WO2025009802A1 - Procédé de pcr pour la détection d'au moins deux acides nucléiques multi-cibles avec un signal unique - Google Patents

Procédé de pcr pour la détection d'au moins deux acides nucléiques multi-cibles avec un signal unique Download PDF

Info

Publication number
WO2025009802A1
WO2025009802A1 PCT/KR2024/008885 KR2024008885W WO2025009802A1 WO 2025009802 A1 WO2025009802 A1 WO 2025009802A1 KR 2024008885 W KR2024008885 W KR 2024008885W WO 2025009802 A1 WO2025009802 A1 WO 2025009802A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
target nucleic
probe
sequence
signal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2024/008885
Other languages
English (en)
Korean (ko)
Inventor
김수옥
김석준
홍선표
조애리
구자현
양승민
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genematrix Inc
Original Assignee
Genematrix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genematrix Inc filed Critical Genematrix Inc
Publication of WO2025009802A1 publication Critical patent/WO2025009802A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the present invention relates to a multiplex detection PCR method for detecting two or more target nucleic acids with a single signal.
  • PCR Polymerase chain reaction
  • Real-time polymerase chain reaction is a technology that detects target nucleic acids simultaneously with amplification, unlike the technology that analyzes the amplified product by electrophoresis after conventional PCR amplification. This allows for more accurate quantitative analysis by accurately knowing the initial concentration of the target nucleic acid, and since amplification and detection are performed in a sealed reaction tube, the possibility of contamination is low, resulting in high detection accuracy and reliability.
  • Real-time polymerase chain reaction uses intercalators such as ethidium bromide, SYBR Green I, and EvaGreen to detect amplification products simultaneously with PCR amplification, or uses hydrolyzable probes such as TaqMan probes (U.S. Patent Nos.
  • hybridization probes such as molecular beacons (Tyagi et al, Nature Biotechnology v14 MARCH 1996), HyBeacons (French DJ et al, Mol Cell Probes, 15(6):363-374, 2001), or two hybridization probes each labeled with a donor and an acceptor (Bernad et al, 147-148 Clin Chem 2000; 46), or Labeled primers such as Sunrise primer (US Patent No. 6,117,635), Scorpion primer (US Patent No. 6,326,145), and TSG primer (WO 2011/078441) are used.
  • Sunrise primer US Patent No. 6,117,635
  • Scorpion primer US Patent No. 6,326,145
  • TSG primer WO 2011/078441
  • intercalators Since intercalators have the property of intercalating into all DNA double strands, melting curve analysis is required after PCR amplification to increase specificity or multiplex detection of target nucleic acids. However, in the case of probe or labeled primer methods, since the probe binds to the target nucleic acid in a sequence-specific manner to generate a signal, not only is the specificity high, but melting curve analysis for multiplex detection of target nucleic acids is not necessarily required.
  • Multiplex detection of target nucleic acids using real-time polymerase chain reaction in the probe mode can simultaneously detect various genes in a single sample with high accuracy, but it requires the use of multiplex probes labeled with different signal molecules as many as the number of target nucleic acids, and the ability to distinguish and detect signals generated from such multiplex probes.
  • signal molecules use fluorescent molecules.
  • the types of fluorescent molecules that can be used are limited by wavelength overlap, that is, only one single fluorescent signal can be detected in one fluorescence detection channel, the number of target nucleic acids that can be detected simultaneously in one reaction tube is limited to 4 to 7.
  • Melting curve analysis is an analytical method that detects two or more target nucleic acids by measuring the change in fluorescence while increasing or decreasing the temperature after PCR reaction using primers or hybridization probes labeled with a single fluorescent molecule.
  • Primers or probes are designed to have a unique melting temperature with the target nucleic acids by controlling the GC content or length, so that a unique melting curve profile is obtained depending on the target nucleic acid.
  • this melting curve analysis requires designing the probe to have a unique melting temperature depending on the target nucleic acid, and detection of multiple target nucleic acids is possible through temperature changes only after the PCR reaction is completed.
  • the present invention also discloses a multiplex detection PCR method capable of real-time detection of two or more target nucleic acids in a single detection channel without requiring data processing such as increase or decrease of signal intensity.
  • An object of the present invention is to provide a PCR method for real-time detection of two or more target nucleic acids in a single detection channel.
  • the PCR method for real-time detection of two or more target nucleic acids in a single detection channel of the present invention detects signals for each target nucleic acid in the annealing step and the extension step among the three steps of PCR, namely, the denaturation step, the annealing step, and the extension step. That is, among the two target nucleic acids, a signal for a first target nucleic acid is detected in the annealing step, and a signal for a second target nucleic acid is detected in the extension step.
  • Each of the detected signals provides qualitative and/or quantitative information for each of the two target nucleic acids.
  • the detection signal is the same single signal for both target nucleic acids, which can be detected in one detection channel.
  • This method of the present invention is possible by using first and second hybridization probes and one quencher probe, which have a structure in which a signal substance and a quencher substance are separated when bound to a target nucleic acid to generate a signal.
  • the first and second hybridization probes have a melting temperature with respect to their target nucleic acid such that the first hybridization probe is between the temperature of the annealing step and the temperature of the extension step, and the other second hybridization probe is between the temperature of the extension step and the temperature of the denaturation step.
  • the quencher probe can quench the second hybridization probe, which has a melting temperature between the temperature of the extension step and the temperature of the denaturation step.
  • the melting temperature of the quencher probe with respect to the second hybridization probe is between the temperature of the annealing step and the temperature of the extension step.
  • the PCR method of the present invention which enables real-time detection of two target nucleic acids in a single detection channel during a PCR process using a single signal, specifically comprises the following steps (a) to (c):
  • a pair of forward and reverse primers for amplifying a first target nucleic acid wherein one of the primer pairs sequentially includes a random nucleic acid sequence that is non-complementary to the first target nucleic acid, a restriction enzyme recognition sequence, and a nucleic acid sequence that is complementary to the first target nucleic acid,
  • a first hybridization probe having a complementary sequence specific to a first artificial target nucleic acid generated depending on a first target nucleic acid among two or more target nucleic acids, wherein the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the artificial target nucleic acid does not bind to the probe, and a melting temperature at which the first hybridization probe dissociates from the artificial target nucleic acid complementary thereto is between the temperature of the annealing step and the temperature of the extension step among a relatively high temperature denaturation step, a relatively low temperature annealing step, and a relatively medium temperature extension step of a PCR reaction, such that in the annealing step, the signal substance and the quencher are separated by binding to its complementary sequence (the first hybridization probe) and a signal is generated, but in the extension step, the signal substance and the quencher are adjacent by dissociating from its complementary sequence (the first hybridization probe) and the signal can be extingu
  • a second hybridization probe having a complementary sequence specific to a second artificial target nucleic acid generated depending on a second target nucleic acid among two or more target nucleic acids, wherein the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the second artificial target nucleic acid does not bind to the probe, and has a single-stranded sequence extended in a terminal direction from the sequence to which the signal substance is bound, and further, a melting temperature at which it (the second hybridization probe) dissociates from the second artificial target nucleic acid complementary thereto is between the temperature of the extension step and the temperature of the denaturation step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of the PCR reaction, such that the second hybridization probe can maintain a binding state with the second artificial target nucleic acid complementary thereto in the annealing step and the extension step; and
  • step (b) placing the mixture of step (a) into a single reaction vessel and performing two or more cycles of PCR reaction of denaturation at a relatively high temperature, hybridization at a relatively low temperature, and extension at a relatively medium temperature;
  • the target nucleic acid means an actual target nucleic acid in a sample or an artificial target nucleic acid generated depending on the actual target nucleic acid, depending on the context; however, unless otherwise specifically stated, the target nucleic acid means an actual target nucleic acid in a sample.
  • PCR method of the present invention three probes, i.e., first and second hybridization probes and one quencher probe, as shown in Fig. 1 are used.
  • Both the first and second hybridization probes have a signal molecule (R) and a quencher (Q), and the signal molecule and the quencher have a structure in which, when the hybridization probe does not bind to a target nucleic acid, they are adjacent to each other and no signal is generated, but when the hybridization probe binds to a target nucleic acid, the signal molecule and the quencher are spaced apart and a signal is generated.
