example 4;
  • Figure 4B is a signal diagram collected at 70°C in Example 4;
  • Figure 4C is a signal diagram collected at 50°C in Example 4;
  • Fig. 5A is a schematic diagram of a variety of target nucleic acid detection devices disclosed in one embodiment of the present application
  • Fig. 5B is a schematic diagram of a variety of target nucleic acid detection devices disclosed in another embodiment of the present application
  • Fig. 5C is a schematic diagram of a variety of target nucleic acid detection devices disclosed in another embodiment of the present application Structural diagram of an orifice plate composed of multiple reaction liquid storage parts.
  • the D600 fully automatic digital PCR analysis system and reagent consumables of Mike Biological Co., Ltd. are used for detection and data analysis.
  • the digital PCR analysis system disperses the reaction system containing samples, primer probe compositions and amplification reagents. Perform amplification and detection in 4 reaction wells (each reaction well contains multiple small droplets, each small droplet is 1 reaction unit), in order to avoid redundancy, the accompanying drawings of the following examples are all provided Droplet results plot for 1 reaction well.
  • the forward primer F1-1 (SEQ ID NO: 2), the reverse primer R1-1 (SEQ ID NO: 3), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F1-1 is 39bp, the 1st to 23rd bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P1 (SEQ ID NO: 1)
  • the 1st to 13th nucleotide sequence at the 5' end of the R1-1 primer is 49 bp in full length, the 1st to 25th nucleotide sequence at the 3' end is the target sequence binding region, and the 6th to 13th nucleotide sequence at the 5' end is the target sequence binding region.
  • the 21st base is the same as the 16th to 31st bases at the 5' end of the probe P1 (SEQ ID NO: 1), and the 1st to 5th bases at the 5' end are a
  • the forward primer F1-2 (SEQ ID NO: 4), the reverse primer R1-2 (SEQ ID NO: 5), are all designed for the specificity of the gene KPC mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2 is 37bp, the 1st to 21st bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are compatible with the 5th base of probe P1 (SEQ ID NO: 1)
  • the 1st to 13th base sequences at the 'end are reverse complementary;
  • the R1-2 primer is 35 bp in length, the 1st to 22nd bases at the 3' end are the target sequence binding region, and the 1st to 10th bases at the 5' end
  • the 32nd to 41st base sequences of the 5' end of the probe probe P1 (SEQ ID NO: 1) are identical.
  • Reagent components concentration 2 ⁇ PCR Reaction Buffer 1 ⁇ DNA Polymerase 2U Forward primer (F1-1) 500nM Reverse primer (R1-1) 100nM Forward primer (F1-2) 500nM
  • Reverse primer 100nM Probe (P1) 400nM Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics 2000 copies Carbapenem-resistant gene KPC mutation nucleic acid template 1000 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 simultaneously use the nucleic acid template of carbapenem-resistant gene VIM mutation and the nucleic acid template of carbapenem-resistant gene KPC mutation as positive samples for detection. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Reaction preparation after the sample preparation is completed, configure the reaction system according to the ratio described in Table 2;
  • Digital PCR amplification and signal collection Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of 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, a total of 45 cycles; the first signal acquisition at 50°C; the second at 68°C Signal Acquisition.
  • Figure 1A is a droplet diagram at the first signal acquisition temperature (50°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background;
  • Figure 1B is at the second signal acquisition temperature (68°C). °C), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared with that in Figure 1A (part of the positive droplets in Figure 1A, in the corresponding position in Figure 1B no positive droplets).
  • the positive droplets that still exist in Figure 1B are the signals of the gene KPC mutation-positive samples resistant to carbapenem antibiotics. By analyzing the number of positive droplets, the content of the gene KPC mutation-positive samples can also be determined; The positive droplets that do not exist in 1B but exist in Figure 1A are the signal of the VIM mutation sample resistant to carbapenem antibiotics. By analyzing the difference between the positive droplets in Figure 1A and Figure 1B, it can also be Determine the content of gene VIM mutation positive samples.
  • the detection method of the present invention can realize the detection of various target nucleic acids.
  • the realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence.
  • the fluorescent recognition of digital PCR has low requirements, simple operation and time saving.
  • the triple primer probe system for detecting carbapenem-resistant gene VIM mutation and carbapenem-resistant gene KPC mutation and carbapenem-resistant gene OXA-48 mutation is as follows: Table 3 shows:
  • the forward primer F2-1 (SEQ ID NO: 2), the reverse primer R2-1 (SEQ ID NO: 3), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2-1 is 39bp, the 1st to 23rd bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P2 (SEQ ID NO: 1)
  • the 1st to 13th nucleotide sequence at the 5' end of the R2-1 primer is 49 bp in full length, and the 1st to 25th nucleotide sequence at its 3' end is the target sequence binding region, and the 6th to 13th nucleotide sequence at its 5' end is
  • the 21st base has the same sequence as the 16th to 31st bases at the 5' end of the probe P2 (SEQ ID NO: 1), and the 1st to 5th bases at the 5' end are amplification retardation district.
  • the forward primer F2-2 (SEQ ID NO: 4), the reverse primer R2-2 (SEQ ID NO: 5), are all designed for the specificity of the gene KPC mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F2-2 is 37bp, the 1st to 21st bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P2 (SEQ ID NO: 1)
  • the 1st to 13th nucleotide sequences at the 5' end of the primer are reverse complementary; the R2-2 primer is 35 bp in length, the 1st to 22nd nucleotides at its 3' end are the target sequence binding region, and the 1st to 13th bases at its 5' end
  • the 10th base is completely identical to the 32nd to 41st base sequence of the 5' end of the probe probe P2 (SEQ ID NO: 1).
  • the forward primer F2-3 (SEQ ID NO: 6), the reverse primer R2-3 (SEQ ID NO: 7), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primers, the full length of F2-3 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P2 (SEQ ID NO: 1)
  • the 1st to 13th nucleotide sequence at the 5' end is reverse complementary;
  • the R2-2 primer is 37 bp in full length, the 1st to 24th nucleotide sequence at its 3' end is the target sequence binding region, and the 5' end nucleotide sequence is
  • the 1st to 10th bases are completely identical to the 1st to 10th bases of the 3' end of the probe probe P2 (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 simultaneously use the nucleic acid template of carbapenem-resistant gene VIM mutation, the nucleic acid template of carbapenem-resistant gene KPC mutation and the nucleic acid template of carbapenem-resistant gene OXA-48 mutation
  • the template is tested as a positive sample. Pure water was used as a no-template control (NTC).
  • Reaction preparation after the sample preparation is completed, configure the reaction system according to the ratio described in Table 4;
  • Digital PCR amplification and signal collection Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of 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; 40°C constant temperature incubation for 3 minutes; 50°C for the first signal acquisition; The second signal acquisition was performed at 68°C; the third signal acquisition was performed at 77°C.
  • Figure 2A is a droplet diagram at the first signal acquisition temperature (50°C), and it can be seen that there are positive droplets that are different from the background (bright spots are positive droplets);
  • Figure 2B is at the second signal acquisition temperature (68°C). °C), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared with that in Figure 2A (part of the positive droplets in Figure 2A, in the corresponding position in Figure 2B There are no positive droplets);
  • Figure 2C is a droplet diagram at the third signal acquisition temperature (77°C), and it can be seen that there are positive droplets that are different from the background, and the number of positive droplets is reduced compared to Figure 2B (Some of the positive droplets in FIG. 2B have no positive droplets in the corresponding positions in FIG. 2C).
  • the positive droplets that still exist in Figure 2C are the signals of carbapenem-resistant gene OXA-48 mutation-positive samples. By analyzing the number of positive droplets, the number of gene OXA-48 mutation-positive samples can also be determined. content; the positive droplets that do not exist in Figure 2C but exist in Figure 2B are the signal of the gene KPC mutation resistant to carbapenem antibiotics, by analyzing the difference between the positive droplets in Figure 2B and Figure 2C , it is also possible to determine the content of gene KPC mutation-positive samples; the positive droplets that do not exist in Figure 2B but exist in Figure 2A are the signals of carbapenem-resistant gene VIM mutation samples. The difference between positive droplets in Figure 2A and Figure 2B can also determine the content of gene VIM mutation positive samples.
  • the detection method of the present invention can realize the detection of various target nucleic acids.
  • the realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and can distinguish between different targets by taking a limited number of photos to distinguish the presence or absence of fluorescence.
  • the fluorescent recognition of digital PCR has low requirements, simple operation and time saving.
  • the forward primer F3-1 (SEQ ID NO: 9), the reverse primer R3-1 (SEQ ID NO: 10), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F3-1 is 39bp, the 1st to 23rd bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P3 (SEQ ID NO: 8)
  • the 1st to 13th base sequence at the 5' end of the primer is reverse complementary;
  • the R3-1 primer is 49 bp in length, the 1st to 25th base at its 3' end is the target sequence binding region, and the 6th to 13th base at its 5' end
  • the 21st base has the same sequence as the 16th to 31st bases at the 5' end of probe P3 (SEQ ID NO: 8), and the 1st to 5th bases at the 5' end are amplification retardation district.
  • the forward primer F3-2 (SEQ ID NO: 11), the reverse primer R3-2 (SEQ ID NO: 12), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primer, the full length of F3-2 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P3 (SEQ ID NO: 8)
  • the 1st to 13th nucleotide sequence at the 5' end is reverse complementary;
  • the R3-2 primer is 38 bp in full length, the 1st to 24th nucleotide sequence at its 3' end is the target sequence binding region, and the 5' end nucleotide sequence is
  • the 1st to 11th bases are completely identical to the 32nd to 42nd bases at the 5' end of the probe probe P3 (SEQ ID NO: 8).
  • 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 simultaneously use the nucleic acid template of the carbapenem-resistant gene VIM mutation and the nucleic acid template of the carbapenem-resistant gene OXA-48 mutation as positive samples for detection. Pure water was used as a no-template control (NTC).
  • NTC no-template control
  • Reaction preparation after the sample preparation is completed, configure the reaction system according to the ratio described in Table 6;
  • Digital PCR amplification and signal collection Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of 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; constant temperature at 52°C for 3 minutes; first signal acquisition at 70°C; °C for the second signal acquisition.
  • Figure 3A is a droplet diagram at the first signal acquisition temperature (70°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background;
  • Figure 3B is at the second signal acquisition temperature (50°C). °C), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets has increased compared to Figure 3A (part of the negative droplets in Figure 3A, and the corresponding position in Figure 3B for positive droplets).
  • the positive droplets in Figure 3A are the signals of the carbapenem-resistant gene OXA-48 mutation-positive samples. By analyzing the number of positive droplets, the content of the gene OXA-48 mutation-positive samples can also be determined.
  • the positive droplets that do not exist in Figure 3A but exist in Figure 3B are the signal of the gene VIM mutation sample resistant to carbapenem antibiotics, by analyzing the difference between the positive droplets in Figure 3A and Figure 3B , can also determine the content of gene VIM mutation positive samples.
  • the detection method of the present invention can realize the detection of various target nucleic acids.
  • the realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and through a limited number of photographs, different targets can be distinguished by distinguishing the presence or absence of fluorescence.
  • the requirements for fluorescent recognition of digital PCR are low, the operation is simple, and time is saved.
  • the forward primer F4-1 (SEQ ID NO: 9), the reverse primer R4-1 (SEQ ID NO: 10), are all designed for the specificity of the gene VIM mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F4-1 is 39bp, the 1st to 23rd bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are linked to the probe P4 (SEQ ID NO: 18)
  • the 1st to 13th nucleotide sequence at the 5' end of the R4-1 primer is 49 bp in full length, the 1st to 25th nucleotides at the 3' end are the target sequence binding region, and the 6th to 13th nucleotides at the 5' end are the target sequence binding region.