  • the structure may include a random coil structure or a stem-loop structure.
  • a hybridization probe having a stem-loop structure has a complementary sequence in the loop region that specifically binds to a target nucleic acid.
  • the second hybridization probe has a single-strand sequence (Extended single strand, ESS) that extends in the terminal direction (i.e., in the opposite direction of the loop structure in the stem-loop structure) from the position where the signal substance is bound.
  • ESS Extended single strand
  • the quenching probe is a probe having a quencher to quench the signal of the second hybridization probe, and has a structure that has a sequence complementary to the extended single-stranded sequence (ESS) of the second hybridization probe and can specifically bind thereto.
  • the quencher is present at a position where, when the quenching probe complementarily binds to the extended single-stranded sequence (ESS) of the second hybridization probe, the quencher of the quenching probe can quench the signal substance of the second hybridization probe by being adjacent to the signal substance.
  • the quencher of the quenching probe will be located closer to the 3' end or the 3' end of the quenching probe if the signal molecule of the second hybridizable probe is located closer to the 5' end than the 3' end (in this case, the quencher of the second hybridizable probe is located closer to the 3' end or the 3' end), and will be located closer to the 5' end or the 5' end of the quenching probe if the signal molecule of the second hybridizable probe is located closer to the 3' end than the 5' end.
  • the signal molecule or quencher being located closer to the 5' end or the 3' end means that the signal molecule or quencher exists within the sequence near the 5' end or the 3' end.
  • the melting temperature with its target nucleic acid is between the temperature of the annealing step and the temperature of the extension step among the three stages of the PCR reaction: the denaturation step at a relatively high temperature, the annealing step at a relatively low temperature, and the extension step at a relatively medium temperature.
  • the denaturation step is performed at 92 to 98°C
  • the annealing step is performed at 50 to 60°C
  • the extension step is performed at 70 to 80°C
  • the melting temperature will be around 65°C.
  • the first hybridization probe has this melting temperature
  • the first hybridization probe binds to the target nucleic acid, thereby separating the signal molecule and the quencher, and thus generating a signal.
  • the first hybridization probe dissociates from the target nucleic acid and assumes the original stem-loop structure, thereby bringing the signal molecule and the quencher into proximity, and thus extinguishing the signal.
  • the second hybridization probe has a melting temperature with respect to its target nucleic acid between the temperature of the extension step and the temperature of the denaturation step among the three stages of the PCR reaction: a relatively high temperature denaturation step, a relatively low temperature annealing step, and a relatively medium temperature extension step. Therefore, the second hybridization probe remains bound to the target nucleic acid in both the annealing step and the extension step, but due to the action of the quenching probe, no signal is generated in the annealing step and only the extension step.
  • the quenching probe has a melting temperature relative to its second hybridization probe that lies between the temperature of the annealing step and the temperature of the extension step among the three stages of the PCR reaction: a denaturation step at a relatively high temperature, an annealing step at a relatively low temperature, and an extension step at a relatively medium temperature. Therefore, as described above, the quenching probe remains bound to the second hybridization probe in the annealing step to quench the signal of the second hybridization probe, but dissociates in the extension step to allow a signal to be generated from the second hybridization probe.
  • the method of the present invention comprises, instead of a primer for amplifying the first or second target nucleic acid, a non-complementary random nucleic acid sequence, a restriction enzyme recognition sequence and a complementary nucleic acid sequence to the first or second target nucleic acid of (i), a first or second target nucleic acid,
  • a pair of forward and reverse primers complementary to a target nucleic acid are used to amplify the target nucleic acid, and in this case, the production of an artificial target nucleic acid is characterized in that a PTO (Probing and Tagging Oligonucleotide) having an artificial random tag sequence together with a complementary probe sequence for the target nucleic acid is used to generate a PTO fragment, which is an artificial random sequence 5' tag consisting only of an artificial random sequence, and this PTO fragment becomes an artificial target nucleic acid.
  • PTO Probing and Tagging Oligonucleotide
  • FIG. 2 A conceptual diagram of the PCR method of the present invention as described above is illustrated in Fig. 2.
  • the two target nucleic acids can be detected by detecting the signals for the two target nucleic acids in the annealing step and the extension step, respectively.
  • the detection signals of the annealing step and the extension step are obtained without interference with each other. That is, the detection signal obtained in the annealing step becomes a detection signal for only the first target nucleic acid among the two target nucleic acids, and the detection signal obtained in the extension step becomes a detection signal for only the second target nucleic acid among the two target nucleic acids.
  • the sequences of the three probes should be designed in consideration of the melting temperature with respect to their complementary target nucleic acids (the quenching probe is the second hybridization probe), etc., and the design of the sequences in consideration of the melting temperature can be accomplished by controlling the GC content, the sequence length, or these together.
  • the design of the probe sequences in consideration of the melting temperature is described in references known in the art [Unit Evol. Genet. 2005; 5:1-9]; [Afr. Jo. Biotechnol. 2003; 2:91-95], [Methods Mol. Biol. 1993; 15:31-40], [PCR Methods Appl. 1993; 3:S30-S37], [Nucleic Acids Res.
  • the second hybridization probe is in a state where its melting temperature with respect to its target nucleic acid is between a relatively medium temperature extension step and a relatively high temperature denaturation step, and is thus in a bound state with the target nucleic acid in the extension step, and when the extension reaction (polymerization reaction) proceeds, it can be hydrolyzed by a polymerase (i.e., by a nuclease activity such as Taq polymerase) like a hydrolyzable probe such as a TaqMan probe.
  • a polymerase i.e., by a nuclease activity such as Taq polymerase
  • the second hybridization probe When the second hybridization probe is in a state where it is bound to the target nucleic acid in the extension step and then hydrolyzed, a signal is generated like a hydrolyzable probe by the hydrolysis, and the generated signal is detected together with the signal of the first hybridization probe in the annealing step of the next cycle of the PCR reaction, so there occurs a problem that the signal of the first hybridization probe does not accurately reflect detection information (i.e., qualitative and quantitative information) on the first target nucleic acid.
  • detection information i.e., qualitative and quantitative information
  • the target nucleic acid to which the second hybridization probe complementarily recognizes and specifically binds must not be the target nucleic acid that is the target of detection in the sample to be actually detected (i.e., the actual target nucleic acid or the second actual target nucleic acid), but must be an artificial target nucleic acid generated depending on such an actual target nucleic acid.
  • Such artificial target nucleic acids be generated quantitatively dependent on actual target nucleic acids, thereby enabling not only qualitative analysis of the actual target nucleic acids but also quantitative analysis.
  • Various techniques for generating artificial target nucleic acids dependent on actual target nucleic acids are known in the art.
  • As a technology for producing such an artificial target nucleic acid for example, the C-TAG (Cleavable Tag) technology disclosed in International Patent Publication No.
  • WO 2017188669 entitled “METHOD FOR DETECTING TARGET NUCLEIC ACID SEQUENCE USING CLEAVED COMPLEMENTARY TAG FRAGMENT AND COMPOSITION THEREOF” and the PCE-SH (PTO Cleavage and Extension-Dependent Signaling) technology disclosed in International Patent Publication No. WO 2013115442 entitled “DETECTION OF TARGET NUCLEIC ACID SEQUENCE BY PTO CLEAVAGE AND EXTENSION-DEPENDENT SIGNALING OLIGONUCLEOTIDE HYBRIDIZATION ASSAY” are disclosed. Examples include oligonucleotide hybridization (OLH) technology.
  • C-TAG technology is a technology that detects target nucleic acids by using a primer introduced together with a sequence that can be cleaved by a restriction enzyme, which is an artificially random sequence, to amplify a target nucleic acid, and when a tag (C-TAG) complementary to the artificially random sequence is generated by the action of the restriction enzyme along with the amplification of the target nucleic acid, the tag is detected with a hybridization probe or the like.
  • the restriction enzyme does not act, so no tag is generated, and if the target nucleic acid exists, a tag is generated in quantitative proportion to it, so that qualitative and quantitative detection of the target nucleic acid is possible through tag detection.
  • the tag itself can be detected as a hybridization probe, or, if the tag is shorter in sequence length than the hybridization probe, it can act as a primer for the hybridization probe so that it is extended by an extension reaction like the target nucleic acid, thereby switching the signal of the hybridization probe from signal-off to signal-on, and thus can be detected.
  • the specific working mode of the C-TAG technology described above can be found in international patent publication WO 2017188669. This document, together with all documents cited herein, is considered a part of this specification. A conceptual diagram of the working mode of these C-TAG technologies is illustrated in Fig. 3.
  • PCE-SH technology uses PTO (Probing and Tagging Oligonucleotide) which has a complementary probe sequence for the target nucleic acid and an artificial random tag sequence while the 3' end is blocked with HEG (hexaethylene glycol), phosphate group, etc., when amplifying the target nucleic acid, and when there is a bifurcated duplex, the PTO fragment consisting only of an artificial random sequence (i.e., a 5' tag of an artificial random sequence) is generated by the action of Taq polymerase which generates a single-stranded 5' tag by endonuclease activity (Proc Natl Acad Sci U S A.
  • PTO Probing and Tagging Oligonucleotide
  • this PTO fragment acts as a primer for CTO (Capturing and Templating Oligonucleotide) so that it extends together during the extension reaction of the target nucleic acid, thereby generating a new extension.
  • CTO Capturing and Templating Oligonucleotide
  • the PTO fragment acts as a primer for the hybridization probe so that it is extended by an extension reaction like the target nucleic acid, thereby switching the signal of the hybridization probe from signal-off to signal-on, so that detection is possible.
  • technologies for generating artificial target nucleic acids include PTOCE (Probing & Tagging Oligonucleotide Cleavage and Extension) technology (International Patent Publication No. WO 2012096523), PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) technology (International Application No. PCT/KR2013/012312), etc.
  • PTOCE technologies PCE-NH technologies, etc., similarly to the PCE-SH technology, all use a PTO (Probing and Tagging Oligonucleotide) having an artificial random tag sequence together with a complementary probe sequence for the target nucleic acid to generate a PTO fragment consisting of only an artificial random sequence (i.e., a 5' tag of the artificial random sequence), and a step of extending this fragment using a CTO (Capturing and Templating Oligonucleotide) as a template to generate a new sequence, thereby detecting the new sequence.
  • a novel sequence extended from a PTO fragment consisting solely of an artificial random sequence becomes an artificial target nucleic acid, and this artificial target nucleic acid is qualitatively and quantitatively generated depending on the actual target nucleic acid of the sample.
  • an artificial target nucleic acid is a nucleic acid of a newly synthesized sequence that is generated only when a target nucleic acid is present in a sample or is generated in proportion to the amount of the target nucleic acid present in the sample.
  • C-TAG refers to a sequence that is newly generated by extending from a tag using a hybridization probe as a template when the tag or the tag acts as a primer for the hybridization probe.
  • PCE-SH technology it refers to a sequence that is newly synthesized by extending from a PTO fragment that acts as a primer using CTO or a hybridization probe as a template.
  • the second hybridization probe must detect an artificial target nucleic acid generated depending on an actual target nucleic acid as described above, but the first hybridization probe is in a dissociated state from the target nucleic acid during the extension step since its melting temperature with respect to the target nucleic acid is between a relatively low temperature annealing step and a relatively medium temperature extension step, so there is no concern about decomposition during the extension reaction. Therefore, the nucleic acid detected by the first hybridization probe may be either an actual target nucleic acid in a sample or an artificial target nucleic acid generated depending on the actual target nucleic acid.
  • the probe is an oligonucleotide for detecting a target nucleic acid (an actual target nucleic acid in a sample or an artificial target nucleic acid), and means a nucleic acid or nucleic acid analogue that contains a base sequence complementary to the target nucleic acid and thus has a specific binding ability to the target nucleic acid.
  • the probe may be RNA as well as DNA as long as it has a specific binding ability to the target nucleic acid (when the probe of the present invention is RNA, "T” in the base sequence of the above sequence number is read as "U"), and may also be a nucleic acid analogue such as PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), HNA (Hexitol Nucleic Acids), ANA (Altritol Nucleic Acids), MNA (Mannitol Nucleic Acids) that are stable to nuclease or heat.
  • the probe may also include a modified nucleotide as well as a natural nucleotide as long as it has a specific binding ability to the target nucleic acid.
  • nucleotides may be modified in the sugar, the phosphate and/or the base.
  • nucleotides modified in the sugar, the phosphate and/or the base are specifically known in the art, including methods for their preparation.
  • nucleotides modified in the sugar include those in which the hydroxyl group (OH group) of the sugar is modified with a halogen group, an aliphatic group, an ether group, an amine group, etc., those in which the sugar ribose or deoxyribose itself is substituted with sugar analogues such as ⁇ -anomeric sugars, arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, etc.
  • modifications in phosphate include modifications of phosphate to P(O)S(thioate), P(S)S(dithioate), P(O)NR2(amidate), P(O)R, P(O)OR', CO or CH2(formacetal).
  • R or R' is H or substituted or unsubstituted alkyl, etc., and when modified in phosphate, the linking group becomes -O-, -N-, -S- or -C-, and adjacent nucleotides are bonded to each other through this linking group.
  • the complementary sequence to the target nucleic acid in the probe need not be 100% complementary, as long as it confers specific binding ability to the target nucleic acid, but may be at least about 80%, preferably at least about 85%, more preferably at least about 90%, 95% or 99%.
  • a hybridization probe refers to a probe having a complementary sequence to a target nucleic acid and a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid does not bind to the probe.
  • the hybridization probe may have a stem-loop structure.
  • a complementary sequence to the target nucleic acid is located in the loop region, and a signal substance and a quencher are located within the stem region (intra-stem) or at the end thereof.
  • a fluorescent substance is used as the signal substance, so such a signal is a fluorescent signal.
  • Various methods are known in the art, including a method of labeling a signal substance and a quencher to an oligonucleotide probe or a method of manufacturing such a probe, such as a direct chemical labeling via solid-phase synthesis and a two-step chemical labeling via solid-phase synthesis, and also described in the literature [Chem. Soc. Rev., 2020, 49, 8749-8773], literature [ACS Cent. Sci., 2017, 3, 701-707], literature [Curr. Opin. Biotechnol., 2015, 31, 42-49], literature [Annual Review of Physiology, ed. D. Julius, 2017, vol. 79, pp. 93-117], etc.
  • Various literatures are known. For specific information regarding the labeling method, refer to these literatures.
  • the first hybridization probe may be a molecular beacon.
  • the sample to be analyzed may be any mixture or solution that contains or is suspected of containing the target nucleic acid to be detected and thus has a need for detection.
  • the sample may be a biological sample obtained from a human or animal, as well as a processed sample obtained by processing such a biological sample to increase the concentration of the target nucleic acid, and further may be a sample that requires inspection, such as water, food, industrial wastewater, etc. that contain or are suspected of containing the target nucleic acid, an environmental pollutant, a toxic agent, an organic matter sample obtained from a food processing device, etc.
  • a sample may include an appropriate diluent, a buffer solution, and when it is desired to detect the presence of bacteria or viruses, it may be a bacterial culture or virus culture containing a medium or a medium component.
  • the sample to be analyzed may preferably be a biological sample obtained from a human or animal or a processed sample thereof.
  • the biological sample may be obtained from a human or animal that contains or is suspected of containing the target nucleic acid to be detected, such as whole blood, serum, plasma, umbilical cord blood, urine, feces, saliva, nasal mucus, semen, amniotic fluid, lavage fluid (bronchial alveolar, stomach, peritoneal, ear, etc.), lymph, sputum, tissue, cell, etc., and thus has a need for detection.
  • the processed sample may be, for example, plasma, serum, a biological sample whose concentration of nucleic acid (DNA and/or RNA) is increased using a nucleic acid extraction kit, a tissue extract, a cell obtained from a tissue, a cell lysate, a cell culture, a bacterial culture, a virus culture, etc.
  • a nucleic acid extraction kit for example, plasma, serum, a biological sample whose concentration of nucleic acid (DNA and/or RNA) is increased using a nucleic acid extraction kit, a tissue extract, a cell obtained from a tissue, a cell lysate, a cell culture, a bacterial culture, a virus culture, etc.
  • the target nucleic acid refers to any nucleic acid that can be useful, such as for the diagnosis or prognosis of a disease, confirmation of a biological indicator, and research on its function or properties, by detecting or measuring the presence and/or amount of the target nucleic acid.
  • the target nucleic acid refers to a single-stranded DNA including a sequence to which a probe (i.e., the first or second hybridization probe) binds, that is, a sequence complementary to the probe, or a double-stranded DNA to which the single-stranded DNA is bound.
  • This single-stranded or double-stranded DNA may be genomic DNA in a sample, particularly a biological sample, cDNA generated by reverse transcription from RNA such as mRNA, rRNA, or microRNA, or an artificial target nucleic acid composed of an artificial arbitrary sequence generated depending on an actual target nucleic acid, as described above.
  • the actual target nucleic acid may be a nucleic acid of a prokaryotic cell such as a pathogenic bacterium, a nucleic acid of a eukaryotic cell derived from or isolated from a human, a nucleic acid of a pathogenic virus, or a viroid nucleic acid.
  • a prokaryotic cell such as a pathogenic bacterium
  • a nucleic acid of a eukaryotic cell derived from or isolated from a human a nucleic acid of a pathogenic virus, or a viroid nucleic acid.
  • pathogenic bacteria include antibiotic-resistant Enterobacteriaceae such as vancomycin or carbapenem, Acinetobacter baumannii that can cause pneumonia or sepsis, Bordetella pertussis that can cause whooping cough, and Klebsiella pneumoniae that can cause pneumonia