  • the 21st base has the same sequence as the 16th to 31st bases at the 5' end of the probe P4 (SEQ ID NO:8), and the 1st to 5th bases at the 5' end are a
  • the forward primer F4-2 (SEQ ID NO: 11), the reverse primer R4-2 (SEQ ID NO: 12), are all designed for the gene OXA-48 mutation target sequence resistant to carbapenem antibiotics Specific primer, the full length of F4-2 is 35bp, the 1st to 19th bases at the 3' end are the target sequence binding region, and the 1st to 13th bases at the 5' end are compatible with the probe P4 (SEQ ID NO: 8)
  • the 1st to 13th nucleotide sequences at the 5' end are reverse complementary;
  • the R4-2 primer is 38 bp in length, the 1st to 24th nucleotides at the 3' end are the target sequence binding region, and the 5' end nucleotides at the 5' end
  • the 1st to 11th bases are identical to the 32nd to 42nd base sequences of the 5' end of the probe probe P4 (SEQ ID NO: 8).
  • the forward primer F4-3 (SEQ ID NO: 13), the reverse primer R4-3 (SEQ ID NO: 14), are all designed for the specificity of the gene IMP mutation target sequence resistant to carbapenem antibiotics Primer, the full length of F4-3 is 40bp, the 1st to 24th bases of its 3' end are the target sequence binding region, and the 1st to 13th bases of its 5' end are connected with the probe P4 (SEQ ID NO: 8)
  • the 1st to 13th nucleotide sequences at the 5' end of the primer are reverse complementary;
  • the R4-3 primer is 35 bp in length, the 1st to 23rd nucleotides at its 3' end are the target sequence binding region, and the 1st to 13th nucleotides at its 5' end
  • the 9th base is identical to the 1st to 9th base sequence of the 3' end of the probe probe P4 (SEQ ID NO: 8).
  • Reagent components concentration 2 ⁇ PCR Reaction Buffer 1 ⁇ DNA Polymerase 2U Forward primer (F4-1) 500nM Reverse primer (R4-1) 100nM Forward primer (F4-2) 500nM
  • Reverse primer R4-2 100nM Forward primer (F4-3) 500nM Reverse primer (R4-3) 100nM Probe (P4) 400nM Gene VIM mutation nucleic acid template resistant to carbapenem antibiotics 10 copies Carbapenem-resistant gene OXA-48 mutation nucleic acid template 15 copies Gene IMP mutation nucleic acid template resistant to carbapenem antibiotics 100 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 simultaneously use the nucleic acid template of the carbapenem-resistant gene VIM mutation, the nucleic acid template of the carbapenem-resistant gene OXA-48 mutation and the nucleic acid template of the carbapenem-resistant gene IPM mutation
  • the template is tested as a positive sample. Pure water was used as a no-template control (NTC).
  • Reaction preparation after the sample preparation is completed, configure the reaction system according to the ratio described in Table 8;
  • Digital PCR amplification and signal collection Seal the cap of the digital PCR tube and mix the sample gently, then centrifuge briefly and let it stand at room temperature for 5 minutes. The digital PCR tube was placed in the hand-held centrifuge again, and after a brief centrifugation, it was transferred to the sample rack of the digital PCR instrument (D600 fully automatic digital PCR analysis system of 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, a total of 45 cycles; constant temperature incubation at 40°C for 3 minutes; first signal acquisition at 77°C; The second signal acquisition was performed at 70°C; the third signal acquisition was performed at 50°C.
  • Figure 4A is a droplet diagram at the first signal acquisition temperature (77°C), and it can be seen that there are positive droplets (bright spots are positive droplets) that are different from the background;
  • Figure 4B is at the second signal acquisition temperature (70°C) °C), it can be seen that there are positive droplets that are different from the background, and the number of positive droplets has increased compared to Figure 4A (some negative droplets in Figure 4A, corresponding positions in Figure 4B is a positive droplet);
  • Fig. 4C is a droplet diagram at the third signal acquisition temperature (50°C), it can be seen that there are positive droplets different from the background, and the number of positive droplets has increased compared to Fig. 4B ( Part of the negative droplet in Figure 4B, the corresponding position in Figure 4C is a positive droplet)
  • the positive droplets present in Figure 4A are the signals of the gene IMP mutation-positive samples resistant to carbapenem antibiotics, and by analyzing the number of positive droplets, the content of the gene IMP mutation-positive samples can also be determined; in Figure 4A
  • the positive droplets that do not exist in but exist in Figure 4B are the signal of the carbapenem-resistant gene OXA-48 mutation sample, by analyzing the difference between the positive droplets in Figure 4A and Figure 4B, and also The content of the gene OXA-48 mutation-positive samples can be determined;
  • the positive droplets that do not exist in Figure 4B but exist in Figure 4C are the signals of the gene VIM mutation samples resistant to carbapenem antibiotics.
  • the difference between positive droplets in Figure 4B and Figure 4C can also determine the content of gene VIM mutation positive samples.
  • the detection method of the present invention can realize the detection of various target nucleic acids.
  • the realization of multiple detection does not only depend on the fluorescent channel, but also uses different melting temperatures in the same fluorescent channel (that is, the same signal channel), and through a limited number of photographs, different targets can be distinguished by distinguishing the presence or absence of fluorescence.
  • the requirements for fluorescent recognition of digital PCR are low, the operation is simple, and time is saved.
  • the embodiment of the present application provides a variety of target nucleic acid detection devices for multiple detection of nucleic acids, which can distinguish the presence or absence of fluorescence through a limited number of photographs to distinguish different targets.
  • the fluorescence recognition requirements for digital PCR Low, easy to operate, save time.
  • 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.
  • a first analyte could be termed a second analyte, and, similarly, a second analyte could be termed a first analyte, without departing from the scope of the present application.
  • Both the first analyte and the second analyte are analytes, but they are not the same analyte.
  • the term “multiple" used in the embodiments of the present application refers to two or more than two.
  • the various target nucleic acid detection devices of this embodiment are used to detect the samples to be tested.
  • the samples to be tested may include one type of component to be tested, or two, three, four, five or even more types of components to be tested.
  • the nucleic acid detection device of this embodiment can realize the detection of various components to be tested in the sample to be tested.
  • the multi-target nucleic acid detection device of this embodiment includes a reaction solution storage unit 1 , a temperature adjustment unit 2 , a signal detection unit 3 , a control unit 4 and a signal analysis unit 5 .
  • the reaction liquid container 1 is used to accommodate the test sample containing the test component and the reaction reagent capable of reacting with the test component, and provides a place for various reactions between the test sample and the reaction reagent.
  • the temperature adjusting part 2 is used to adjust the temperature of the mixed liquid in the reaction liquid containing part 1, so that the sample to be tested and the reaction reagents 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 signal analyzing part 5 is used for analyzing the signal collected by the signal detecting part 3 .
  • the sample to be tested is body fluid from a human body or an animal body, and the sample to be tested 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 to be detected contains different nucleic acid molecules to be detected, for example, the first analyte (the first target nucleic acid to be detected), the second analyte (the second target nucleic acid to be detected), the third analyte (the third target nucleic acid to be detected), the fourth analyte (the fourth target nucleic acid to be detected), the fifth analyte (the fifth target nucleic acid to be detected) and the like.
  • the first analyte the first target nucleic acid to be detected
  • the second analyte the second target nucleic acid to be detected
  • the third analyte the third target nucleic acid to be detected
  • the fourth analyte the fourth target nucleic acid to be detected
  • the fifth target nucleic acid to be detected the fifth target nucleic acid to be detected
  • the reaction reagents include amplification substances for amplifying nucleic acids and labeling substances for labeling nucleic acids.
  • the amplification substances can amplify different nucleic acid molecules.
  • the labeling substances can combine with nucleic acids at a certain temperature to generate detectable signals. .
  • 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 amplifying substances for amplifying different nucleic acids to be tested are different, and the labeling substances for marking different nucleic acids to be tested may be the same or different. In some embodiments, different nucleic acids to be detected are labeled with the same labeling substance.
  • the reaction reagent includes a primer-probe composition and an amplification reagent: wherein, the amplification reagent includes nucleic acid polymerase and dNTPs; wherein, the primer-probe composition includes a probe and a primer mixture.
  • the primer mixture includes at least two primer sets, and different primer sets specifically bind to different target nucleic acids respectively; after the primer sets in the primer mixture specifically bind to their corresponding target nucleic acids, a pre-product is generated, and the pre-product contains the A probe-specific single-stranded pre-product, the single-stranded pre-product specifically binds to the first probe and extends ⁇ 0 bases to form a double-stranded product, the formation of the double-stranded product causes a detectable Signal changes.
  • the single-stranded pre-products produced by different primer sets in the first primer mixture and their corresponding target nucleic acids are different, and the different single-stranded pre-products are different from the double-stranded products formed by the first probe.
  • the melting temperatures of the double-stranded products are different; in some embodiments, the melting temperatures of different double-stranded products differ by more than 4°C.
  • 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. Included in each microfluid is at most one unit of nucleic acid molecule and sufficient reagents to amplify and label multiple target nucleic acids.
  • each first analyte and each second analyte are Distributed to different micro-fluids, in one micro-fluid, there is enough reagent, which can not only meet the needs of amplification and labeling of the first analyte in the micro-liquid, but also meet the needs of the second analyte in the amplification and labeling of the micro-liquid.
  • the requirements of the test substance are two kinds of target nucleic acids to be detected in the sample to be tested, that is, the first analyte and the second analyte.
  • 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.
  • micro-sections on the container, and add the micro-liquid of the sample to be tested and the reaction reagent into several micro-sections, so that there is at most 1 unit of nucleic acid molecule in each micro-section.
  • the reaction liquid 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 liquid 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 microfluids containing the nucleic acid to be tested and reagents are wrapped in more than 500, for example, about 20,000 microfluids, and each microfluid contains one or no target to be detected Nucleic acid molecules, regardless of the number of analytes in the sample to be tested, are all distributed into different microfluids (this is an ideal state, and there may be more than one target nucleic acid molecule to be detected in a small amount of microfluidics).
  • 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 parts 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 solution storage unit 1 within the range of 4°C to 105°C.
  • FIG. 5A 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 to 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. 5A 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 control the temperature adjustment unit and the signal detection unit at the same time.
  • the signal analysis unit 5 and the signal detection unit 3 are communicably connected.
  • the signal analysis part is used to analyze the signal collected by the signal detection part to obtain the signal of the target nucleic acid to be tested, so as to obtain information such as whether the sample to be tested contains the target nucleic acid to be tested, the type and quantity of the target nucleic acid to be tested.
  • the sample to be tested contains the first analyte (the first target nucleic acid to be detected) and the second analyte (the second target nucleic acid to be detected), and it is required
  • the first associated product to be tested produced by the first analyte (the target nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage etc., produces its corresponding
  • the associated product to be detected generally, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; combined.
  • the first analyte in a certain temperature mode, produces the first analyte associated product, which binds to the marker substance, and at the same time, in this temperature mode, the second analyte produces the second analyte associated product product and binds to the labeled substance.
  • the separation temperatures of the two are different: the first correlation product to be detected can be separated from the labeled substance in the reagent in the first separation temperature range, and the second correlation product to be detected can be separated from the label in the reagent in the second separation temperature range.
  • the minimum temperature of the first separation temperature range and the second separation temperature range are different. In this embodiment, 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 t1
  • 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 t2, wherein the temperature t2 is higher than the temperature t1.
  • the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces an associated product to be tested (the nucleic acid to be tested comprises the first nucleic acid to be tested, the second nucleic acid to be tested) Nucleic acid is detected, but exists in different microfluids); during this process, the microfluidics will go through multiple temperature cycles, and in each temperature cycle, the microfluidics will change to several different temperatures and continue at that temperature In a certain period of time, the number of cycles may be multiple, such as 20-60, for example, about 40.
  • the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature).
  • annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature.
  • the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected.
  • the amount of the nucleic acid to be detected and/or the related product to be detected increases, and combines with the labeling substance in the reagent, so that several microparticles containing the first analyte or the second analyte The liquid simultaneously produces a signal.
  • the microfluid Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.
  • the control part controls the temperature adjustment part to adjust the temperature of the micro-liquid to t1, and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte generate signals , forming the first mixed signal.
  • the second analyte or the associated product of the second analyte and the label are in a combined state to generate a signal, and the first analyte or the associated product of the first analyte and the label are also in a state of Combining states, while generating signals.
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected or the second nucleic acid to be detected.
  • the microfluid containing the first nucleic acid to be detected and the microfluid containing the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • control part controls the temperature regulating part to adjust the temperature of the micro-liquids to t2 (t2>t1), and only a few micro-liquids containing the second analyte generate signals to form a second signal.
  • the first analyte, or an associated product of the first analyte is separated from the label and thus produces no detectable signal, but the second analyte, or an associated product of the second analyte, is separated from the label Still in the bound state, a signal is generated.
  • this nucleic acid molecule may be the first nucleic acid to be detected or the second nucleic acid to be detected.
  • this nucleic acid molecule may be the first nucleic acid to be detected or the second nucleic acid to be detected.
  • the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • the 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°C -15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.
  • the signal detection part detects the signal generated in the micro-fluid
  • the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the first mixed signal contains the signals of the first analyte and the second analyte, but the second signal only contains the signal of the first analyte).
  • the signal analysis department performs the following operations:
  • a first signal is calculated according to the first mixed signal and the second signal collected by the signal detection unit.
  • the signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal.
  • the signal analyzing part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the first signal.
  • the first signal is a fluorescent signal generated by the microfluid containing the first analyte
  • the second signal is a fluorescent signal generated by the microfluid containing the second analyte
  • the first The mixed signal is the fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte.
  • the fluorescence signal generated by the micro-liquid containing the second analyte can be directly obtained.
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected, or the second nucleic acid to be detected.
  • the quantity of the analyte; the total quantity of the first analyte and the second analyte in the first mixed signal is subtracted from the quantity of the second analyte in the second signal to obtain the quantity of the first analyte.
  • the sample to be tested contains the first analyte (the first target nucleic acid to be detected), the second analyte (the second target nucleic acid to be detected) and the third The analyte (the third analyte target nucleic acid).
  • the first associated product to be tested produced by the first analyte (the target nucleic acid to be tested undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage, etc., produces its corresponding
  • the associated product to be detected usually, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; the second related product to be tested produced by the second analyte can be combined with the labeling substance in the reagent, The third analyte-associated product produced by the third analyte can be combined with the labeling substance in the reagent.
  • the first analyte in a certain temperature mode, produces the first analyte associated product, which binds to the marker substance, and at the same time, in this temperature mode, the second analyte produces the second analyte associated product
  • the product is combined with the labeled substance
  • the third analyte produces a third analyte-related product, which is combined with the labeled substance.
  • the separation temperatures of the three are different: the first associated product to be detected can be separated from the labeled substance in the reagent within the first separation temperature range, and the second associated product to be detected can be separated from the labeled substance in the reagent within the second separation temperature range.
  • the third associated product to be detected can be separated from the labeled substance in the reagent within the third separation temperature range, the minimum temperature of the first separation temperature range, the second separation temperature range and the third separation temperature range are different.
  • the lowest temperature in the third separation temperature range is lower than the lowest temperature in the first separation temperature range, and the lowest temperature in the first separation temperature range is lower than the lowest temperature in the second 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 t1
  • 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 t2
  • the third associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t2. It can be separated from the marker substance when the temperature is higher than or equal to the temperature t3, wherein, the temperature t3 ⁇ t1 ⁇ t2.
  • the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces an associated product to be tested (the target nucleic acid to be tested comprises the first analyte, the second analyte and a third analyte, but present in different microfluids); during this process, the microfluidics undergoes multiple temperature cycles, and in each temperature cycle, the microfluidics changes to several different temperatures , and continue at this temperature for a certain period of time, the number of cycles can be multiple, such as 20-60, for example, about 40.
  • the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature).
  • annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature.
  • the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected.
  • the amount of the nucleic acid to be tested and/or the associated product to be tested increases, and binds to the labeling substance in the reagent, so that the nucleic acid containing the first analyte or the second analyte or the third analyte Several microfluidics of the analyte generate signals simultaneously.
  • the microfluid Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.
  • control part controls the temperature adjustment part to adjust the temperature of the micro-liquids to t3, several micro-liquids containing the first analyte, several micro-liquids containing the second analyte, and The several microfluids of the third analyte all generate signals to form a second mixed signal.
  • the second analyte or the associated product of the second analyte and the marker are in a combined state to generate a signal
  • the first analyte or the associated product of the first analyte and the marker are also in a state of In the combined state, a signal is generated at the same time
  • the third analyte or the associated product of the third analyte and the marker are also in a combined state, and a signal is also generated.
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected, or the second nucleic acid to be detected or the second nucleic acid to be detected.
  • the microfluids containing the first nucleic acid to be detected, the microfluids containing the second nucleic acid to be detected, and the microfluids containing the third nucleic acid to be detected can all generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t1 (t1>t3), and several micro-liquids containing the first analyte and several micro-liquids containing the second analyte are produced. signal, forming the first mixed signal.
  • the second analyte or the associated product of the second analyte and the label are in a combined state to generate a signal, and the first analyte or the associated product of the first analyte and the label are also in a state of In the combined state, a signal is generated at the same time, however, the third analyte or the associated product of the third analyte and the label are also in a separated state, and no detectable signal is generated.
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected or the second nucleic acid to be detected.
  • the microfluid containing the first nucleic acid to be detected and the microfluid containing the second nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • control part controls the temperature regulating part to adjust the temperature of the micro-liquids to t2 (t2>t1), and only a few micro-liquids containing the second analyte generate signals to form a second signal.
  • the first analyte or the associated product of the first analyte is separated from the label, and the third analyte or the associated product of the third analyte is separated from the label, therefore, no detectable signal, but the second analyte or the associated product of the second analyte is still in a combined state with the label to generate a signal.
  • a single nucleic acid molecule is distributed into a microfluid, that is, there is only one nucleic acid molecule in a microfluid, at temperature t2, only a few microfluids where the second nucleic acid to be detected is located can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • the 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°C -18°C, 4-15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.
  • the difference between t1 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, 4-5°C or 4°C, etc.
  • the signal detection part detects the signal generated in the micro-fluid
  • the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the second mixed signal Contains the signals of the first analyte, the second analyte and the third analyte, the first mixed signal contains the signals of the first analyte and the second analyte, but the second signal only contains the first signal of the analyte).
  • the signal analysis department performs the following operations:
  • a third signal is calculated according to the first mixed signal and the second mixed signal collected by the signal detection unit.
  • the signal analysis unit subtracts the second mixed signal from the first mixed signal to obtain the third signal. Specifically, the signal analysis part subtracts the second mixed signal generated by the micro-liquid from the micro-liquid containing the first analyte and the micro-liquid containing the second analyte at the same position. The first mixed signal is used to obtain a third signal.
  • the second mixed signal is the fluorescence generated by the microfluid containing the first analyte, the microfluid containing the second analyte, and the microfluid containing the third analyte signal;
  • the first mixed signal is the fluorescence signal generated by the micro-liquid containing the first analyte and the micro-liquid containing the second analyte;
  • the third signal is the micro-liquid containing the third analyte The fluorescent signal generated by the micro-fluid of the test object;
  • a first signal is calculated according to the first mixed signal and the second signal collected by the signal detection unit.
  • the signal analysis unit subtracts the first mixed signal from the second signal to obtain the first signal.
  • the signal analyzing part subtracts the fluorescent signal generated by the microliquid containing the second analyte at the same position from the first mixed signal generated by the microliquid to obtain the first signal.
  • the first signal is a fluorescent signal generated by the microfluid containing the first analyte
  • the second signal is a fluorescent signal generated by the microfluid containing the second analyte
  • the first The mixed signal is a fluorescent signal generated by the microfluid containing the first analyte and the microfluid containing the second analyte;
  • the fluorescence signal generated by the micro-liquid containing the second analyte can be directly obtained;
  • the nucleic acid molecule to be detected may be the first nucleic acid to be detected, the second nucleic acid to be detected or the third nucleic acid to be detected.
  • the total quantity of the first analyte, the second analyte and the third analyte can be obtained; analyze the amount of microfluids that generate signals in the first mixed signal Quantity, that is, the total quantity of the first analyte and the second analyte; analyze the quantity of the microfluid that generates the signal in the second signal, that is, obtain the quantity of the second analyte; the first analyte in the second mixed signal
  • the total quantity of the analyte, the second analyte and the third analyte is subtracted from the total quantity of the first analyte and the second analyte in the first mixed signal to obtain the quantity of the third analyte;
  • the total amount of the first analyte and the second analyte in the first mixed signal is subtracted from the amount of the second analyte in the second signal to obtain the amount of the amount of the
  • the sample to be tested contains the fourth analyte (the fourth target nucleic acid to be detected) and the fifth analyte (the fifth target nucleic acid to be detected).
  • the fourth associated product to be tested produced by the fourth analyte undergoes operations such as polymerase chain reaction (PCR) and/or nucleic acid hybridization and/or nuclease cleavage etc., produces its corresponding
  • the associated product to be detected usually, the associated product to be detected is a nucleic acid), which can be combined with the labeling substance in the reagent; similarly, the fifth associated product to be detected produced by the fifth analyte can also be combined with the reagent in the binding of labeled substances.
  • the fourth analyte in a certain temperature mode, produces the fourth analyte-related product, which is combined with the marker substance, and at the same time, in this temperature mode, the fifth analyte produces the fifth analyte-related product product and binds to the labeled substance.
  • the separation temperatures of the two are different: the fourth to-be-measured associated product can be separated from the labeled substance in the reagent in the fourth separation temperature range, and the fifth to-be-measured associated product can be separated from the labeled substance in the reagent in the fifth separation temperature range.
  • the fourth separation temperature range and the minimum temperature of the fourth separation temperature range are different.
  • the lowest temperature in the fourth separation temperature range is higher than the lowest temperature in the fifth separation temperature range.
  • the fourth associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t4
  • the fifth associated product to be detected can be separated from the labeled substance when the temperature is higher than or equal to the temperature t5, wherein the temperature t4 is higher than the temperature t5.
  • the sample to be tested and The mixed solution of the reaction reagent (comprising the primer probe composition and the amplification reagent) is added to the reaction solution containing part to form several micro-liquids, and the control part performs the following control:
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid in the reaction liquid containing part, so that the target nucleic acid to be tested in the sample to be tested produces a related product to be tested (the nucleic acid to be tested includes the fourth nucleic acid to be tested, the fifth nucleic acid to be tested, and the target nucleic acid to be tested).
  • Nucleic acid is detected, but exists in different microfluids); during this process, the microfluidics will go through multiple temperature cycles, and in each temperature cycle, the microfluidics will change to several different temperatures and continue at that temperature In a certain period of time, the number of cycles may be multiple, such as 20-60, for example, about 40.
  • the microfluidics will reach a high temperature state, the double-stranded nucleic acid becomes single-stranded nucleic acid, and then the microfluidics reaches a low temperature state, and the single-stranded nucleic acid undergoes annealing and extension processes (annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature).
  • annealing and extension can also be carried out at different temperatures, and extension temperature higher than the annealing temperature.
  • the number of different target nucleic acids to be detected and/or related products to be detected in the sample to be tested increased (that is, the amount of target nucleic acids to be detected and/or related products to be detected in each microfluid Both increase, but the type of nucleic acid to be tested remains unchanged), and it is easier to be detected.
  • the number of the nucleic acid to be detected and/or the related product to be detected increases, and combines with the labeling substance in the reagent, so that several microparticles containing the fourth analyte or the fifth analyte The liquid simultaneously produces a signal.
  • the microfluid Before performing 40 temperature cycles, the microfluid can be heated to temperature t0 to pre-denature the nucleic acid to be detected in the sample for a period of time (about 2-5 minutes). Pre-denaturing for a period of time is to change the target nucleic acid from a double-stranded state to a single-stranded state.
  • the control part controls the temperature adjustment part to adjust the temperature of the micro liquid to t4, and only a few micro liquids containing the fourth analyte generate signals to form a fourth signal.
  • the fifth analyte or the associated product of the fifth analyte is separated from the label and thus produces no detectable signal, but the fourth analyte or the associated product of the fourth analyte is separated from the label Still in the bound state, a signal is generated.
  • this nucleic acid molecule may be the fourth nucleic acid to be detected, or the fifth nucleic acid to be detected.