  • pathogenic viruses include viruses that cause gastrointestinal diseases (Rotavirus A, Astrovirus, Adenovirus F40, Adenovirus F41, Norovirus GI, Norovirus GII, etc.), human papillomavirus, and respiratory disease agents (Influenza A/H1N1, Influenza A/H3N2, Influenza A/H1N1/2009pdm, Influenza B, Parainfluenza 1, Parainfluenza 3, Respiratory syncytial virus A, Respiratory These include syncytial virus B, human metapneumovirus, and adenovirus.
  • the primer is an oligonucleotide capable of inducing the initiation of synthesis of a target nucleic acid, and is composed of a forward primer and a reverse primer for each target nucleic acid.
  • the primer is designed to specifically bind to the target nucleic acid (the actual target nucleic acid in the sample or the artificial target nucleic acid generated depending on the target nucleic acid) and to have a melting temperature sufficient to maintain binding to the target nucleic acid in the extension reaction of the PCR reaction.
  • the extension reaction is generally performed in the extension step, but it is not necessarily so.
  • the first hybridization probe may bind to either the actual target nucleic acid or the artificial target nucleic acid in the sample and detect it, and when the first hybridization probe detects the artificial target nucleic acid, if the artificial target nucleic acid is shorter than the first hybridization probe, it may act as a primer in the annealing step in which it binds complementarily to the first hybridization probe, thereby separating the signal substance and the quencher of the first hybridization probe and extending it.
  • the forward and reverse primers for the target nucleic acid are designed so that their melting temperatures are at least higher than the temperature of the annealing step, and if an extension reaction for a target nucleic acid occurs in the extension step, the forward and reverse primers for the target nucleic acid are designed so that their melting temperatures are at least higher than the temperature of the extension step.
  • This design can usually be achieved by adjusting the length of the primer while making the sequence complementary to the target nucleic acid.
  • Primers can have various lengths, but are usually 10 to 50 nucleotides long, and similar to probes, some sequences of the primer can be nucleic acid analogs such as PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid), HNA (Hexitol Nucleic Acids), ANA (Altritol Nucleic Acids), MNA (Mannitol Nucleic Acids), etc., as long as it does not affect the synthesis initiation inducing function of the primer, and may also include modified nucleotides as well as natural nucleotides.
  • PNA Peptide Nucleic Acid
  • LNA Locked Nucleic Acid
  • HNA Heexitol Nucleic Acids
  • ANA Altritol Nucleic Acids
  • MNA Merannitol Nucleic Acids
  • the mixture for the PCR reaction of the step (a) includes, in addition to the primer set and the three probes described above, four NTPs, DNA polymerase, and other factors, such as Tris-HCl as a pH stabilizer, MgCl2 or MgSO4 or (NH4)2SO4 as cofactors for promoting enzyme activity, BSA (Bovine Serum Albumin), gelatin, glycerol or PEG 6000 as factors for stabilizing the enzyme, KCl as a factor for promoting binding of the primer to the target nucleic acid, Tween 20 or Triton X-100 as nonionic surfactants for suppressing nonspecific amplification, spermidine for reducing nonspecific binding of DNA polymerase to nucleic acid, urea, DMSO (dimethylsulfoxide) or DMF (dimethylformamid) as factors for promoting annealing of the primer to the target nucleic acid, Betaine, sodium dodecyl sulfate (S)), sodium do
  • the DNA polymerase uses a thermostable polymerase.
  • thermostable polymerases include Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, Thermus antranikianii, and Thermus caldophilus.
  • DNA polymerase may be used in a modified form.
  • Modified polymerases include those in which some sequences have been artificially modified or naturally modified (mutated). Examples of such modified polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46E L329A D640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ⁇ Z05 polymerase, ⁇ Z05-Gold polymerase, ⁇ Z05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, and E678G TMA-30 polymerase.
  • the DNA polymerase is preferably a DNA polymerase from which 5'->3' exonuclease activity has been eliminated.
  • Commercially available polymerases include AptaTaq exo DNA Polymerase (Roche, Switzerland), Top DNA Polymerase (Bioneer, Korea), Vent® (exo-) DNA Polymerase (NEB, USA), Phusion® High-Fidelity DNA Polymerase (NEB, USA), and KlenTaq-1 DNA polymerase (an analogue of the Klenow fragment in Taq polymerase).
  • the PCR reaction is performed by repeatedly performing three stages of denaturation, annealing, and extension for two or more cycles, usually 15 to 50 cycles, to amplify the template nucleic acid.
  • An initial denaturation step may be included before performing these three-stage cycles. This initial denaturation step is performed to activate DNA polymerase and denature the template DNA. This initial denaturation step may be performed within a range of 30 seconds to 20 minutes, taking into account the GC content of the template DNA, etc.
  • an extension step (final elongation step) may be included.
  • This extension step is for the DNA polymerase to complete the final extension reaction, and is usually performed at the same temperature as the above 3-step extension step for about 1 to 20 minutes.
  • a signal such as a fluorescent signal, a luminescent signal, or a colorimetric signal may be used without any special limitation, but it is preferable to use a fluorescent signal.
  • any fluorescent material for generating the signal can be appropriately selected and used from among those known in the art, taking into consideration the detection wavelength of each detection channel of the PCR device, etc.
  • Known fluorescent substances include, for example, Cy2TM (506), YO-PROTM-1 (509), YOYOTM-1 (509), Calcein (517), FITC (518), FluorXTM (519), AlexaTM (520), Rhodamine 110 (520), Oregon GreenTM 500 (522), Oregon GreenTM 488 (524), RiboGreenTM (525), Rhodamine GreenTM (527), Rhodamine 123 (529), Magnesium GreenTM (531), Calcium GreenTM (533), TO-PROTM-1 (533), TOTO1 (533), JOE (548), BODIPY530/550 (550), Dil (565), BODIPY TMR (568), BODIPY558/568 (568), BODIPY564/570 (570), Cy3TM (570), AlexaTM 546 (570),
  • a quencher is a substance that absorbs, generates heat, and extinguishes fluorescence emitted by a fluorescent substance when it is adjacent to the fluorescent substance.
  • Many such quenchers are known in the art. Among these known quenchers, one having an appropriate absorption wavelength can be selected and used in consideration of the emission wavelength of the fluorescent substance used.
  • Known quenchers include Dabcyl (453), QSY 35 (475), BHQ-0 (495), Eclipse (530), BHQ-1 (534), QSY 7 (560), QSY 9 (562), BHQ-2 (579), ElleQuencher (630), Iowa Black (651), QSY 21 (661), BHQ-3 (672), etc.
  • the numbers in parentheses are the maximum absorption wavelengths in nanometers.
  • Cy5/BHQ-2, Cy5/BHQ-1, TexRed/BHQ-2, TexRed/QSY 7, TexRed/BHQ-1, TexRed/Dabcyl, Cy3/BHQ2, Cy3/QSY 7, Cy3/BHQ-1, Cy3/Dabcyl, TET/BHQ-2, TET/QSY 7, TET/TAMRA, TET/BHQ-1, FAM/BHQ-2, FAM/QSY 7, FAM/TAMRA, FAM/BHQ-1, FAM/Dabcyl are known to have high quenching efficiency ( Nucliec Acids Res. 30, e122, 2002), so it may be desirable to select and use these pairs.
  • a single signal refers to a signal detected through one detection channel. Therefore, even if two signal generating substances are used, if the signals generated by the two substances are detected in one detection channel, it is a single signal.
  • Cy2TM 506
  • the fluorescent signal generated by it is naturally a single signal, but even if two fluorescent substances, AlexaTM (520) and Rhodamine 110 (520), are used as signal substances, if their detection wavelengths are the same and detected in one fluorescence detection channel, the signals by these two fluorescent substances also become a single signal.
  • signal detection is performed in the annealing step and the extension step of the PCR reaction.
  • the signal of the first hybridization probe which specifically binds to the first target nucleic acid (the actual target nucleic acid in the sample or the artificial target nucleic acid generated depending on the actual target nucleic acid) and generates a signal by separating the signal substance and the quencher, is detected, thereby detecting the first target nucleic acid
  • the signal of the second hybridization probe which specifically binds to the second target nucleic acid (which is an artificial target nucleic acid) and generates a signal by separating the signal substance and the quencher, is detected, thereby detecting the second target nucleic acid.
  • the signal of the second hybridization probe is generated by dissociation of the quencher probe.
  • Signal detection is usually performed by irradiating the sample with light from a light source (lamp, laser, LED) equipped in the PCR device at the end of each step and detecting the light emitted from the sample in a detection channel.
  • a light source lamp, laser, LED
  • Signal detection can be performed in some or all cycles of the amplification reaction, but it is preferable that it be performed in all cycles.
  • the detection results for the first target nucleic acid and/or the second target nucleic acid are derived as a PCR result graph consisting of signal intensities and the number of PCR cycles.
  • the PCR result graph can be derived as a single graph showing two amplification curves for the two target nucleic acids, or as separate graphs showing amplification curves for the two target nucleic acids, respectively.
  • the PCR result graph will appear as an initiation phase, an exponential phase, and a plateau phase, through which detection information (qualitative and/or quantitative information) for the two target nucleic acids can be easily derived.
  • the first and second hybridization probes complementarily bind to target nucleic acids, thereby generating a signal by separating the signal substance and the quencher.
  • the target nucleic acid is an artificial target nucleic acid and the artificial target nucleic acid is shorter than the hybridization probe, the artificial target nucleic acid may act as a primer for each of the first and second hybridization probes and be extended, thereby separating the signal substance and the quencher of the hybridization probe.