  • this nucleic acid molecule may be the fourth nucleic acid to be detected, or the fifth nucleic acid to be detected.
  • the fourth nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • the control part controls the temperature regulating part to adjust the temperature of the micro-liquid to t5 (t5 ⁇ t4), and several micro-liquids containing the fourth analyte and several micro-liquids containing the fifth analyte are produced.
  • signal to form a third mixed signal.
  • the fourth analyte or the associated product of the fourth analyte and the marker are in a combined state to generate a signal
  • the fifth analyte or the associated product of the fifth analyte and the marker are also in a state of Combines the state, while generating the signal.
  • this nucleic acid molecule to be detected may be the fourth nucleic acid to be detected or the fifth nucleic acid to be detected.
  • both the microfluid containing the fourth nucleic acid to be detected and the microfluid containing the fifth nucleic acid to be detected can generate signals. If multiple microfluids overlap, the signal sent by such microfluids is an invalid signal; the signal sent by a single microfluid that does not overlap is a valid signal.
  • the difference between t4 and t5 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°C -15°C, 4-12°C, 4-10°C, 4-8°C, 4-6°C, 4-5°C or 4°C, etc.
  • the signal detection part detects the signal generated in the micro-fluid
  • the number of target nucleic acid species contained in the signals obtained by two adjacent signal acquisitions differs by 1 (that is, in the fourth signal, only contains the signal of the fourth analyte, and the third mixed signal contains the signals of the fourth analyte and the fifth analyte).
  • the signal analysis department performs the following operations:
  • a fifth signal is calculated based on the fourth signal and the third mixed signal collected by the signal detection unit.
  • the signal analysis unit subtracts the third mixed signal from the fourth signal to obtain the fifth signal.
  • the signal analysis part subtracts the fluorescent signal generated by the microfluid containing the fourth analyte at the same position from the third mixed signal generated by the microfluid to obtain the fifth signal.
  • the fourth signal is the fluorescence signal generated by the micro-liquid containing the fourth analyte
  • the fifth signal is the fluorescence signal generated by the micro-liquid containing the fifth analyte
  • the third The mixed signal is a fluorescent signal generated by the microfluid containing the fourth analyte and the microfluid containing the fifth analyte;
  • the fluorescence signal generated by the micro-liquid containing the fourth analyte can be directly obtained;
  • the nucleic acid molecule to be detected may be the fourth nucleic acid to be detected, or the fifth nucleic acid to be detected.
  • the quantity of the analyte subtracting the quantity of the fourth analyte in the fourth signal from the total quantity of the fourth analyte and the fifth analyte in the third mixed signal, the quantity of the fifth analyte is obtained.
  • 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 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

涉及分子生物学领域具体涉及用于单个样品容器中多种靶核酸检测的方法。特别是涉及一种在数字PCR上进行多重检测的方法，包括: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；将所述反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件，获得反应产物；将反应产物进行信号采集。采用上述检测方法，能够实现对多种靶核酸的检测，并且操作简单，节省时间。

Description

多种靶核酸检测的方法
相关申请的交叉引用
本申请要求享有于2021年12月27日提交的名称为“一种用于检测的引物探针、引物探针组及其应用”的中国专利申请CN 202111617233.2的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本发明涉及分子生物学领域，具体涉及用于单个样品容器中多种靶核酸检测的方法。
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。
为了实现数字PCR的多重检测，常用的方法是针对每个靶标设计1种探针，通过不同探针的荧光不同，实现对不同靶标的检测。但探针种类太多，不仅成本高，且荧光背景高，降低了检测的灵敏性。
发明内容
为解决上述问题，本发明提供一种用于多种靶核酸检测的方法，包括如下内容：
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；所述扩增试剂包括核酸聚合酶和dNTPs；
将所述反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件，获得反应产物；
将反应产物置于n种不同的信号采集温度共进行n次信号采集，每种所述信号采集温度进行1次信号采集；每次信号采集包括至少1次信号通道采集；
分析同一信号通道下，相邻两次所述信号通道采集获得的信号是否存在差异，确定待测样本中存在或不存在靶核酸；和/或，
分析同一信号通道下，采用的信号采集温度最高的所述信号通道采集所获得的信号的有或无，确定待测样本中存在或不存在靶核酸；
其中，所述n为≥2的整数，且n≤靶核酸的种类数。
采用上述的检测方法，能够实现对多种靶核酸的检测(可以在数字PCR上实现至少8重反应)。多重检测的实现并不仅仅依赖荧光通道，而且在同一个荧光通道(即，同一信号通道)中利用不同熔解温度，通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
某些实施例中，每种信号采集温度进行1次信号采集；采用n种不同的信号采集温度，则进行了n次信号采集。某些实施例中，n即为信号采集的次数；n≤靶核酸的种类数，即，信号采集的次数≤靶核酸的种类数。
某些实施例中，所述同一信号通道下，相邻两次信号通道采集所获得的信号中所含的靶核酸种类数最多相差1种；某些实施例中，所述同一信号通道下，相邻两次信号通道采集获得的信号中所含的靶核酸种类数相差1种；某些实施例中，采用的信号采集温度最高的所述信号通道采集获得的信号中，最多含有1种靶核酸。某些实施例中，所述“1种靶核酸”还包括分类不分型的1类靶核酸。
某些实施例中，“信号采集”包括如下操作：将反应产物在第一温度下进行第一次信号采集，然后升温到第二温度下进行第二次信号采集，然后再继续升温到第三温度下进行第三次信号采集，然后再继续升温和信号采集，直到待测样本中的靶核酸全部被检出。也可以包括连续降温中进行信号采集的操作，例如，将反应产物在第一温度下进行第一次信号采集， 然后降温到第二温度下进行第二次信号采集，然后再继续降温到第三温度下进行第三次信号采集，然后再继续降温和信号采集，直到待测样本中的靶核酸全部被检出。
某些实施例中，引物探针组合物中只有1种探针(1种探针，是指一类修饰的检测标记或荧光基团相同，其碱基序列可以相同、也可以不同的1类探针)，则每次信号采集只包括1次信号通道采集，即，每次信号采集为在某个信号通道(荧光通道)下进行1次信号采集。此时，相邻两次信号通道采集即为相邻两次信号采集，相邻两次信号通道采集获得的信号中所含的靶核酸种类数相差1个，则信号采集的次数n等于靶核酸的种类数。例如，待测样本中含有3种靶核酸，在某个荧光通道下，将反应产物在第一温度下进行第一次信号采集(相当于信号通道采集)，获得的信号中含有3种靶标，然后升温到第二温度下进行第二次信号采集，获得的信号中含有2种靶标，然后再继续升温到第三温度下进行第三次信号采集，获得的信号中含有1种靶标。根据比对相邻两次信号采集的信号情况，即可确定靶核酸的存在情况。例如，相比第一次信号采集，第二次信号采集时，有部分信号消失，则消失的信号即为第一靶标；相比第二次信号采集，第三次信号采集时，有另一部分信号消失，则消失的另一部分信号即为第二靶标，第三次信号采集中依然存在的信号即为第三靶标，如此，即可将三种靶标是否存在全部确定出来。此外，还可以根据信号消失/存在的数量多少，确定每种靶标的含量。
某些实施例中，引物探针组合物中有2种探针(2种探针，是指修饰的检测标记或荧光基团不同，并且碱基序列不同的2类探针)，则至少有1次信号采集包括2次信号通道采集。其中，2次信号通道采集，即，在两个信号通道(荧光通道)下分别进行的1次信号采集。本领域技术人员根据引物探针组合物的设计情况，可以预期反应产物的熔解温度，并进一步预期代表靶核酸的反应产物产生可检测信号所需的温度、以及所在的荧光通道。因此，本领域技术人员可以根据引物探针组合物的设计情况，选择合适的信号采集温度、以及在合适的信号通道(荧光通道)进行信号采集。如此，在该实施例中，可在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，通过不同的信号采集温度，可以检测不同的靶标；或使用不同的荧光通道中，即可实现荧光通道数量与信号采集温度特征之乘积的靶标种类检测。
某些实施例中，所述扩增试剂还可以包括一些促进PCR反应的试剂，例如KCl、MgCl2、Tris-HCl、二硫苏糖醇(DTT)、(NH4)2SO4等。某些实施例中，所述扩增试剂除了含有具有聚合活性的聚合酶之外，还包括一些作用于核酸的其他酶，例如，核酸外切酶、核酸内切酶等。
某些实施例中，同一信号通道下，相邻两次信号通道采集，是指两次信号通道采集所采用的信号采集温度相近。例如，某些实施例中，在同一信号通道下，将反应产物置于第一温度下进行第一次信号通道采集，然后升温到第二温度下进行第二次信号通道采集，然后再继 续升温到第三温度下进行第三次信号通道采集，则，第一次信号通道采集和第二次信号通道采集为相邻两次信号通道采集，第二次信号通道采集和第三次信号通道采集为相邻两次信号通道采集，但是，第一次信号通道采集和第三次信号通道采集不为相邻两次信号通道采集。
某些实施例中相邻两次信号通道采集所采用的信号采集温度相差4℃以上，以实现同一信号通道下相邻两次信号通道采集获得的信号中所含的靶核酸种类数相差1个，避免相邻两次信号通道采集获得的靶核酸信号无差异的情况，从而减少信号通道采集的次数，简化操作步骤。
某些实施例中，信号采集温度为0-95℃。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。
某些实施例中，每次信号采集包括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。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，则存在信号采集温度相邻的两次信号采集获得的信号中所含的靶核酸种类数相差0个的情况。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。
在某些实施例中，上述多种靶核酸检测的方法为多种靶核酸的数字PCR检测方法。上述将反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件之前，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个待测样本的靶核酸或不含有待测样本的靶核酸。某些实施例中，所述信号采集为通过相机对荧光信号进行采集。某些实施例中，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。
某些实施例中，将引物探针组合物、待测样本(含有2种靶标)和扩增试剂混合，获得反应体系，然后将反应体系分配到500个以上的反应单元中，形成无数个小液滴，每个小液滴中最多含有1个待测样本的靶核酸，但含有适合核酸扩增所需的成分，例如，引物探针组合物和核酸聚合酶等扩增试剂。然后将所有小液滴置于允许核酸聚合酶进行杂交及延伸反应 的条件(某些实施例中，即，PCR扩增条件)，得到的反应产物(扩增产物)在第一温度下进行第一次拍照，有部分小液滴发光，获得2种靶标的阳性信号，然后升温到第二温度下进行第二次拍照，发现第一次拍照的小液滴中出现某些小液滴不发光、某些小液滴依然发光。则，第二次拍照中依然发光的小液滴为第一靶标，第二次拍照中不发光、但在第一次拍照中发光的小液滴为第二靶标。根据两次拍照所采用的温度和荧光通道，即可分析出2种靶标的具体类型，并通过分析第二次拍照中发光小液滴的数量，获得第一靶标的数量，分析第一次拍照和第二次拍照中发光小液滴的数量差值，即可得到第二靶标的数量。
某些实施例中，上述允许核酸聚合酶进行杂交及延伸反应的条件包括：：约85℃-约105℃预变性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。
某些实施例中，上述允许核酸聚合酶进行杂交及延伸反应的条件包括：85℃-105℃预变性0-15分钟；85℃-105℃变性2-60秒，40℃-75℃退火及延伸10-90秒，20-60个循环。某些实施例中，当待测样本中靶核酸含有RNA时，所述扩增试剂还包括逆转录酶，所述允许核酸聚合酶进行杂交及延伸反应的条件包括：30-65℃逆转录2-30分钟；85℃-105℃预变性0-15分钟；85℃-105℃变性2-60秒，40℃-75℃退火及延伸10-90秒，20-60个循环。
某些实施例中，用于多种靶核酸检测的方法中，上述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。
某些实施例中，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，不同的单链预产物与第一探针形成的双链产物不同，不同的双链产物的熔解温度不同。某些实施例中，不同的双链产物的熔解温度相差4℃以上。
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。
在该实施例中，针对某一靶核酸的引物组，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(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。
寡核苷酸的熔解温度，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。因此，本领域技术人员可根据实际情况，通过调整引物和/或探针的序列组成，使不同引物形成的单链预产物不同，不同的单链预产物与第一探针特异性结合的位置不同，从而形成的不同双链产物的熔解温度不同。也就是说，本领域技术人员可根据实际情况，使代表不同靶标的双链产物不同，而不同的双链产物具有不同熔解温度，从而使得不同信号采集温度下获得的不同靶核酸的信号。某些实施例中，可以通过对引物探针的设计，使得代表不同靶标的不同双链产物的熔解温度相差4℃以上。
某些实施例中，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。
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。某些实施例中，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。
某些实施例中，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。
某些实施例中，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，不同的单链预产物与第二探针形成的双链产物不同，不同的双链产物的熔解温度不同。某些实施例中，不同的双链产物的熔解温度相差4℃以上。
某些实施例中，所述引物探针组合物中，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。
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；
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。
“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。
某些实施例中，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，然后将所述反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件，获得反应产物，具体包括以下内容：
在允许核酸聚合酶进行杂交及延伸反应的条件下，进行PCR扩增，获得预产物：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)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’)；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’)；
将上述预产物置于允许核酸聚合酶进行杂交及延伸反应的条件(恒温孵育、或若干个高低温度循环的PCR扩增条件)，获得反应产物：此时，探针(P)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(P)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(P)形成的杂交双链产物(D1)，其熔解温度为T1，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(P)或靶序列的任意部分互补；此时探针(P)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(P)的部分或全部反向互补序列，即完成以单链预扩增产物(S2)为引物，以探针(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)，其熔解温度为T2，T2>T1；任选的，如果存在正向引物(F3)和反向引物(R3)，此时探针(P)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(P)的5’端，得到探针(P)的部分或全部反向互补序列，即完成以单链预扩增产物(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)，其熔解温度为T3，T3>T2。
某些实施例中，将反应产物置于n种不同的信号采集温度下进行n次信号采集，然后分析同一信号通道下，信号采集温度相邻的两次信号采集获得的信号是否存在有或无的差异，确定待测样本中存在或不存在靶核酸，具体包括以下内容：
在第一信号采集温度(t1，t1≤T1)下进行第一次信号采集，由于单链扩增产物S1，S2和S3分别与探针(P)形成了全部或部分双链结构D1，D2，D3，相比于PCR之前探针(P)的状态，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；再将温度升温至第二信号采集温度(t2，T1＜t2≤T2)下进行第二次信号采集，单链预扩增产物(S1)与探针(P)形成的扩增双链产物(D1)由于第二信号采集温度(t2)高于扩增双链产物(D1)的熔解温度而无法形成双链结构，没有荧光信号的产生，而单链预扩增产物(S2)与探针(P)形成的扩增双链产物(D2)及单链预扩增产物(S3)与探针(P)形成的扩增双链产物(D3)依旧是双链状态，可被仪器检测到荧光信号；任选的，如果存在正向引物(F3)和反向引物(R3)，再将温度升温至第三信号采集温度(t3，T2＜t3≤T3)下进行第三次信号采集，单链预扩增产物(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，没有荧光信号的产生，而单链预扩增产物(S3)与探针(P)形成的扩增双链产物(D3)依旧是双链状态，可被仪器检测到荧光信号。
某些实施例中，将反应产物置于n种不同的信号采集温度下进行n次信号采集，然后分析同一信号通道下，信号采集温度相邻的两次信号采集获得的信号是否存在有或无的差异，确定待测样本中存在或不存在靶核酸，具体包括以下内容：
任选的，如果存在正向引物(F3)和反向引物(R3)，在第一信号采集温度(t3，T2＜t3≤T3)下进行第一次信号采集，单链预扩增产物(S1)与探针(P)形成的扩增双链产物(D1)和单链预扩增产物(S2)与探针(P)形成的扩增双链产物(D2)由于第一信号采集温度(t3)高于扩增双链产物(D1和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(P)形成的扩增双链产物(D3)相比于PCR之前探针(P)的状态，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；再将温度降温至第二信号采集温度(t2，T1＜t2≤T2)下进行第二次信号采集，单链预扩增产物(S1)与探针(P)形成的扩增双链产物(D1)由于第二信号采集温度(t2)高于扩增双链产物(D1)的熔解温度而无法形成双链结构，没有荧光信号的产生，而单链预扩增产物(S2)与探针(P)形成的扩增双链产物(D2)，相比于PCR之前探针(P)的状态，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，单链预扩增产物(S3)与探针(P)形成的扩增双链产物(D3)依旧是双链状态，可被仪器检测到荧光信号；再将温度降温至第三信号采集温度(t1，t1≤T1)下进行第三次信号采集，单链预扩增产物(S1)与探针(P)形成的扩增双链产物(D1)， 相比于PCR之前探针(P)的状态，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，单链预扩增产物(S3)与探针(P)形成的扩增双链产物(D3)和单链预扩增产物(S2)与探针(P)形成的扩增双链产物(D2)依旧是双链状态，可被仪器检测到荧光信号。
本发明的第二方面，提供一种用于多种靶核酸检测的装置，包括：
反应液容纳部，用于容纳数个微液体，每个所述微液体中包含有反应试剂，部分所述微液体中还包含第一待测物或第二待测物中的一种；
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调节所述反应液容纳部中微液体的温度，使含有第一待测物或第二待测物的数个微液体同时产生信号；
所述控制部控制所述温度调节部调节所述微液体温度至t1，含有所述第一待测物的数个微液体和含有所述第二待测物的数个微液体产生信号，形成第一混合信号；
所述控制部控制所述温度调节部调节所述微液体温度至t2，仅含有所述第二待测物的数个微液体产生信号，形成第二信号；
其中，t1＜t2；所述控制部控制所述信号检测部在温度t1和t2时，采集所述微液体产生的信号，并输出所述第一混合信号和第二信号；
信号分析部，所述信号分析部根据所述信号检测部采集的所述第一混合信号和第二信号，计算出第一信号。
某些实施例中，所述控制部控制所述温度调节部调节所述反应液容纳部中微液体的温度至t3，包含有所述第一待测物的数个微液体、含有所述第二待测物的数个微液体和含有第三待测物的数个微液体产生信号，形成第二混合信号，其中，t3＜t1，所述t3和t1相差4℃以上。
某些实施例中，所述t1和t2相差4℃以上。
某些实施例中，所述t1和t2相差4℃。
某些实施例中，所述信号分析部将所述第一混合信号与所述第二信号相减，获得所述第一信号。
某些实施例中，在采集所述第一混合信号和第二信号时，数个所述微液体平铺在所述反应液容纳部底部，且始终保持相同的位置状态。
某些实施例中，所述第一信号为包含有所述第一待测物的微液体产生的荧光信号，所述第二信号为包含有所述第二待测物的微液体产生的荧光信号，所述第一混合信号为包含有所述第一待测物的微液体和包含有所述第二待测物的微液体产生的荧光信号。
某些实施例中，所述信号分析部将所述微液体产生的第一混合信号，减去在相同位置上包含有所述第二待测物的微液体产生的所述荧光信号，获得所述第一信号。
某些实施例中，所述信号分析部将所述微液体产生的第二混合信号，减去在相同位置上包含有所述第一待测物的微液体和含有所述第二待测物的微液体产生的所述第一混合信号，获得第三信号，所述第三信号为包含有所述第三待测物的微液体所产生的荧光信号。某些实施例中，每个所述微液体中包含有反应试剂，所述反应试剂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。
某些实施例中，所述反应试剂包括引物探针组合物和扩增试剂；所述扩增试剂包括核酸聚合酶和dNTPs；所述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；
某些实施例中，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，不同的单链预产物与第一探针形成的双链产物不同，不同的双链产物的熔解温度不同；某些实施例中，不同的双链产物的熔解温度相差4℃以上。
某些实施例中，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；
某些实施例中，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；
某些实施例中，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，不同的单链预产物与第二探针形成的双链产物不同，不同的双链产物的熔解温度不同；优选地，不同的双链产物的熔解温度相差4℃以上。
某些实施例中，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；
优选地，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；
优选地，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。
有益效果
本发明的引物探针组合物及数字PCR检测方法，与现有技术相比，其优点在于：
(1)方法简便：本发明所述方法可以在数字PCR上实现至少8重反应，并且通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间；
(2)多重检测：本发明所述方法可在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，通过不同的信号采集温度，可以检测不同的靶标；或使用不同的荧光通道中，即可实现荧光通道数量与信号采集温度特征之乘积的靶标种类检测；
(3)荧光背景低：每个荧光通道只使用1种探针，即可通过扩增产物的不同熔解温度进行区分，大大降低了PCR反应中的荧光背景，提高了反应灵敏度；
(4)熔解温度可调整：本发明所述方法是利用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，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)形成的二次扩增双链产物的熔解温度；
(5)包容性好：本发明所述引物探针设计方法与靶序列有两部分反向互补配对，即正向引物(F)和反向引物(R)，相比Taqman水解探针法需要三部分与模板反向互补配对，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)成本低：在试剂中减少了荧光探针的数量，大大降低了试剂成本，使得大规模推广成为可能；
(7)单管反应：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；
(8)操作简便：仅需使用数字PCR仪进行检测，无后续步骤如毛细管电泳或核酸杂交等，同时配制简单，上机后无需操作；
(9)不容易造成污染：本发明所述方法在加样完成后即完全封闭，无需开盖进行后续检测，大大降低了对实验环境造成污染的可能性；
(10)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；
(11)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。
附图说明
图1A为实施例1中在50℃采集的信号图；图1B为实施例1中在68℃采集的信号图；
图2A为实施例2中在50℃采集的信号图；图2B为实施例2中在68℃采集的信号图；图2C为实施例2中在77℃采集的信号图；
图3A为实施例3中在70℃采集的信号图；图3B为实施例3中在50℃采集的信号图；
图4A为实施例4中在77℃采集的信号图；图4B为实施例4中在70℃采集的信号图；图4C为实施例4中在50℃采集的信号图；
图5A是本申请一实施例公开的多种靶核酸检测装置的示意图；图5B是本申请又一实施例公开的多种靶核酸检测装置的示意图；图5C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。
具体实施方式
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，均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和数据分析，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KPC突变的双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 PCTCN2022142473-appb-000001
Figure PCTCN2022142473-appb-000002
上述引物探针中:
所述正向引物F1-1(SEQ ID NO:2)，反向引物R1-1(SEQ ID NO:3)，皆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，F1-1全长39bp，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13个碱基与探针P1(SEQ ID NO:1)的5’端的第1至第13个碱基序列反向互补；R1-1引物全长49bp，其3’端第1至第25个碱基为靶序列结合区，其5’端的第6至第21个碱基与探针探针P1(SEQ ID NO:1)的5’端的第16至第31个碱基序列相同，其5’端的第1至第5个碱基为扩增阻滞区。
所述正向引物F1-2(SEQ ID NO:4)，反向引物R1-2(SEQ ID NO:5)，皆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，F2全长37bp，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13个碱基与探针P1(SEQ ID NO:1)的5’端的第1至第13个碱基序列反向互补；R1-2引物全长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个碱基与探针探针P1(SEQ ID NO:1)的5’端的第32至第41个碱基序列完全相同。