  • the extension reaction is preferably performed in the annealing step, since the first hybridization probe binds to the target nucleic acid in the annealing step at a relatively low temperature to generate a signal, but dissociates in the extension step at a relatively medium temperature to quench the signal, and the second hybridization probe binds to the target nucleic acid in the annealing step at a relatively low temperature, but the signal is quenched by the quenching probe, and then the quenched signal reappears when the quenching probe dissociates in the extension step at a relatively medium temperature.
  • Taq DNA polymerase is known to exhibit the most optimal activity at the typical temperature of 72-75°C in the extension step, but it is known to have activity sufficient to cause the extension reaction at a sufficient speed at the typical temperature of 50-65°C in the annealing step. If an artificial target nucleic acid acts as a primer and an extension reaction occurs in the annealing step, it is desirable to maintain the annealing step for an appropriate amount of time (typically 30 seconds to 2 minutes) to ensure that the extension reaction occurs sufficiently.
  • the first target nucleic acid detected by the first hybridization probe in the annealing step at a relatively low temperature may be an actual target nucleic acid in the sample or an artificial target nucleic acid generated depending on the target nucleic acid, but the second target nucleic acid detected by the second hybridization probe in the extension step at a relatively medium temperature must be an artificial target nucleic acid. If the nucleic acid to which the second hybridization probe binds and detects is an actual target nucleic acid in the sample, as described above, the hybridization probe may be decomposed by the exonuclease activity of DNA polymerase, like the TaqMan probe.
  • the nucleic acid detected by the first hybridization probe and the second hybridization probe in the present invention the detection step thereof, and the step in which the extension reaction of the actual target nucleic acid, etc. is performed can be summarized as follows:
  • the first target nucleic acid detected is an actual target nucleic acid in the sample and not an artificial target nucleic acid.
  • the first actual target nucleic acid undergoes an extension reaction in the extension step, and is detected by the first hybridization probe that binds complementarily to the first target nucleic acid in the annealing step of the next cycle.
  • the second target nucleic acid detected at this time is an artificial target nucleic acid that is generated dependently on the actual second target nucleic acid in the sample, and this artificial target nucleic acid is generated dependently on the actual second target nucleic acid in the sample as the actual second target nucleic acid is extended in the extension step, binds to the second hybridization probe in the annealing step of the next cycle, but the signal is quenched and not detected as the quenching probe binds together with the second hybridization probe, and is then detected as the quenching probe dissociates as the temperature rises in the next step, the extension step.
  • the first target nucleic acid detected is an artificial target nucleic acid generated based on the actual target nucleic acid in the sample.
  • the first real target nucleic acid undergoes an extension reaction in the extension step, and during this extension reaction, an artificial target nucleic acid is generated depending on the first real target nucleic acid, and is detected by the first hybridization probe that binds complementarily to the first target nucleic acid in the annealing step of the next cycle.
  • Detection of the second target nucleic acid is the same as in case (1) above.
  • first and second artificial target nucleic acids are shorter than each hybridization probe, they can act as primers using each hybridization probe as a template, and when they act as primers, the extension reaction of the primers is performed in the annealing step.
  • the present invention relates to a method for detecting a target nucleic acid using the hybridization probe and the quenching probe together as described above.
  • the hybridization probe is a probe having a complementary sequence specific to a target nucleic acid
  • the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid is not bound to the probe, and has a single-stranded sequence extended in the terminal direction from the sequence to which the signal substance is bound.
  • the hybridization probe and the quenching probe are designed such that the melting temperature at which the hybridization probe dissociates from its complementary target nucleic acid is higher than the melting temperature at which the quenching probe dissociates from its complementary hybridization probe.
  • the hybridization probe binds to its target nucleic acid, the signal substance and the quencher are separated, but no signal is generated by the quenching probe.
  • the quenching probe dissociates, the quenching effect by the quenching probe disappears, and a signal is generated from the hybridization probe.
  • the target nucleic acid may be an actual target nucleic acid in the sample or an artificial target nucleic acid generated depending thereon.
  • the description of the second hybridization probe and the quenching probe in relation to the PCR method for real-time detection of two target nucleic acids with a single signal of the present invention can be applied as is to the hybridization probe and the quenching probe.
  • the method of the present invention may be applied to a PCR method for real-time detection of a target nucleic acid.
  • This method is similar to the case where the first hybridization probe is not used in the PCR method for real-time detection of two target nucleic acids with a single signal as described above, and only the second hybridization probe and the quenching probe are used.
  • the target nucleic acid is preferably an artificial target nucleic acid generated depending on the actual target nucleic acid in the sample for the reasons described above.
  • the PCR method of the present invention is configured to include steps (a) to (c) below.
  • a hybridization probe having a complementary sequence specific to an artificial target nucleic acid generated depending on a target nucleic acid, wherein the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid is not bound to the probe, and has a single-stranded sequence extended in the terminal direction from the sequence to which the signal substance is bound, and the melting temperature at which it (the hybridization probe) dissociates from the artificial target nucleic acid complementary thereto is between the temperature of the extension step and the temperature of the denaturation step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of the PCR reaction, so that the hybridization probe can maintain a binding state with the artificial target nucleic acid complementary thereto in the annealing step and the extension step; and
  • a quenching probe having a quenching substance capable of complementarily binding to the extended single-stranded sequence of the hybridizable probe and quenching the signal substance of the hybridizable probe, wherein when the quenching probe complementarily binds to the hybridizable probe and the single-stranded sequence extended toward the end thereof (hybridizable probe), the quenching substance of the probe has a structure capable of quenching the signal substance of the hybridizable probe by being adjacent to the signal substance of the hybridizable probe, and the melting temperature at which the quenching probe dissociates from the sequence complementary thereto is between the temperature of the annealing step and the temperature of the extension step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of a PCR reaction, such that in the annealing step, the probe can quench the signal of the hybridizable probe by binding to the hybridizable probe, but in the extension step, the quenching substance of
  • step (b) a step of placing the mixture of step (a) into a single reaction vessel and performing two or more cycles of PCR reaction of denaturation at a relatively high temperature, hybridization at a relatively low temperature, and extension at a relatively medium temperature;
  • the PCR method for real-time detection of two target nucleic acids with a single signal of the present invention can be applied as is.
  • the present invention relates to a set of (a) hybridization probes and (b) quenching probes.
  • a probe having a complementary sequence specific to a target nucleic acid as a self-hybridizing probe wherein the hybridizing probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid is not bound to the probe, and a probe having a single-stranded sequence extending in a terminal direction from the sequence to which the signal substance is bound;
  • a quenching probe having a quenching substance capable of complementarily binding to the extended single-stranded sequence of the hybridization probe and quenching the signal substance of the hybridization probe, wherein the quenching probe has a structure in which, when complementarily binding to the single-stranded sequence extended toward the terminal of the hybridization probe, the quenching substance of the probe is adjacent to the signal substance of the hybridization probe and can quench the signal substance of the hybridization probe;
  • the hybridization probe and the quenching probe are designed such that the melting temperature at which the hybridization probe dissociates from its complementary target nucleic acid is higher than the melting temperature at which the quenching probe dissociates from its complementary hybridization probe.
  • the description of the second hybridization probe and the quenching probe with respect to the PCR method for real-time detection of two target nucleic acids with a single signal of the present invention can also be applied to the probe set of the present invention.
  • the present invention relates to a kit for real-time detection of two or more target nucleic acids with a single signal during PCR performance.
  • the kit of the present invention comprises the primer set of (i) and the probes of (ii) to (iv) below.
  • a first hybridization probe having a complementary sequence specific to the first target nucleic acid among two target nucleic acids, or (ii-b) a first artificial target nucleic acid generated depending on the first target nucleic acid