(2)利用上述引物探针体系进行2种靶核酸的检测，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(F1-1) 500nM
反向引物(R1-1) 100nM
正向引物(F1-2) 500nM
反向引物(R1-2) 100nM
探针(P1) 400nM
耐碳青霉烯类抗生素的基因VIM突变核酸模板 2000拷贝
耐碳青霉烯类抗生素的基因KPC突变核酸模板 1000拷贝
超纯水 加至20μL
注：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
(3)检测时的具体操作步骤如下：
样本制备：同时使用耐碳青霉烯类抗生素的基因VIM突变的核酸模板和耐碳青霉烯类抗生素的基因KPC突变的核酸模板作为阳性样本进行检测。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：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(迈克生物股份有限公司的D600全自动数字PCR分析系统)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；50℃进行第一次信号采集；68℃进行第二次信号采集。
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1A为在第一信号采集温度(50℃)时的液滴图，可以看到有区别于背景的阳性液滴(亮点即为阳性液滴)；图1B为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，且阳性液滴数目相较于图1A减少了(在图1A中的部分阳性液滴，在图1B中的相应位置并无阳性液滴)。
图1B中依然存在的阳性液滴，即为耐碳青霉烯类抗生素的基因KPC突变阳性样本的信号，通过分析阳性液滴的个数，还可以确定基因KPC突变阳性样本的含量；在图1B中不存在、但在图1A中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因VIM突变样本的信号，通过分析图1A和图1B中阳性液滴的差值，还可以确定基因VIM突变阳性样本的含量。
从图1A和图1B可以看出，采用本发明的检测方法，能够实现对多种靶核酸的检测。多重检测的实现并不仅仅依赖荧光通道，而且在同一个荧光通道(即，同一信号通道)中利用不同熔解温度，通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
实施例2
(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KPC突变和耐碳青霉烯类抗生素的基因OXA-48突变的三重引物探针体系如表3所示：
表3实施例2引物探针的碱基序列
Figure PCTCN2022142473-appb-000003
上述引物探针中:
所述正向引物F2-1(SEQ ID NO:2)，反向引物R2-1(SEQ ID NO:3)，皆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，F2-1全长39bp，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13个碱基与探针P2(SEQ ID NO:1)的5’端的第1至第13个碱基序列反向互补；R2-1引物全长49bp，其3’端第1至第25个碱基为靶序列结合区，其5’端的第6至第21个碱基与探针探针P2(SEQ ID NO:1)的5’端的第16至第31个碱基序列相同，其5’端的第1至第5个碱基为扩增阻滞区。
所述正向引物F2-2(SEQ ID NO:4)，反向引物R2-2(SEQ ID NO:5)，皆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，F2-2全长37bp，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13个碱基与探针P2(SEQ ID NO:1)的5’端的第1至第13个碱基序列反向互补；R2-2引物全长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个碱基与探针探针P2(SEQ ID NO:1)的5’端的第32至第41个碱基序列完全相同。
所述正向引物F2-3(SEQ ID NO:6)，反向引物R2-3(SEQ ID NO:7)，皆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，F2-3全长35bp，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13个碱基与探针P2(SEQ ID NO:1)的5’端的第1至第13个碱基序列反向互补；R2-2引物全长37bp，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10个碱基与探针探针P2(SEQ ID NO:1)的3’端的第1至第10个碱基序列完全相同。
(2)利用上述引物探针体系进行3种靶核酸的检测，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(F2-1) 500nM
反向引物(R2-1) 100nM
正向引物(F2-2) 500nM
反向引物(R2-2) 100nM
正向引物(F2-3) 500nM
反向引物(R2-3) 100nM
探针(P2) 400nM
耐碳青霉烯类抗生素的基因VIM突变核酸模板 2400拷贝
耐碳青霉烯类抗生素的基因KPC突变核酸模板 80拷贝
耐碳青霉烯类抗生素的基因OXA-48突变核酸模板 800拷贝
超纯水 加至20μL
注：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
(3)检测时的具体操作步骤如下：
样本制备：同时使用耐碳青霉烯类抗生素的基因VIM突变的核酸模板，耐碳青霉烯类抗生素的基因KPC突变的核酸模板和耐碳青霉烯类抗生素的基因OXA-48突变的核酸模板作为阳性样本进行检测。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：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(迈克生物股份有限公司的D600全自动数字PCR分析系统)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；40℃恒温孵育3min；50℃进行第一次信号采集；68℃进行第二次信号采集；77℃进行第三次信号采集。
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为在第一信号采集温度(50℃)时的液滴图，可以看到有区别于背景的阳性液滴(亮点即为阳性液滴)；图2B为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，且阳性液滴数目相较于图2A减少了(在图2A中的部分阳性液滴，在图2B中的相应位置并无阳性液滴)；图2C为在第三信号采集温度(77℃)时的液滴图，可以看到有区别于背景的阳性液滴，且阳性液滴数目相较于图2B减少了(在图2B中的部分阳性液滴，在图2C中的相应位置并无阳性液滴)。
图2C中依然存在的阳性液滴，即为耐碳青霉烯类抗生素的基因OXA-48突变阳性样本的信号，通过分析阳性液滴的个数，还可以确定基因OXA-48突变阳性样本的含量；在图2C中不存在、但在图2B中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因KPC突变的信号，通过分析图2B和图2C中阳性液滴的差值，还可以确定基因KPC突变阳性样本的含量；在图2B中不存在、但在图2A中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因VIM突变样本的信号，通过分析图2A和图2B中阳性液滴的差值，还可以确定基因VIM突变阳性样本的含量。
从图2A-图2C可以看出，采用本发明的检测方法，能够实现对多种靶核酸的检测。多重检测的实现并不仅仅依赖荧光通道，而且在同一个荧光通道(即，同一信号通道)中利用不同熔解温度，通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
实施例3
(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5所示：
表5实施例3引物探针的碱基序列
Figure PCTCN2022142473-appb-000004
上述引物探针中：
所述正向引物F3-1(SEQ ID NO:9)，反向引物R3-1(SEQ ID NO:10)，皆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，F3-1全长39bp，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13个碱基与探针P3(SEQ ID NO:8)的5’端的第1至第13个碱基序列反向互补；R3-1引物全长49bp，其3’端第1至第25个碱基为靶序列结合区，其5’端的第6至第21个碱基与探针探针P3(SEQ ID NO:8)的5’端的第16至第31个碱基序列相同，其5’端的第1至第5个碱基为扩增阻滞区。
所述正向引物F3-2(SEQ ID NO:11)，反向引物R3-2(SEQ ID NO:12)，皆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，F3-2全长35bp，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13个碱基与探针P3(SEQ ID NO:8)的5’端的第1至第13个碱基序列反向互补；R3-2引物全长38bp，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11个碱基与探针探针P3(SEQ ID NO:8)的5’端的第32至第42个碱基序列完全相同。
(2)利用上述引物探针体系进行2种靶核酸的检测，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(F3-1) 500nM
反向引物(R3-1) 100nM
正向引物(F3-2) 500nM
反向引物(R3-2) 100nM
探针(P3) 400nM
耐碳青霉烯类抗生素的基因VIM突变核酸模板 10拷贝
耐碳青霉烯类抗生素的基因OXA-48突变核酸模板 100拷贝
超纯水 加至20μL
注：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
(3)检测时的具体操作步骤如下：
样本制备：同时使用耐碳青霉烯类抗生素的基因VIM突变的核酸模板和耐碳青霉烯类抗生素的基因OXA-48突变的核酸模板作为阳性样本进行检测。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：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(迈克生物股份有限公司的D600全自动数字PCR分析系统)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；52℃恒温3min；70℃进行第一次信号采集；50℃进行第二次信号采集。
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为在第一信号采集温度(70℃)时的液滴图，可以看到有区别于背景的阳性液滴(亮点即为阳性液滴)；图3B为在第二信号采集温度(50℃)时的液滴图，可以看到有区别于背景的阳性液滴，且阳性液滴数目相较于图3A增加了(在图3A中的部分阴性液滴，在图3B中的相应位置为阳性液滴)。
图3A中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因OXA-48突变阳性样本的信号，通过分析阳性液滴的个数，还可以确定基因OXA-48突变阳性样本的含量；在图3A中不存在、但在图3B中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因VIM突变样本的信号，通过分析图3A和图3B中阳性液滴的差值，还可以确定基因VIM突变阳性样本的含量。
从图3A和图3B可以看出，采用本发明的检测方法，能够实现对多种靶核酸的检测。多重检测的实现并不仅仅依赖荧光通道，而且在同一个荧光通道(即，同一信号通道)中利用不同熔解温度，通过有限次数的拍照，进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
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突变和耐碳青霉烯类抗生素的基因IMP突变的三重引物探针体系如表7所示：
表7实施例4引物探针的碱基序列
Figure PCTCN2022142473-appb-000005
Figure PCTCN2022142473-appb-000006
所述正向引物F4-1(SEQ ID NO:9)，反向引物R4-1(SEQ ID NO:10)，皆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，F4-1全长39bp，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13个碱基与探针P4(SEQ ID NO:18)的5’端的第1至第13个碱基序列反向互补；R4-1引物全长49bp，其3’端第1至第25个碱基为靶序列结合区，其5’端的第6至第21个碱基与探针探针P4(SEQ ID NO:8)的5’端的第16至第31个碱基序列相同，其5’端的第1至第5个碱基为扩增阻滞区。
所述正向引物F4-2(SEQ ID NO:11)，反向引物R4-2(SEQ ID NO:12)，皆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，F4-2全长35bp，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13个碱基与探针P4(SEQ ID NO:8)的5’端的第1至第13个碱基序列反向互补；R4-2引物全长38bp，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11个碱基与探针探针P4(SEQ ID NO:8)的5’端的第32至第42个碱基序列相同。
所述正向引物F4-3(SEQ ID NO:13)，反向引物R4-3(SEQ ID NO:14)，皆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，F4-3全长40bp，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13个碱基与探针P4(SEQ ID NO:8)的5’端的第1至第13个碱基序列反向互补；R4-3引物全长35bp，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9个碱基与探针探针P4(SEQ ID NO:8)的3’端的第1至第9个碱基序列相同。
(2)利用上述引物探针体系进行3种靶核酸的检测，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
试剂组分 浓度
2×PCR Reaction Buffer 1×
DNA Polymerase 2U
正向引物(F4-1) 500nM
反向引物(R4-1) 100nM
正向引物(F4-2) 500nM
反向引物(R4-2) 100nM
正向引物(F4-3) 500nM
反向引物(R4-3) 100nM
探针(P4) 400nM
耐碳青霉烯类抗生素的基因VIM突变核酸模板 10拷贝
耐碳青霉烯类抗生素的基因OXA-48突变核酸模板 15拷贝
耐碳青霉烯类抗生素的基因IMP突变核酸模板 100拷贝
超纯水 加至20μL
注：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
(3)检测时的具体操作步骤如下：
样本制备：同时使用耐碳青霉烯类抗生素的基因VIM突变的核酸模板，耐碳青霉烯类抗生素的基因OXA-48突变的核酸模板和耐碳青霉烯类抗生素的基因IPM突变的核酸模板作为阳性样本进行检测。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：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(迈克生物股份有限公司的D600全自动数字PCR分析系统)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；40℃恒温孵育3min；77℃进行第一次信号采集；70℃进行第二次信号采集；50℃进行第三次信号采集。
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为在第一信号采集温度(77℃)时的液滴图，可以看到有区别于背景的阳性液滴(亮点即为阳性液滴)；图4B为在第二信号采集温度(70℃)时的液滴图，可以看到有区别于背景的阳性液滴，且阳性液滴数目相较于图4A增加了(在图4A中的部分阴性液滴，在图4B中的相应位置为阳性液滴)；图4C为在第三信号采集温度(50℃)时的液滴图，可以看到有区别于背景的阳性液滴，且阳性液滴数目相较于图4B增加了(在图4B中的部分阴性液滴，在图4C中的相应位置为阳性液滴)
图4A中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因IMP突变阳性样本的信号，通过分析阳性液滴的个数，还可以确定基因IMP突变阳性样本的含量；在图4A中不存在、但在图4B中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因OXA-48突变样本的信号，通过分析图4A和图4B中阳性液滴的差值，还可以确定基因OXA-48突变阳性样本的含量；在图4B中不存在、但在图4C中存在的阳性液滴，即为耐碳青霉烯类抗生素的基因VIM突 变样本的信号，通过分析图4B和图4C中阳性液滴的差值，还可以确定基因VIM突变阳性样本的含量。
从图4A-图4B可以看出，采用本发明的检测方法，能够实现对多种靶核酸的检测。多重检测的实现并不仅仅依赖荧光通道，而且在同一个荧光通道(即，同一信号通道)中利用不同熔解温度，通过有限次数的拍照，进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
具体实施方式2
本申请实施例提供了一种用于对核酸进行多重检测的多种靶核酸检测装置，能够通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标，对于数字PCR的荧光识别要求低，操作简单，节省时间。
为了使本技术领域的人员更好地理解本申请方案，下面将结合本申请实施例中的附图，对本申请实施例中的技术方案进行描述，显然，所描述的实施例仅仅是本申请一部分的实施例，而不是全部的实施例。基于本申请中的实施例，都应当属于本申请保护的范围。
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，可以将第一待测物称为第二待测物，且类似地，可将第二待测物称为第一待测物。第一待测物和第二待测物两者都是待测物，但其不是同一个待测物。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。
本实施方式的多种靶核酸检测装置用于对待测样本进行检测，待测样本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，通过本实施方式的核酸检测装置可以实现对待测样本中的多种待测成分进行检测。
请参阅图5A和图5B本实施方式的多种靶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和信号分析部5。其中，反应液容纳部1用于容纳包含待测成分的待测样本和能够与待测成分反应的反应试剂，为待测样本与反应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，以使待测样本与反应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。其中，信号分析部5用于分析信号检测部3采集的信号。
Ⅰ、待测样本
待测样本为来自人体或者动物体的体液，待测样本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待测样本，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待测样本，也可以不执行核酸提取纯化操作。待测样本中包含不同的待测核酸分子，例如，第一待测物(第一待测靶核酸)、第二待测物(第二待测靶核酸)、第三待测物(第三待测靶核酸)、第四待测物(第四待测靶核酸)、第五待测物(第五待测靶核酸)等。
Ⅱ、反应试剂
反应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。某些实施例中，采用相同的标记物质标记不同的待测核酸。
某些实施例中，反应试剂包括引物探针组合物和扩增试剂：其中，扩增试剂包括核酸聚合酶和dNTPs；其中，引物探针组合物包括探针和引物混合物。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；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。某些实施例中，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，不同的单链预产物与第一探针形成的双链产物不同，不同的双链产物的熔解温度不同；某些实施例中，不同的双链产物的熔解温度相差4℃以上。
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反应试剂。例如，若待测样本中有两种待测靶核酸，即第一待测物、第二待测物，经过微分区处理后，每个第一待测物、每个第二待测物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物的需求，也可以满足扩增和标记微液体中第二待测物的需求。
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，通过将待测样本和反应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5A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5B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。
为了实现对待测靶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500个以上，例如，20000个左右的微液体中，每个微液体中含有一个或不含待检靶核酸分子，无论待测样本中有几种待测物，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靶核酸分子)。
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1C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5A和图5B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℃～105℃的范围内变化。如图5A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5B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5A所示的上方)。如图5B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5A和图5B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。
Ⅷ、信号分析部
如图所示，信号分析部5与信号检测部3可通讯连接。信号分析部用于根据信号检测部采集的信号，进行分析，获得待测靶核酸的信号，从而获得待测样本中是否含有待测靶核酸、含有的待测靶核酸的种类及数量等信息。
实施例1
该实施例示例性地描述了二重检测的装置及方法：待测样本中包含第一待测物(第一待测靶核酸)和第二待测物(第二待测靶核酸)，需要说明的是，由第一待测物产生的第一待测关联产物(待测靶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)，可与试剂中的标记物质结合；由第二待测物产生的第二待测关联产物，也可与试剂中的标记物质结合。本实施例中，在某一温度模式下，第一待测物产生第一待测关联产物，并与标记物质结合，同时，在该温度模式下，第二待测物产生第二待测关联产物，并与标记物质结合。但是，两者的分离温度不同：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t1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t2时能够与标记物质分离，其中温度t2高于温度t1。
为了实现对待测样本中的不同待测核酸进行多重检测，并且能够通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标(靶核酸)，本实施例中，将待测样本和反应试剂(包含引物探针组合物和扩增试剂)的混合液加入反应液容纳部形成数个微液体，控制部执行以下控制：
控制部控制温度调节部调节反应液容纳部中微液体的温度，使待测样本中的待测靶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，双链核酸变成单链核酸，然后微液体达到低温状态，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，待测样本中的不同待测靶核酸和/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靶核酸和/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，待测核酸和/或待测关联产物的数量增加，并与试剂中的标记物质结合，使含有第一待测物或第二待测物的数个微液体同时产生信号。
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(约2-5分钟)。预变性一段时间，是为了将靶核酸从双链状态变成单链状态。
40个循环完成后，控制部控制温度调节部调节所述微液体温度至t1，含有所述第一待测物的数个微液体和含有所述第二待测物的数个微液体产生信号，形成第一混合信号。在温度t1时，第二待测物或第二待测物的关联产物与标记物处于结合状态，产生信号，并且，第一待测物或第一待测物的关联产物与标记物也处于结合状态，同时产生信号。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，也可能是第二待测核酸，在温度t1时，含有第一待测核酸的微液体和含有第二待测核酸的微液体都能够产生信号。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。
接着，控制部控制所述温度调节部调节所述微液体温度至t2(t2＞t1)，仅含有所述第二待测物的数个微液体产生信号，形成第二信号。在温度t2时，第一待测物或第一待测物的关联产物与标记物分离，因此不产生可检测的信号，但第二待测物或第二待测物的关联产物与标记物仍然处于结合状态，产生信号。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，也可能是第二待测核酸，在温度t2时，仅第二待测核酸所在的数个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。
为更好地区分第一混合信号和第二信号，t1和t2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℃、4-5℃或4℃等。
为获得更准确的第一信号，在采集所述第一混合信号和第二信号时，数个所述微液体平 铺在所述反应液容纳部底部，且始终保持相同的位置状态。
通过控制部的上述控制，可以实现信号检测部检测微液体中产生的信号时，相邻两次信号采集所获得的信号中所含的靶核酸种类数相差1种(即，第一混合信号中含有第一待测物和第二待测物的信号，但第二信号中仅含有第一待测物的信号)。
为了获得待测样本中靶核酸的种类及数量，实现对待测样本中的不同待测核酸进行多重检测，信号分析部进行以下操作：
根据所述信号检测部采集的所述第一混合信号和第二信号，计算出第一信号。信号分析部将所述第一混合信号与所述第二信号相减，即可获得所述第一信号。具体地，信号分析部将所述微液体产生的第一混合信号，减去在相同位置上包含有所述第二待测物的微液体产生的所述荧光信号，获得所述第一信号。其中，第一信号为包含有所述第一待测物的微液体产生的荧光信号，所述第二信号为包含有所述第二待测物的微液体产生的荧光信号，所述第一混合信号为包含有所述第一待测物的微液体和包含有所述第二待测物的微液体产生的荧光信号。
根据所述信号检测部采集的第二信号，可直接获得包含有第二待测物的微液体产生的荧光信号。
由于单个待测核酸分子被分配到一个微液体中，即一个微液体中最多有一个待测核酸分子(有些微液体中不含有待测核酸分子，则在每个信号采集温度下均不产生信号)，这个待测核酸分子可能是第一待测核酸，也可能是第二待测核酸。通过分析第一混合信号中产生信号的微液体的数量，即得第一待测物和第二待测物的总数量；分析第二信号中产生信号的微液体的数量，即得第二待测物的数量；第一混合信号中第一待测物和第二待测物的总数量，减去第二信号中第二待测物的数量，即得第一待测物的数量。
实施例2
该实施例示例性地描述了三重检测的装置及方法：待测样本中包含第一待测物(第一待测靶核酸)、第二待测物(第二待测靶核酸)和第三待测物(第三待测靶核酸)。同样的，由第一待测物产生的第一待测关联产物(待测靶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)，可与试剂中的标记物质结合；由第二待测物产生的第二待测关联产物，可与试剂中的标记物质结合，由第三待测物产生的第三待测关联产物，可与试剂中的标记物质结合。本实施例中，在某一温度模式下，第一待测物产生第一待测关联产物，并与标记物质结合，同时，在该温度模式下，第二待测物产生第二待测关联产物，并与标记物质结合，第三待测物产生第三待测关联产物，并与标记物质结合。但是，三者的分离温度不同：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，第三待测关联产物可在第三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、第二分离温度范围和第三分离温度范围的最低温度各不同。本实施例中，第三分离温度范围的最低温度低于第一分离温度范围的最低温度，第一分离温度范围的最低温度低于第二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t1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t2时能够与标记物质分离，第三待测关联产物在温度高于或等于温度t3时能够与标记物质分离，其中，温度t3＜t1＜t2。
为了实现对待测样本中的不同待测核酸进行三重检测，并且能够通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标(靶核酸)，本实施例中，将待测样本和反应试剂(包含引物探针组合物和扩增试剂)的混合液加入反应液容纳部形成数个微液体，控制部执行以下控制：
控制部控制温度调节部调节反应液容纳部中微液体的温度，使待测样本中的待测靶核酸产生与之关联的待测关联产物(待测靶核酸包含第一待测物、第二待测物和第三待测物，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，双链核酸变成单链核酸，然后微液体达到低温状态，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，待测样本中的不同待测靶核酸和/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靶核酸和/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，待测核酸和/或待测关联产物的数量增加，并与试剂中的标记物质结合，使含有第一待测物或第二待测物或第三待测物的数个微液体同时产生信号。
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(约2-5分钟)。预变性一段时间，是为了将靶核酸从双链状态变成单链状态。
40个循环完成后，控制部控制温度调节部调节所述微液体温度至t3，含有所述第一待测物的数个微液体、含有所述第二待测物的数个微液体和含有所述第三待测物的数个微液体，均产生信号，形成第二混合信号。在温度t3时，第二待测物或第二待测物的关联产物与标记物处于结合状态，产生信号，并且，第一待测物或第一待测物的关联产物与标记物也处于结合状态，同时产生信号，第三待测物或第三待测物的关联产物与标记物也处于结合状态，也产生信号。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，也可能是第二待测核酸或第三待测核酸，在温度t3时，含有第一待测核酸的微液体、含有第二待测核酸的微液体和含有第三待测核酸的微液体都能够产生信号。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。
接着，控制部控制温度调节部调节所述微液体温度至t1(t1＞t3)，含有所述第一待测物的数个微液体和含有所述第二待测物的数个微液体产生信号，形成第一混合信号。在温度t1时，第二待测物或第二待测物的关联产物与标记物处于结合状态，产生信号，并且，第一待测物或第一待测物的关联产物与标记物也处于结合状态，同时产生信号，但是，第三待测物或第三待测物的关联产物与标记物也处于分离状态，不产生可检测的信号。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，也可能是第二待测核酸，在温度t1时，含有第一待测核酸的微液体和含有第二待测核酸的微液体都能够产生信号。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。