  • the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid is not bound to the probe, and the melting temperature at which it (the first hybridization probe) dissociates from the target nucleic acid or the artificial target nucleic acid complementary thereto is between the temperature of the annealing step and the temperature of the extension step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of the PCR reaction, so that in the annealing step, the signal substance and the quencher are separated by binding to the complementary sequence of it (the first hybridization probe) and a signal is generated, but in the extension step, the signal substance and the quencher are separated
  • a second hybridization probe having a complementary sequence specific to a second artificial target nucleic acid generated depending on a second target nucleic acid among two or more target nucleic acids, wherein the hybridization probe has a structure in which a signal substance and a quencher are adjacent to each other and no signal is generated when the target nucleic acid is not bound to the probe, and has a single-stranded sequence extended in a terminal direction from the sequence to which the signal substance is bound, and further, a melting temperature at which it (the second hybridization probe) dissociates from the second artificial target nucleic acid complementary thereto is between the temperature of the extension step and the temperature of the denaturation step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of the PCR reaction, such that the second hybridization probe can maintain a binding state with the second artificial target nucleic acid complementary thereto in the annealing step and the extension step; and
  • a quenching probe having a quenching substance capable of complementarily binding to the extended single-stranded sequence of the second hybridization probe and quenching the signal substance of the second hybridization probe, wherein when the quenching probe complementarily binds to the second hybridization probe and the single-stranded sequence extended toward the end thereof (the second hybridization probe), the quenching substance of the probe has a structure capable of quenching the signal substance of the second hybridization probe by being adjacent to the signal substance of the second hybridization probe, and further, the melting temperature at which the quenching probe dissociates from the sequence complementary thereto is between the temperature of the annealing step and the temperature of the extension step among the relatively high temperature denaturation step, the relatively low temperature annealing step, and the relatively medium temperature extension step of the PCR reaction, so that in the annealing step, the signal of the second hybridization probe can be quenched by binding to the second hybridization probe, but in the extension step, the quenching probe A probe capable of causing
  • the kit of the present invention may further include, in addition to the primer set and the three probes, four NTPs, DNA polymerase, and other factors, such as Tris-HCl as a pH stabilizer, MgCl2 or MgSO4 or (NH4)2SO4 as cofactors for promoting enzyme activity, BSA (Bovine Serum Albumin) or gelatin or glycerol or PEG 6000 as factors for stabilizing the enzyme, KCl as a factor for promoting binding of the primer to the target nucleic acid, Tween 20 or Triton X-100 as nonionic surfactants for suppressing nonspecific amplification, spermidine for reducing nonspecific binding of DNA polymerase to nucleic acid, urea or DMSO (dimethylsulfoxide) or DMF (dimethylformamid) as factors for promoting annealing of the primer to the target nucleic acid, betaine, and a chelating agent for preventing aggregation of DNA polymerase.
  • the kit of the present invention may further include instructions for using the kit.
  • the instructions may teach a PCR method for real-time detection of two target nucleic acids with a single signal of the present invention as described above.
  • a PCR method for detecting two or more target nucleic acids with a single signal can be provided.
  • the method of the present invention can detect signals for two target nucleic acids in the annealing step and the extension step, respectively, without interference with each other, even when a single signal is used during the PCR process.
  • These detection signals according to the method of the present invention are obtained using the same single signal, so there is no need for data processing such as addition and subtraction between them, and therefore, it provides the effect of being able to immediately and easily check whether two target nucleic acids are detected (qualitative information) and the degree of detection (quantitative information) in a PCR result graph composed of signal intensity and the number of PCR cycles.
  • Figure 1 illustrates the structure of three probes according to the present invention, namely, first and second hybridization probes and one quenching probe.
  • Figure 2 is a conceptual diagram of a PCR method for real-time detection of two target nucleic acids with a single signal of the present invention.
  • Figure 3 is a conceptual diagram of C-TAG (Cleavable Tag) technology for generating artificial target nucleic acids.
  • Figure 4 shows the experimental results showing that in the case of the second hybridization probe (DaO), signals are generated in both the annealing step and the extension step, but when combined with the quenching probe (QO), signals are generated only in the extension step.
  • DaO second hybridization probe
  • QO quenching probe
  • Figure 5 shows the results of confirming the melting temperature of the second hybridization probe (DaO) with its target nucleic acid and the melting temperature of the quenching probe (QO) with the second hybridization probe (DaO).
  • Figure 6 is a result showing that real-time detection of target nucleic acids H3 and 09pdm is possible during PCR with a single signal according to the method of the present invention.
  • Figure 7 is a result showing that individual analysis of multiple target nucleic acids is possible through fluorescence channels and detection temperatures in a multi-fluorescence channel configuration according to the method of the present invention through real-time detection of SARS-CoV-2 and Flu A/H3N2.
  • Figure 8 is a result showing that individual analysis of multiple target nucleic acids is possible through fluorescence channels and detection temperatures in a multi-fluorescence channel configuration according to the method of the present invention through real-time detection of FluA/H1N1pdm and Flu B.
  • FIG. 9 is a result showing that individual analysis of multiple target nucleic acids is possible through fluorescence channels and detection temperatures in a multi-fluorescence channel configuration according to the method of the present invention, through real-time detection of adenovirus (ADV) and a control (NC) containing no target nucleic acids.
  • ADV adenovirus
  • NC control
  • the cTAG primer is a primer applied to the C-TAG technology, and refers to a primer that enables the generation of an artificial target nucleic acid depending on a target nucleic acid.
  • the DaO (Dual-annealing oligonucleotide) probe in this embodiment refers to a second hybridization probe according to the present invention, which has a sequence that enables complementary binding with a target nucleic acid and a sequence that enables complementary binding with a quenching probe.
  • the QO (Queching oligonucleotide) probe in this embodiment is a quenching probe according to the present invention, which has a structure that can complementarily bind to the DaO and quench a signal of the DaO.
  • This experiment was conducted to confirm that the DaO probe binds to a target nucleic acid at a temperature lower than the melting temperature of the QO probe, and that the signal of the DaO probe is quenched by QO when the QO probe binds to it.
  • the penton DNA of adenovirus which is the causative agent of respiratory disease, was used as an actual target nucleic acid, and an artificial target nucleic acid generated by C-TAG technology from the actual target nucleic acid was detected using the DaO probe.
  • the primer and probe sequences used are as follows.
  • Primer 1 5'- GCATCTCCGTGAGCCAGA CCAGG CACCGTCAGTGAAAACGTTCC-3' (SEQ ID NO: 1)
  • Primer 2 5'-ATGCCCAGGGCCTTGTAAAC-3' (SEQ ID NO: 2)
  • QO #1 5'-CCGGCGGTCGACGCAGGTC[BHQ2]-3' (SEQ ID NO: 4)
  • C-TAG sequence 1 5'-TCTGGCTCACGGAGATGC-3' (SEQ ID NO: 5)
  • the generated C-TAG sequence is hybridized and extended to the probe or DAO, and the extended sequence is as follows.
  • C-TAG extension sequence 1 TCTGGCTCACGGAGATGCGACGTCGCGGCG (SEQ ID NO: 6)
  • the melting temperature of DaO #1 with the target nucleic acid (C-TAG extension sequence 1) is designed to be 78°C, and the melting temperature of QO #1 with DaO #1 is designed to be 70°C.
  • primer 1 a sequence complementary to 5'- GCATCTCCGTGAGCCAGA -3' (SEQ ID NO: 7), an artificial random sequence at the 5' end generated by Psp GI that acts only in the presence of a target nucleic acid, complementarily binds to the C-TAG sequence at a position identical to the random sequence in the DaO probe sequence, thereby acting as a primer for the DaO probe and being extended at an annealing step at 60°C, thereby separating the quencher molecule and the fluorescent signal molecule of the DaO probe from each other.
  • the extended C-TAG sequence is designed to have a dissociation temperature of 78°C so that the binding is maintained even at the extension step at 75°C, thereby separating the quencher molecule and the fluorescent signal molecule.
  • Fig. 4 when the QO probe is not present, the DaO probe is bound to its complementary sequence in both the 60°C annealing step and the 75°C extension step, so its quenching molecule and the fluorescent signal molecule are separated from each other, and a fluorescent signal is detected; however, when the QO probe is present, it complementarily binds to the DaO probe in the 60°C annealing step, so that the quenching molecule of the QO probe and the fluorescent signal molecule of the DaO probe are adjacent to each other, and no signal is generated; however, in the 75°C extension step, the QO probe melts and separates from the DaO probe, so that a signal of the DaO probe is generated.
  • the change in fluorescence signal according to the melting temperature of the DaO probe (DaO #1) and QO probe (QO #1) of Example 1 was confirmed through melting peak analysis.