接着，控制部控制所述温度调节部调节所述微液体温度至t2(t2＞t1)，仅含有所述第二待测物的数个微液体产生信号，形成第二信号。在温度t2时，第一待测物或第一待测物的关联产物与标记物分离，第三待测物或第三待测物的关联产物与标记物分离，因此，均不产生可检测的信号，但第二待测物或第二待测物的关联产物与标记物仍然处于结合状态，产生信号。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，在温度t2时，仅第二待测核酸所在的数个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。
为更好地区分第二混合信号、第一混合信号和第二信号，t1和t2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℃、4-5℃或4℃等。t1和t3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℃、4-5℃或4℃等。
为获得更准确的第一信号和第三信号，在采集所述第二混合信号、第一混合信号和第二信号时，数个所述微液体平铺在所述反应液容纳部底部，且始终保持相同的位置状态。
通过控制部的上述控制，可以实现信号检测部检测微液体中产生的信号时，相邻两次信号采集所获得的信号中所含的靶核酸种类数相差1种(即，第二混合信号中含有第一待测物、第二待测物和第三待测物的信号，第一混合信号中含有第一待测物和第二待测物的信号，但第二信号中仅含有第一待测物的信号)。
为了获得待测样本中靶核酸的种类及数量，实现对待测样本中的不同待测核酸进行多重检测，信号分析部进行以下操作：
根据所述信号检测部采集的所述第一混合信号和第二混合信号，计算出第三信号。信号分析部将所述第二混合信号与所述第一混合信号相减，即可获得所述第三信号。具体地，信号分析部将所述微液体产生的第二混合信号，减去在相同位置上包含有所述第一待测物的微液体和含有所述第二待测物的微液体产生的所述第一混合信号，获得第三信号。其中，所述第二混合信号为包含有所述第一待测物的微液体、包含有所述第二待测物的微液体和包含有 所述第三待测物的微液体产生的荧光信号；所述第一混合信号为包含有所述第一待测物的微液体和包含有所述第二待测物的微液体产生的荧光信号；第三信号为包含有所述第三待测物的微液体产生的荧光信号；
根据所述信号检测部采集的所述第一混合信号和第二信号，计算出第一信号。信号分析部将所述第一混合信号与所述第二信号相减，即可获得所述第一信号。具体地，信号分析部将所述微液体产生的第一混合信号，减去在相同位置上包含有所述第二待测物的微液体产生的所述荧光信号，获得所述第一信号。其中，第一信号为包含有所述第一待测物的微液体产生的荧光信号，所述第二信号为包含有所述第二待测物的微液体产生的荧光信号，所述第一混合信号为包含有所述第一待测物的微液体和包含有所述第二待测物的微液体产生的荧光信号；
根据所述信号检测部采集的第二信号，可直接获得包含有第二待测物的微液体产生的荧光信号；
由于单个待测核酸分子被分配到一个微液体中，即一个微液体中最多有一个待测核酸分子(有些微液体中不含有待测核酸分子，则在每个信号采集温度下均不产生可检测的信号)，这个待测核酸分子可能是第一待测核酸，也可能是第二待测核酸或第三待测核酸。通过分析第二混合信号中产生信号的微液体的数量，即得第一待测物、第二待测物和第三待测物的总数量；分析第一混合信号中产生信号的微液体的数量，即得第一待测物和第二待测物的总数量；分析第二信号中产生信号的微液体的数量，即得第二待测物的数量；第二混合信号中第一待测物、第二待测物和第三待测物的总数量，减去第一混合信号中第一待测物和第二待测物的总数量，即得第三待测物的数量；第一混合信号中第一待测物和第二待测物的总数量，减去第二信号中第二待测物的数量，即得第一待测物的数量。
实施例3
该实施例示例性地描述了二重检测的装置及方法：待测样本中包含第四待测物(第四待测靶核酸)和第五待测物(第五待测靶核酸)，需要说明的是，由第四待测物产生的第四待测关联产物(待测靶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)，可与试剂中的标记物质结合；同样地，由第五待测物产生的第五待测关联产物，也可与试剂中的标记物质结合。本实施例中，在某一温度模式下，第四待测物产生第四待测关联产物，并与标记物质结合，同时，在该温度模式下，第五待测物产生第五待测关联产物，并与标记物质结合。但是，两者的分离温度不同：第四待测关联产物可在第四分离温度范围内与试剂中的标记物质分离，第五待测关联产物可在第五分离温度范围内与试剂中的标记物质分离，第四分离温度范围和第四分离温度范围的最低温度不同。本实施例中，第四分离温度范围的最低温度高于第五分离温度范围的最低温度。例如，第四待测关联产物在温度高于或等于温度t4时能够与标记物 质分离，第五待测关联产物在温度高于或等于温度t5时能够与标记物质分离，其中温度t4高于温度t5。
为了实现对待测样本中的不同待测核酸进行多重检测，并且能够通过有限次数的拍照进行荧光的有或无的区分即可区别不同靶标(靶核酸)，本实施例中，将待测样本和反应试剂(包含引物探针组合物和扩增试剂)的混合液加入反应液容纳部形成数个微液体，控制部执行以下控制：
控制部控制温度调节部调节反应液容纳部中微液体的温度，使待测样本中的待测靶核酸产生与之关联的待测关联产物(待测核酸包含第四待测核酸、第五待测核酸，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，双链核酸变成单链核酸，然后微液体达到低温状态，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，待测样本中的不同待测靶核酸和/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靶核酸和/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，待测核酸和/或待测关联产物的数量增加，并与试剂中的标记物质结合，使含有第四待测物或第五待测物的数个微液体同时产生信号。
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(约2-5分钟)。预变性一段时间，是为了将靶核酸从双链状态变成单链状态。
40个循环完成后，控制部控制所述温度调节部调节所述微液体温度至t4，仅含有所述第四待测物的数个微液体产生信号，形成第四信号。在温度t4时，第五待测物或第五待测物的关联产物与标记物分离，因此不产生可检测的信号，但第四待测物或第四待测物的关联产物与标记物仍然处于结合状态，产生信号。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四待测核酸，也可能是第五待测核酸，在温度t4时，仅第四待测核酸所在的数个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。
接着，控制部控制温度调节部调节所述微液体温度至t5(t5＜t4)，含有所述第四待测物的数个微液体和含有所述第五待测物的数个微液体产生信号，形成第三混合信号。在温度t5时，第四待测物或第四待测物的关联产物与标记物处于结合状态，产生信号，并且，第五待测物或第五待测物的关联产物与标记物也处于结合状态，同时产生信号。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四待测核酸，也可能是第五待测核酸，在温度t5时，含有第四待测核酸的微液体和 含有第五待测核酸的微液体都能够产生信号。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。
为更好地区分第四信号和第三混合信号，t4和t5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℃、4-5℃或4℃等。
为获得更准确的第五信号，在采集所述第四信号和第三混合信号时，数个所述微液体平铺在所述反应液容纳部底部，且始终保持相同的位置状态。
通过控制部的上述控制，可以实现信号检测部检测微液体中产生的信号时，相邻两次信号采集所获得的信号中所含的靶核酸种类数相差1种(即，第四信号中仅含有第四待测物的信号，而第三混合信号中含有第四待测物和第五待测物的信号)。
为了获得待测样本中靶核酸的种类及数量，实现对待测样本中的不同待测核酸进行多重检测，信号分析部进行以下操作：
根据所述信号检测部采集的所述第四信号和第三混合信号，计算出第五信号。信号分析部将所述第三混合信号与所述第四信号相减，即可获得所述第五信号。具体地，信号分析部将所述微液体产生的第三混合信号，减去在相同位置上包含有所述第四待测物的微液体产生的所述荧光信号，获得所述第五信号。其中，第四信号为包含有所述第四待测物的微液体产生的荧光信号，所述第五信号为包含有所述第五待测物的微液体产生的荧光信号，所述第三混合信号为包含有所述第四待测物的微液体和包含有所述第五待测物的微液体产生的荧光信号；
根据所述信号检测部采集的第四信号，可直接获得包含有第四待测物的微液体产生的荧光信号；
由于单个待测核酸分子被分配到一个微液体中，即一个微液体中最多有一个待测核酸分子(有些微液体中不含有待测核酸分子，则在每个信号采集温度下均不产生信号)，这个待测核酸分子可能是第四待测核酸，也可能是第五待测核酸。通过分析第三混合信号中产生信号的微液体的数量，即得第四待测物和第五待测物的总数量；分析第四信号中产生信号的微液体的数量，即得第四待测物的数量；第三混合信号中第四待测物和第五待测物的总数量，减去第四信号中第四待测物的数量，即得第五待测物的数量。
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省时、高效的四重核酸检测(对待测样本中的4种靶核酸进行检测)、五重核酸检测(对待测样本中的5种靶核酸进行检测)、六重核酸检测(对待测样本中的6种靶核酸进行检测)或更多重的核酸检测等。
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用于多种靶核酸检测的装置、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 (18)

  1. 一种用于多种靶核酸检测的方法，其特征在于，包括如下内容：
    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；所述扩增试剂包括核酸聚合酶和dNTPs；
    将所述反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件，获得反应产物；
    将反应产物置于n种不同的信号采集温度共进行n次信号采集，每种所述信号采集温度进行1次信号采集；每次信号采集包括至少1次信号通道采集；
    分析同一信号通道下，相邻两次所述信号通道采集所获得的信号是否存在差异，确定待测样本中存在或不存在靶核酸；和/或，
    分析同一信号通道下，采用的信号采集温度最高的所述信号通道采集所获得的信号的有或无，确定待测样本中存在或不存在靶核酸；
    其中，所述n为≥2的整数，且所述n≤靶核酸的种类数。
  2. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述同一信号通道下，相邻两次信号通道采集所获得的信号中所含的靶核酸种类数最多相差1种；优选地，所述同一信号通道下，相邻两次信号通道采集获得的信号中所含的靶核酸种类数相差1种；优选地，采用的信号采集温度最高的所述信号通道采集获得的信号中，最多含有1种靶核酸。
  3. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述同一信号通道下，相邻两次信号通道采集所采用的信号采集温度相差4℃以上。
  4. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述每次信号采集包括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。
  5. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述将反应体系置于允许核酸聚合酶进行杂交及延伸反应的条件之前，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个待测样本的靶核酸或不含有待测样本的靶核酸；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。
  6. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述允许核酸聚合酶进行杂交及延伸反应的条件包括：约85℃-约105℃预变性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。
  7. 根据权利要求1所述的用于多种靶核酸检测的方法，其特征在于，所述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；
    优选地，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，不同的单链预产物与第一探针形成的双链产物不同，不同的双链产物的熔解温度不同；优选地，不同的双链产物的熔解温度相差4℃以上。
  8. 根据权利要求6所述的用于多种靶核酸检测的方法，其特征在于，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；
    优选地，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，不同的单链预产物与第二探针形成的双链产物不同，不同的双链产物的熔解温度不同；优选地，不同的双链产物的熔解温度相差4℃以上。
  9. 根据权利要求7或8所述的用于多种靶核酸检测的方法，其特征在于，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；
    优选地，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；
    优选地，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。
  10. 一种用于多种靶核酸检测的装置，包括：
    反应液容纳部，用于容纳数个微液体，每个所述微液体中包含有反应试剂，部分所述微液体中还包含第一待测物或第二待测物中的一种；
    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调节所述反应液容纳部中微液体的温度，使含有第一待测物或第二待测物的数个微液体同时产生信号；
    所述控制部控制所述温度调节部调节所述微液体温度至t1，含有所述第一待测物的数个微液体和含有所述第二待测物的数个微液体产生信号，形成第一混合信号；
    所述控制部控制所述温度调节部调节所述微液体温度至t2，仅含有所述第二待测物的数个微液体产生信号，形成第二信号；
    其中，t1＜t2；所述控制部控制所述信号检测部在温度t1和t2时，采集所述微液体产生的信号，并输出所述第一混合信号和第二信号；
    信号分析部，所述信号分析部根据所述信号检测部采集的所述第一混合信号和第二信号，计算出第一信号。
  11. 根据权利要求10所述的用于多种靶核酸检测的装置，其特征在于，所述控制部控制所述温度调节部调节所述反应液容纳部中微液体的温度至t3，包含有所述第一待测物的数个微液体、含有所述第二待测物的数个微液体和含有第三待测物的数个微液体产生信号，形成第二混合信号，其中，t3＜t1，所述t3和t1相差4℃以上。
  12. 根据权利要求10或11所述的用于多种靶核酸检测的装置，其特征在于，所述t1和t2相差4℃以上。
  13. 根据权利要求10或11所述的用于多种靶核酸检测的装置，其特征在于，所述t1和t2相差4℃。
  14. 根据权利要求10或11所述的用于多种靶核酸检测的装置，其特征在于，所述信号分析部将所述第一混合信号与所述第二信号相减，获得所述第一信号。
  15. 根据权利要求10或11所述的用于多种靶核酸检测的装置，其特征在于，在采集所述第一混合信号和第二信号时，数个所述微液体平铺在所述反应液容纳部底部，且始终保持相同的位置状态。
  16. 根据权利要求14或15所述的用于多种靶核酸检测的装置，其特征在于，所述第一信号为包含有所述第一待测物的微液体产生的荧光信号，所述第二信号为包含有所述第二待测物的微液体产生的荧光信号，所述第一混合信号为包含有所述第一待测物的微液体和包含有所述第二待测物的微液体产生的荧光信号。
  17. 根据权利要求16所述的用于多种靶核酸检测的装置，其特征在于，所述信号分析部将所述微液体产生的第一混合信号，减去在相同位置上包含有所述第二待测物的微液体产生的所述荧光信号，获得所述第一信号。
  18. 根据权利要求11所述的用于多种靶核酸检测的装置，其特征在于，所述信号分析部将所述微液体产生的第二混合信号，减去在相同位置上包含有所述第一待测物的微液体和含有所述第二待测物的微液体产生的所述第一混合信号，获得第三信号，所述第三信号为包含有所述第三待测物的微液体所产生的荧光信号。
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