  • the cycle was performed 40 times under the same conditions as Example 1, and the amplification product was extended for 10 minutes in the last 40th cycle. Then, the temperature of the obtained amplification product was increased by 0.5°C per 5 seconds from 55°C to 90°C, and the change in fluorescence signal according to the temperature change was observed.
  • This experiment was conducted to demonstrate that detection of two different target nucleic acids within a single reaction tube is possible using a single fluorescence channel.
  • This example was conducted targeting RNA of influenza A virus H3N2 subtype and 09pdm subtype, which are causative agents of respiratory diseases, and PCR using C-TAG technology was performed in the detection process described below.
  • Extension of forward and reverse primers was performed using Taq DNA polymerase, and a DNA fragment (C-TAG) complementary to the C-TAG probe or DaO was generated from the amplified product through a restriction enzyme.
  • the generated DNA fragment hybridizes to the complementary binding site within the C-TAG probe/DaO sequence and is extended by the polymerase to change the structure of the C-TAG probe/DaO.
  • quenching of DaO occurs by QO below the melting temperature of QO, and a fluorescence signal is detected above the melting temperature.
  • the primer information and target sequence information generated during the amplification process are as follows.
  • Primer 3 5'- ACACATCGACCCGTACGATG CCAGG CCGACAGTCCTCACCGAATCC -3' (SEQ ID NO: 8)
  • Primer 4 5'- GCTGTAAGCTTTGCTGCGTT -3' (SEQ ID NO: 9)
  • Primer 5 5'- GTGCCTCAGGAACGCTCG CCAGG AGCAAGAAGTTCAAGCCGGA -3' (SEQ ID NO: 10)
  • C-TAG probe 1 5'- ATAATTAAGAA[T(FAM)] ACACATCGACCCGTACGATG [BHQ1] -3' (SEQ ID NO: 12)
  • the bold and slanted sequences of the above primers 3 and 5 represent the recognition sequence of the restriction enzyme PspGI, and the underlined sequence is the complementary sequence of the artificial target nucleic acid generated thereby.
  • the parentheses in the probes, DaO and QO represent the positions of the base sequences where the fluorescent reporter, fluorescent quencher or C3 spacer is located.
  • the C3 spacer represents an aliphatic linker consisting of three carbons.
  • the C-TAG sequence generated from the amplicon is as follows.
  • C-TAG sequence 3 5'-CGAGCGTTCCTGAGGCAC-3' (SEQ ID NO: 16)
  • C-TAG extension sequence 3 5'-CGAGCGTTCCTGAGGCACCCGCGGCG-3' (SEQ ID NO: 18)
  • the melting temperature of C-TAG probe 1 is designed to be 66°C
  • the melting temperature with the target nucleic acid of DaO #2 is designed to be 77°C
  • the melting temperature of QO #2 with DaO #2 is designed to be 68°C.
  • Primers 3 and 4 react with the H3N2 gene sequence, primers 5 and 6 react with the 09pdm gene sequence, and the C-TAG sequence 2 generated during the H3N2 gene amplification process of primers 3 and 4 reacts with probe 1, and the C-TAG sequence 3 generated during the 09pdm gene amplification process of primers 5 and 6 reacts with DaO #2.
  • QO #2 reacts with the QO binding site of DaO #2.
  • the tube containing the above reaction solution was subjected to reverse transcription reaction at 55°C for 30 minutes, denatured at 95°C for 15 minutes, and then subjected to 45 cycles of a denaturation step at 95°C for 30 seconds, an annealing step at 60°C for 30 seconds, and an extension step at 75°C for 30 seconds. Fluorescence signals were detected at 60°C and 75°C for each cycle.
  • Fig. 6 The results are shown in Fig. 6. As can be confirmed in Fig. 6, in the H3 template alone condition, the amplification curve is confirmed only at 60°C and no fluorescence signal is confirmed at 75°C. In the 09pdm template alone condition, the amplification curve is not confirmed at 60°C and only at 75°C. In the mixed condition of H3 and 09pdm, the amplification curve of H3 is confirmed at 60°C and the amplification curve of 09pdm is confirmed at 75°C.
  • the PCR result graph confirmed at each temperature in the mixed condition of H3 and 09pdm is confirmed to have the same pattern as the PCR result graph detected in the H3 or 09pdm alone condition where the same concentration of template is added, confirming that the detection results for each target nucleic acid in the detection of two target nucleic acids are obtained by a single fluorescence detection channel without interference with each other.
  • This experiment was conducted to demonstrate that individual analysis of multiple target nucleic acids via fluorescence channels and detection temperatures is possible within a single reaction tube using multiple fluorescence channels.
  • This example was conducted targeting nucleic acids of SARS-CoV-2, Influenza A virus/H3N2, Influenza A virus/09pdm, Influenza B virus, and Adenovirus, which are causative agents of respiratory diseases, and PCR using C-TAG technology was performed in the detection process described below.
  • Extension of forward and reverse primers was performed using Taq DNA polymerase, and a DNA fragment (C-TAG) complementary to the C-TAG probe or DaO was generated from the amplified product through a restriction enzyme.
  • the generated DNA fragment hybridizes to the complementary binding site within the C-TAG probe/DaO sequence and is extended by the polymerase to change the structure of the C-Tag probe/DaO.
  • quenching of DaO occurs by QO below the melting temperature of QO, and a fluorescence signal is detected above the melting temperature.
  • the primer information and target sequence information generated during the amplification process are as follows.
  • Primer 7 5'- ACACATCGACCCGCGATG CCAGG ACCCGCAATCCTGCTAACAA-3' (SEQ ID NO: 19)
  • Primer 8 5'-ACGAGAAGAGGCTTGACTGC-3' (SEQ ID NO: 20)
  • Primer 9 5'- GTGCCTCAGGAACGCTCG CCAGG AATAGAGCTCGCACCGTAGC-3' (SEQ ID NO: 21)
  • Primer 11 5'- CGCACGCGAAGCGTCT CCAGG CGCAGCAAAGCTTACAGCAA-3' (SEQ ID NO: 23)
  • Primer 12 5'-AAACTCCAGGGTGCCTGATG-3' (SEQ ID NO: 24)
  • Primer 13 5'- GACGACGCTGGCCACC CCAGG CAACCGCAAATGCAGACACA-3' (SEQ ID NO: 25)
  • Primer 14 5'-ACCCAAATGCAATGGGGCTA-3' (SEQ ID NO: 26)
  • Primer 15 5'- GCGACCCTGCGACGAG CCAGG ACGCCAGAATGCAACTGAGA-3' (SEQ ID NO: 27)
  • Primer 16 5'-TTCTGGATCAACCGCCCTTC-3' (SEQ ID NO: 28)
  • Primer 17 5'- CTGGCGTGCACGGGTC CCAGG AGACATCAAGGCCAAGACGG-3' (SEQ ID NO: 29)
  • Primer 18 5'-TGTCTGCAATCCCTGGGTTC-3' (SEQ ID NO: 30)
  • Primer 20 5'-CCCAGGGCCTTGTAAACGTA-3' (SEQ ID NO: 32)
  • C-TAG probe 2 5'-ATAATTAAGAA[T(FAM)]ACACATCGACCCGTACGATG[BHQ1]-3' (SEQ ID NO: 33)
  • C-TAG probe 3 5'-ATTATTATAA[T(HEX)] CGCACGCGAAGCGTCT [BHQ1]-3' (SEQ ID NO: 34)
  • C-TAG probe 4 5'-CTATTATTA[T(Cy5)] GCGACCCTGCGACGAG [BHQ2]-3' (SEQ ID NO: 35)
  • the bold and slanted sequences of the above primers represent the recognition sequence of the restriction enzyme PspGI, and the underlined sequences are the complementary sequences of the artificial target nucleic acid generated thereby.
  • the parentheses represent the positions of the base sequences where the quencher molecule, fluorescent signal molecule, or C3 spacer is located, and the C3 spacer represents an aliphatic linker consisting of three carbons.
  • the C-TAG sequence generated from the amplicon is as follows.
  • C-TAG sequence 8 5'- CTCGTCGCAGGGTCGC -3' (SEQ ID NO: 48)
  • the generated C-TAG sequence is hybridized and extended to the probe or DAO, and the extended sequence is as follows.
  • C-TAG extension sequence 4 5’-CATCGTACGGGTCGATGTGTATTCTTAATTAT-3’ (SEQ ID NO: 51)
  • C-TAG extension sequence 6 5’-AGACGCTTCGCGTGCGATTATAATAAT-3’ (SEQ ID NO: 53)
  • C-TAG extension sequence 7 5’-GGTGGCCAGCGTCGTCCTGCCCGCG-3’ (SEQ ID NO: 54)
  • C-TAG extension sequence 8 5’-CTCGTCGCAGGGTCGCATAATAATAG-3’ (SEQ ID NO: 55)
  • C-TAG extension sequence 9 5’-GACCCGTGCACGCCAGGTCGCGGCC-3’ (SEQ ID NO: 56)
  • C-TAG extension sequence 10 5’-CTGCCTGCGCTGCCAGGAGGCCGGG-3’ (SEQ ID NO: 57)
  • Primers 7 and 8, 9 and 10 react with the N and RDRP genes of SARS-CoV-2, respectively; primers 11 and 12, 13 and 14 react with the HA gene of influenza A virus H3N2 and H1N1pdm, respectively; primers 15 and 16 react with the NP gene of influenza A virus; primers 17 and 18 react with the NP gene of influenza B virus; and primers 19 and 20 react with the penton gene of adenovirus.
  • QO #3, 4, 5, and 6 react with the QO binding sites of DaO #3, 4, 5, and 6, respectively.
  • the melting temperature of C-TAG probes 2 to 5 is designed to be 66 to 67°C
  • the melting temperature with each target nucleic acid of DaO #3 to 6 is designed to be 77 to 78°C
  • the melting temperature with DaO including each binding site of QO #3 to 6 is designed to be 68 to 70°C.
  • the tube containing the above reaction solution was subjected to reverse transcription reaction at 55°C for 30 minutes, denatured at 95°C for 15 minutes, and then subjected to 45 cycles of a denaturation step at 95°C for 30 seconds, an annealing step at 60°C for 30 seconds, and an extension step at 75°C for 30 seconds. Fluorescence signals were detected at 60°C and 75°C for each cycle.
  • Fig. 7 As can be confirmed in Fig. 7, under the SARS-CoV-2 template reaction conditions, only the amplification curves by C-TAG probe 2 and DaO #3 are confirmed in the FAM channel at 60°C and 75°C, and no fluorescence signals are confirmed in other channels. Under the H3N2 template reaction conditions, only the amplification curves by C-TAG probe 3 and DaO #4 are confirmed in the HEX channel at 60°C and in CalRed 610 at 75°C, and no fluorescence signals are confirmed in other channels.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention divulgue un procédé PCR pour la détection en temps réel de deux acides nucléiques cibles avec un signal unique. Le procédé de la présente invention détecte un signal pour chaque acide nucléique cible dans une étape de recuit et une étape d'extension parmi les trois étapes de PCR comprenant la dénaturation, l'étape de recuit et les étapes d'extension. En d'autres termes, parmi les deux acides nucléiques cibles, un signal pour un premier acide nucléique cible est détecté lors d'une étape d'hybridation, et un signal pour un deuxième acide nucléique cible est détecté lors d'une étape d'extension. Chaque signal détecté fournit des informations qualitatives et/ou quantitatives concernant chacun des deux acides nucléiques cibles. À ce moment, le signal de détection est le même signal unique pour les deux acides nucléiques cibles, qui peut être détecté dans un canal de détection.
PCT/KR2024/008885 2023-07-03 2024-06-26 Procédé de pcr pour la détection d'au moins deux acides nucléiques multi-cibles avec un signal unique Ceased WO2025009802A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2023-0085510 2023-07-03
KR1020230085510A KR102651224B1 (ko) 2023-07-03 2023-07-03 단일 신호로 2가지 이상의 다중 표적핵산을 검출하기 위한 pcr 방법

Publications (1)

Publication Number Publication Date
WO2025009802A1 true WO2025009802A1 (fr) 2025-01-09

Family

ID=90472840

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2024/008885 Ceased WO2025009802A1 (fr) 2023-07-03 2024-06-26 Procédé de pcr pour la détection d'au moins deux acides nucléiques multi-cibles avec un signal unique

Country Status (2)

Country Link
KR (1) KR102651224B1 (fr)
WO (1) WO2025009802A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118879840A (zh) * 2024-08-19 2024-11-01 赵锦祥 一种基于pcr的多色荧光通道检测方法及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102651224B1 (ko) * 2023-07-03 2024-03-26 (주)진매트릭스 단일 신호로 2가지 이상의 다중 표적핵산을 검출하기 위한 pcr 방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007515956A (ja) * 2003-12-10 2007-06-21 バイオトローブ， インコーポレイテッド 改良された選択的ライゲーションおよび増幅アッセイ
CN109576352A (zh) * 2018-11-25 2019-04-05 江苏宏微特斯医药科技有限公司 单管检测多个待测目标核酸序列的方法、探针及其试剂盒
KR102651224B1 (ko) * 2023-07-03 2024-03-26 (주)진매트릭스 단일 신호로 2가지 이상의 다중 표적핵산을 검출하기 위한 pcr 방법

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007515956A (ja) * 2003-12-10 2007-06-21 バイオトローブ， インコーポレイテッド 改良された選択的ライゲーションおよび増幅アッセイ
CN109576352A (zh) * 2018-11-25 2019-04-05 江苏宏微特斯医药科技有限公司 单管检测多个待测目标核酸序列的方法、探针及其试剂盒
KR102651224B1 (ko) * 2023-07-03 2024-03-26 (주)진매트릭스 단일 신호로 2가지 이상의 다중 표적핵산을 검출하기 위한 pcr 방법

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ARTIKA I MADE, DEWI YORA PERMATA, NAINGGOLAN ITA MARGARETHA, SIREGAR JOSEPHINE ELIZABETH, ANTONJAYA UNGKE: "Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis", GENES, MDPI AG, US, vol. 13, no. 12, 1 December 2022 (2022-12-01), US , pages 2387, XP093014826, ISSN: 2073-4425, DOI: 10.3390/genes13122387 *
BRIAN W KIRK, FEINSOD, FAVIS, KLIMAN, BARANY: "Single nucleotide polymorphism seeking long term association with complex disease", 1 June 2002 (2002-06-01), XP055039969, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC137089/pdf/gkf466.pdf> [retrieved on 20121004], DOI: 10.1093/nar/gkf466 *
TABARA KENTARO; WATANABE KAZUNORI; SHIGETO HAJIME; YAMAMURA SHOHEI; KISHI TAKAMASA; KITAMATSU MIZUKI; OHTSUKI TAKASHI: "Fluorophore–PNA–Quencher/Quencher–DNA probe for miRNA detection", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, ELSEVIER, AMSTERDAM NL, vol. 51, 14 September 2021 (2021-09-14), Amsterdam NL , XP086826307, ISSN: 0960-894X, DOI: 10.1016/j.bmcl.2021.128359 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118879840A (zh) * 2024-08-19 2024-11-01 赵锦祥 一种基于pcr的多色荧光通道检测方法及应用

Also Published As

Publication number Publication date
KR102651224B1 (ko) 2024-03-26

Similar Documents

Publication Publication Date Title
WO2025009802A1 (fr) Procédé de pcr pour la détection d&#39;au moins deux acides nucléiques multi-cibles avec un signal unique
WO2013168924A1 (fr) Détection d&#39;acide nucléique par des sondes oligonucléotidiques clivées à la fois par une exonucléase et une endonucléase
WO2013133680A1 (fr) Composition pour une réaction de transcription inverse à démarrage à chaud ou une réaction en chaîne de la polymérase de transcription inverse à démarrage à chaud
WO2011149255A2 (fr) Rnase h modifiée et détection d&#39;amplification d&#39;acides nucléiques
WO2012030136A2 (fr) Oligonucléotides permettant la détection de bactéries du genre listeria et leur utilisation
US20200048693A1 (en) Method for nucleic acid amplification
WO2015126078A1 (fr) Procédé de détection d&#39;acide nucléique au moyen d&#39;une amplification isotherme asymétrique d&#39;acide nucléique et d&#39;une sonde signal
WO2022265463A1 (fr) Détection de plusieurs acides nucléiques cibles à l&#39;aide de températures de détection multiples
EP4176090A1 (fr) Procédé de réalisation d&#39;une préparation pour la détection d&#39;une séquence d&#39;acide nucléique cible dans un échantillon
WO2019013451A1 (fr) Adn polymérase présentant une spécificité de mutation génique accrue et composition tampon de pcr permettant d&#39;accroître l&#39;activité de ladite adn polymérase
WO2021133088A1 (fr) Molécules d&#39;acide nucléique double brin et procédé d&#39;élimination de l&#39;adaptateur de verre dans une banque d&#39;adn au moyen de celles-ci
WO2015105336A1 (fr) Procédé de test de gène mutant par réaction en chaîne de la polymérase en temps réel utilisant de l&#39;adn polymérase ayant une activité inhibée d&#39;endonuclease 5&#39;flap
WO2023106868A1 (fr) Procédé de génération d&#39;un brin unique et procédé de détection d&#39;une mutation l&#39;utilisant
WO2020145711A1 (fr) Adn polymérase pour la détection de mutation d&#39;egfr et trousse comprenant celle-ci
WO2020145715A1 (fr) Adn polymérase pour détecter des mutations tert et trousse comprenant cette derniere
WO2024186140A1 (fr) Procédé de détection d&#39;acide nucléique cible à l&#39;aide d&#39;acide nucléique artificiel
WO2023106866A1 (fr) Amorce à boucle marquée comprenant un système de double marquage interactif simple brin, et procédé d&#39;amplification isotherme d&#39;acide nucléique l&#39;utilisant
EP4301880A1 (fr) Méthode pour la détection de mutations du sars-cov-2
WO2018016683A1 (fr) Méthode de détection de séquence d&#39;acide nucléique cible utilisant une amplification nichée de signal de type amplification multiple
WO2023146243A1 (fr) Procédé de détection d&#39;acides nucléiques cibles dans un échantillon
WO2013081198A1 (fr) Kit de détection du virus de l&#39;hépatite c et procédé de détection du virus de l&#39;hépatite c l&#39;utilisant
WO2026005334A1 (fr) Méthode destinée à l&#39;analyse de l&#39;activité de la 5&#39;-nucléase
WO2024005458A1 (fr) Procédé d&#39;amplification d&#39;acide nucléique cible à l&#39;aide d&#39;acide bêta-hydroxy
US20120052483A1 (en) Kit for detecting hiv-1 and method for detecting hiv-1 using the same
WO2025116499A1 (fr) Procédé d&#39;analyse d&#39;activité de polymérase d&#39;acide nucléique à l&#39;aide d&#39;amorce auto-dimère

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24836250

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE