WO2023071788A1 - Procédé de pcr par fluorescence pour la détection d'acides nucléiques - Google Patents

Procédé de pcr par fluorescence pour la détection d'acides nucléiques Download PDF

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WO2023071788A1
WO2023071788A1 PCT/CN2022/124790 CN2022124790W WO2023071788A1 WO 2023071788 A1 WO2023071788 A1 WO 2023071788A1 CN 2022124790 W CN2022124790 W CN 2022124790W WO 2023071788 A1 WO2023071788 A1 WO 2023071788A1
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blocking
nucleotide
primer
nucleic acid
fluorescent
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谭若颖
张奉武
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上海翔琼生物技术有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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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
    • C12Q1/68—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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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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
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Definitions

  • the present disclosure relates to the field of molecular biology, in particular to methods for amplifying and detecting nucleic acids by polymerase chain reaction. Specifically, the present disclosure relates to a fluorescent PCR method for detecting nucleic acid by using primer-activated polymerization reaction, such as pyrophosphorolysis activated polymerization (Pyrophosphorolysis activated polymerization, PAP).
  • primer-activated polymerization reaction such as pyrophosphorolysis activated polymerization (Pyrophosphorolysis activated polymerization, PAP).
  • Polymerase chain reaction is a nucleic acid amplification reaction used to amplify specific DNA fragments, and is widely used in the diagnosis of genetic diseases, criminal investigation, gene cloning, and DNA sequence determination.
  • a conventional PCR reaction system includes: template DNA, nucleic acid polymerase, primers, dNTP, Mg 2+ , and buffer.
  • the conventional PCR reaction includes a series of cyclical temperature-changing steps. Each cycle starts with denaturing the template DNA into a single strand at a high temperature, and then combines the primers with the single-stranded template DNA at a low temperature according to the principle of base complementary pairing, and then adjusts the temperature.
  • the DNA polymerase synthesizes a complementary strand along the direction from the phosphate to the five-carbon sugar (5'-3'), so that the amount of template DNA doubles in each cycle. Due to the certain error tolerance of primers and single-stranded template DNA complementarity, conventional PCR reactions will produce false positives in the presence of strong background DNA (that is, a large number of sequences that are very close to the template DNA sequence, such as a sequence that differs by only one nucleotide). Positive, that is, the reaction product is the product of background DNA amplification. Therefore, it is often not possible to use conventional PCR reactions to directly and accurately detect template DNA in samples in the presence of strong background DNA, such as detecting small amounts of mutant sequence in a sample in which a large amount of wild-type sequence is present.
  • Primer-activated polymerization is a special class of nucleic acid amplification reactions that can accurately detect template DNA in samples in the presence of strong background DNA.
  • the primer-activated polymerization reaction system uses specifically modified blocking primers, such as the use of dideoxynucleotides at the 3' end of the primers, so that the nucleic acid polymerase cannot mediate DNA strands when the blocking primers are not unblocked (that is, not activated). synthesis. Therefore, before the polymerase mediates the synthesis reaction of the DNA chain, it is first necessary to unblock the blocking primer to trigger the polymerization reaction.
  • the primer-activated polymerization reaction ensures that the unblocking reaction can only occur when there is a template DNA sequence in the system. Therefore, the primer-activated polymerization reaction can ensure that the amplified product is not produced by the combination of the primer and the background DNA sequence, which greatly reduces the false positive of the reaction.
  • Typical primer-activated polymerization reactions include pyrophosphorolysis activated polymerization (Pyrophosphorolysis activated polymerization, PAP).
  • PAP uses 3' end-blocking primers (such as dideoxynucleotides) to perform nucleic acid amplification using pyrophosphorolysis tandem polymerization reactions of DNA polymerases (Liu Q, Sommer SS, Biotechniques 2000, 29:1072-1076, 1078, 1080).
  • the 3' end of the blocking primer cannot undergo pyrophosphorolysis reaction in the absence of a template or in a state that is not complementary to the template, resulting in the inability of DNA polymerase to extend.
  • the amplification product of the PCR reaction can be detected by fluorescence method.
  • Fluorescent methods currently used to detect nucleic acid amplification can be divided into two main types: 1) non-specific fluorescent label-dye methods (such as SYBR Green I); and 2) specific fluorescent label-probe methods (such as TaqMan probes )
  • SYBR Green I is a DNA double-strand minor groove binding dye, which emits very weak light in the free state, and the fluorescence intensity is significantly enhanced only after binding to double-stranded DNA, and this binding is non-specific.
  • the advantage of the SYBR Green I method is that it is low in cost, and it can determine several products generated by PCR and the presence or absence of primer dimers through the melting curve function. However, this method has high requirements on the specificity of primers, and it is easy to combine with non-specific double-stranded DNA to generate fluorescent signals, resulting in false positives. It has no template specificity, can only detect a single target nucleic acid, and cannot be used for multiplex PCR amplification to distinguish multiple target nucleic acids.
  • the 5' end of the TaqMan probe is covalently linked to a reporter fluorescent group, and the 3' terminal nucleotide is linked to a quencher group.
  • the fluorescent energy emitted by the reporter fluorescent group is absorbed by the quencher group and there is no fluorescence.
  • PCR amplification when the DNA polymerase extends the primer, its 5'-3' exonuclease activity will hydrolyze the probe bound to the template, releasing the fluorophore and emitting fluorescence.
  • the main advantages of TaqMan probes include high specificity, high sensitivity, and good repeatability. It can also screen multiple genes in the same system at the same time. Different genes correspond to different probes, and different probes correspond to different fluorescent labels.
  • TaqMan probes are not suitable for PAP PCR reactions because the DNA polymerases used in classic PAP PCR reactions often do not have 5’-3’ exonuclease activity.
  • One aspect of the present disclosure provides a nucleic acid detection method combining a primer-activated polymerization reaction and a specific fluorescent label-probe.
  • the method includes:
  • a primer pair for amplifying the target sequence to generate an amplicon comprising a blocking primer comprising
  • a blocking nucleotide located at the 3' end of the blocking primer is A blocking nucleotide located at the 3' end of the blocking primer
  • the fluorescent signal of the fluorescent group is quenched by the quenching group when the blocking primer is not hybridized to the target sequence or the amplicon, wherein the blocking nucleotide can block the Nucleic acid polymerase extension;
  • a deblocking agent capable of removing the blocking nucleotide from the blocking primer when the blocking nucleotide hybridizes to the target sequence or the amplicon such that all said nucleic acid polymerase is capable of extending from said blocking primer
  • the present disclosure provides a method for multiplex nucleic acid detection, the method comprising:
  • each primer pair comprises a respective blocking primer comprising
  • a blocking nucleotide located at the 3' end of the blocking primer is A blocking nucleotide located at the 3' end of the blocking primer
  • said fluorophore is quenched by said quencher when said respective blocking primer is not hybridized to its corresponding target sequence or amplicon, wherein said blocking nucleotide can block said nucleic acid polymerase extend,
  • blocking primers contain the same or different fluorescent groups
  • a deblocking agent capable of removing said blocking nucleotide from said respective blocking primer when said blocking nucleotide hybridizes to its corresponding target sequence or amplicon removal on the blocking primer such that the nucleic acid polymerase is able to extend from the blocking primer;
  • the multiple amplicons can be distinguished by the difference in the size of the fluorescent group, amplification Ct value, melting curve, melting peak or amplicon .
  • the blocking nucleotides are 2',3'-dideoxynucleotides, ribonucleotide residues, 2',3'SH nucleotides or 2'-O-PO 3 Nucleotides.
  • the deblocking agent is selected from the group consisting of ampliTaq or KlenTaq polymerase, pyrophosphate, tripolyphosphate, RNase H2 and CS5 DNA polymerase with F667Y mutation, wherein the CS5 DNA polymerase Has a mutation selected from G46E, L329A, Q601R, D640G, I669F, S671F, E678G or a combination of these mutations.
  • the first nucleotide in the blocking primer is located upstream or downstream of the second nucleotide.
  • the first nucleotide or the second nucleotide is selected from deoxyribonucleotide thymine (dTMP), deoxyribonucleotide adenine (dAMP), deoxyribonucleotide guanine Purine (dGMP), deoxyribonucleotide cytosine (dCMP), deoxyribonucleotide uracil (dUMP), including modified above-mentioned nucleotides.
  • dTMP deoxyribonucleotide thymine
  • dAMP deoxyribonucleotide adenine
  • dGMP deoxyribonucleotide guanine Purine
  • dCMP deoxyribonucleotide cytosine
  • dUMP deoxyribonucleotide uracil
  • the sequence on the blocking primer between the first nucleotide and the second nucleotide is at least partially complementary to a target sequence. In certain embodiments, the sequence between the first nucleotide and the second nucleotide on the blocking primer is not complementary to the target sequence.
  • the target sequence comprises a mutant nucleotide
  • the blocking nucleotide is complementary to the mutant nucleotide
  • the sequence between the first nucleotide and the second nucleotide on the blocking primer forms a stem-loop structure.
  • the fluorescent group is selected from the group consisting of FAM, VIC, JOE, NED, TET, HEX, TAMRA, ROX, TEXASRED, CY3, CY5, CY5.5, and CY7; wherein the quencher
  • the killing group is selected from the group consisting of BHQ1, BHQ2, BHQ3, Dabcyl, MGB and TAMARA.
  • the blocking primer further comprises mismatched nucleotides that are not complementary to the target sequence when the blocking primer hybridizes to the target sequence.
  • the mismatched nucleotide is 2-18 nucleotides away from the blocking nucleotide.
  • the blocking primer further comprises ribonucleotides.
  • the distance between the ribonucleotide and the blocking nucleotide is 2-18 nucleotides, and the ribonucleotide is between the blocking nucleotide and the target sequence or the extension.
  • the accumulator can be excised by RNase H2 during hybridization.
  • the blocking primer is 8 to 70 nucleotides in length.
  • the nucleic acid polymerase lacks 5'-3' exonuclease activity.
  • the method further comprises detecting the amplification Ct value of the amplicon.
  • the method further comprises detecting a melting curve or melting peak of the amplicon.
  • the method further comprises detecting the size of the amplicon.
  • the nucleic acid sample comprises modified or unmodified single-stranded DNA, double-stranded DNA, RNA, cDNA, or combinations thereof.
  • Figure 1 shows the principle of linear fluorophore-quencher double-labeled blocking primers for primer-activated polymerization fluorescent PCR amplification of nucleic acids.
  • the dideoxynucleotides at the 3' ends of the two corresponding blocking primers are shown as non-overlapping, but can also be designed to overlap.
  • Figure 2 shows the principle of stem-loop fluorophore-quencher double-labeled blocking primers for primer-activated polymerization fluorescent PCR amplification of nucleic acids.
  • the dideoxynucleotides at the 3' ends of the two corresponding blocking primers are shown as non-overlapping, but can also be designed to overlap.
  • Figure 3 shows the results of single or double PAP fluorescent PCR detection of deafness gene mutations with stem-loop fluorophore-quencher double-labeled blocking primers.
  • Figure 3A is the detection result of stem-loop type A fluorescent group-quencher double-labeled blocking primer used in single-plex PAP fluorescent PCR
  • Figure 3B is the detection result of stem-loop type B fluorescent group-quencher double-labeled blocking primer The detection results of primers used in single-plex PAP fluorescent PCR
  • Figure 3C is the detection result of stem-loop A-type fluorophore-quencher double-labeled blocking primers used in dual-PAP fluorescent PCR
  • Figure 3D is the detection result of stem-loop B-type fluorophore
  • the group-quencher double-labeled blocking primer is used for the detection of double PAP fluorescent PCR.
  • Figure 4 shows the results of double PAP fluorescent PCR melting curve detection of deafness gene mutations using stem-loop fluorophore-quencher double-labeled blocking primers.
  • the present disclosure provides a novel fluorescent PCR method for detecting nucleic acid, including single-plex fluorescent PCR and multiplex fluorescent PCR.
  • the single-plex fluorescent PCR combines primer-activated polymerization reaction and at least one fluorophore-quencher double-labeled blocking primer, which can detect the corresponding target sequence with high selectivity and high specificity.
  • Multiplex fluorescent PCR can be performed using multiple fluorophore-quencher double-labeled blocking primers, and multiple target sequences can be detected using different fluorophores, amplification Ct values, melting curves or melting peaks.
  • a and “the” are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article.
  • a protein means one protein or more than one protein.
  • PCR reaction refers to a nucleic acid amplification reaction used to amplify a nucleic acid comprising a specific sequence.
  • a conventional PCR reaction system includes: template DNA, nucleic acid polymerase, primers, dNTP, Mg 2+ , and buffer.
  • a conventional PCR reaction consists of a series of cyclic temperature-varying steps, each cycle starting with denaturing the template DNA into a single strand at a high temperature (often around 95°C), and then denaturing the primers with the single strand at a low temperature (often around 60°C).
  • the template DNA is combined according to the principle of complementary base pairing, and then adjusted to the optimum reaction temperature of DNA polymerase (about 72°C), so that DNA polymerase synthesizes complementary DNA along the direction from phosphate to five-carbon sugar (5'-3'). strands, thereby doubling the amount of template DNA with each cycle.
  • fluorescent PCR reaction refers to a method that combines PCR reaction with fluorescence detection technology, so that nucleic acid amplification can be quantitatively monitored according to the intensity of the monitored fluorescent signal.
  • fluorophore and “fluorescent molecule” are used interchangeably and refer to a group or molecule that can generate fluorescence. When the fluorophore absorbs short-wavelength light energy, it can emit longer-wavelength fluorescence. Each fluorophore has a characteristic absorption spectrum and a characteristic emission spectrum. The specific wavelength at which a fluorophore absorbs energy most efficiently is referred to as peak absorption, while the wavelength at which the fluorophore most efficiently fluoresces is referred to as peak emission.
  • the fluorophore is selected from the group consisting of FAM, VIC, JOE, NED, TET, HEX, TAMRA, ROX, TEXASRED, CY3, CY5, CY5.5, and CY7.
  • quencher group As used herein, the terms “quencher group”, “quencher molecule” or “quencher” are used interchangeably and refer to a group or molecule that reduces the output fluorescence intensity of a fluorophore. It has a characteristic absorption spectrum and absorption peaks. In order for the mechanism via fluorescence resonance energy transfer (FRET) to work, the absorption spectrum of the quencher must overlap the emission spectrum of the fluorophore and be sufficiently close to the fluorophore, e.g. no more than 30 nucleosides acid.
  • the quenching group is selected from the group consisting of BHQ1, BHQ2, BHQ3, Dabcyl, MGB, and TAMARA.
  • pyrophosphorylation or "pyrophosphorylation” is the reverse reaction of deoxyribonucleic acid polymerization.
  • the polymerase in the presence of pyrophosphate, removes the 3' terminal nucleotide from double-stranded DNA to generate a nucleoside triphosphate and a double nucleotide from which this nucleotide is removed at the 3' end.
  • Stranded DNA [dNMP] n + PPi ⁇ [dNMP] n-1 + dNTP.
  • primer is a macromolecule with a specific nucleotide sequence used to stimulate synthesis at the initiation of a nucleic acid amplification reaction (or nucleotide polymerization).
  • the primers appear in pairs (i.e., primer pairs), which are usually two artificially synthesized oligonucleotide sequences, one primer is complementary to a DNA template strand at one end of the region to be amplified, and the other primer is complementary to a DNA template strand at one end of the region to be amplified.
  • the other DNA template strand at the other end of the region to be amplified is complementary, and its function is as the starting point of nucleotide polymerization, and the nucleic acid polymerase can start to synthesize a new nucleic acid strand from its 3' end.
  • the length of each primer in the primer pair is independently 8 to 70 nucleotides; preferably, 8 to 50 nucleotides; most preferably, 8 to 40 nucleosides acid.
  • nucleic acid and “polynucleotide” are used interchangeably to refer to a polymeric form of nucleotides (eg, deoxyribonucleotides or ribonucleotides) of any length, or analogs thereof.
  • a polynucleotide can have any three-dimensional structure, and can have any known or unknown function.
  • Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant Polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, regulatory regions, isolated RNA of any sequence, nucleic acid probes and primers. Nucleic acid molecules can be linear or circular.
  • nucleic acid comprising a target sequence As used herein, the terms “nucleic acid comprising a target sequence”, “target nucleic acid”, “test nucleic acid” or “target nucleic acid” are used interchangeably and refer to a nucleic acid fragment that is specifically amplified in a fluorescent PCR method, or A nucleic acid fragment that can trigger a detectable signal in a PCR reaction system, or a nucleic acid fragment that can be specifically detected by a nucleic acid detection method.
  • the nucleic acid to be tested in the present disclosure may be a nucleic acid fragment with a specific site mutation, or a trace specific nucleic acid fragment in a complex background.
  • the test nucleic acid is not a single test nucleic acid fragment, it may include N different test nucleic acids, where 2 ⁇ N ⁇ 250.
  • the nucleic acid to be tested in the present disclosure includes: modified or unmodified single-stranded DNA, double-stranded DNA, RNA, cDNA or a combination thereof.
  • the target nucleic acid comprises wild-type or mutant.
  • nucleotide is the basic constituent unit of nucleic acid. Nucleotides are composed of a nitrogenous base, a five-carbon sugar and one or more phosphate groups. There are five nitrogenous bases, namely adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Nucleotides whose five-carbon sugar is deoxyribose are called deoxyribonucleotides (monomers of DNA), and nucleotides whose five-carbon sugars are ribose are called ribonucleotides (monomers of RNA).
  • nucleotides include, but are not limited to, deoxyribonucleotide thymine (dTMP), deoxyribonucleotide adenine (dAMP), deoxyribonucleotide guanine (dGMP), deoxyribonucleotide Cytosine (dCMP), deoxyribonucleotide uracil (dUMP), or modified nucleotides of the above.
  • dTMP deoxyribonucleotide thymine
  • dAMP deoxyribonucleotide adenine
  • dGMP deoxyribonucleotide guanine
  • dCMP deoxyribonucleotide Cytosine
  • dUMP deoxyribonucleotide uracil
  • nucleic acid polymerase is a nucleic acid polymerase used in a primer activated polymerization system for polymerizing or extending deoxyribonucleic acid.
  • the nucleic acid polymerase may be a nucleic acid polymerase that does not have 5'-3' exonuclease activity.
  • One aspect of the present disclosure provides a nucleic acid detection method combining a primer-activated polymerization reaction and a specific fluorescent label-probe. Since primer-activated polymerization reactions, such as classic PAP reactions, generally require the use of DNA polymerases without 5'-3' exonuclease activity for polymerization reactions, and commonly used specific fluorescent label-probes, such as Taqman probe reactions, require The fluorescent signal is generated by the hydrolysis of the probe by a DNA polymerase with 5'-3' exonuclease activity, so primer-activated polymerization generally cannot be combined with Taqman probes.
  • the method provided by the present disclosure not only ensures the specificity of amplification, but also maintains the amplification efficiency of fluorescent PCR by combining primers to activate the polymerization reaction and specific fluorescent marker-probes.
  • Primer-activated polymerization is a polymerase chain reaction in which a specifically modified blocking primer is included in the system. Nucleic acid polymerase cannot mediate DNA strand synthesis when the blocking primer is not unblocked (ie not activated). Therefore, before the polymerase mediates the synthesis reaction of the DNA chain, it is first necessary to unblock the blocking primer to trigger the polymerization reaction.
  • the primer-activated polymerization reaction ensures that the unblocking reaction can only occur when there is a template DNA sequence in the system. Therefore, the primer-activated polymerization reaction can ensure that the amplified product is not produced by the combination of the primer and the background DNA sequence (ie, a sequence very close to the target sequence), which greatly reduces the false positive of the reaction.
  • the primer activated polymerization is Pyrophosphorolysis activated polymerization (PAP).
  • PAP uses 3' end-blocking primers (such as dideoxynucleotides) to perform nucleic acid amplification using pyrophosphorolysis tandem polymerization reactions of DNA polymerases (Liu Q, Sommer SS, Biotechniques 2000, 29:1072-1076, 1078, 1080).
  • the 3' end of the blocking primer cannot undergo pyrophosphorolysis reaction in the absence of a template or in a state that is not complementary to the template, resulting in the inability of DNA polymerase to extend.
  • the present disclosure provides a nucleic acid detection method that combines PAP and a specific fluorescent label-probe.
  • the primer activated polymerization reaction is RNase H dependent PCR.
  • RNase H-dependent PCR also uses 3' end-blocking primers and contains a ribonucleotide near the 3' end.
  • the RNase H-dependent PCR system contains RNase H2, which can cut the primer at the ribonucleotide when the primer and the DNA template hybridize, and this enzymatic cleavage activity is less efficient when there is a mismatch near the ribonucleotide . Therefore, the 3' end of the blocking primer cannot undergo RNase H2-dependent enzyme cleavage in the absence of a template or in a non-complementary state to the template, resulting in the inability of DNA polymerase to extend.
  • the present disclosure provides a nucleic acid detection method combining RNase H-dependent PCR and specific fluorescent label-probes.
  • a blocking primer refers to a primer whose 3' end is blocked to block extension by nucleic acid polymerase.
  • the 3' end of the blocking primer is blocked by a blocking nucleotide.
  • a blocking nucleotide is any nucleotide nucleic acid with a specific structure capable of blocking the extension of a nucleic acid polymerase. Examples of blocking nucleotides include, but are not limited to, dideoxynucleotides (such as 2',3'-dideoxynucleotides), ribonucleotides, 2',3'SH nucleotides and 2'-O- PO 3 nucleotides.
  • the blocking primer can be deblocked (i.e. activated) by a deblocking agent under specific conditions (for example, the 3' terminal blocking nucleotide is complementary to the template DNA).
  • the deblocking agent can be any component capable of removing the blocking nucleotide from the blocking primer when the blocking nucleotide hybridizes to the target sequence.
  • deblocking agents include, but are not limited to, ampliTaq or KlenTaq polymerase with the F667Y mutation, pyrophosphate, tripolyphosphate, RNase H2, and CS5 DNA polymerase with specific mutations (the mutations include G46E, L329A, Q601R, D640G, I669F, S671F, E678G or a combination of these mutations).
  • the primer activation step of the present disclosure is achieved by pyrophosphorylation.
  • the deblocking agent included KlenTaq polymerase, pyrophosphate, with the F667Y mutation.
  • the nucleic acid detection method provided by the present disclosure uses a double-labeled blocking primer with a fluorescent group-quencher group, which can detect the target sequence in the system through a fluorescent signal.
  • the nucleic acid detection method provided by the present disclosure can be understood through the illustrative embodiments shown in FIGS. 1 and 2 .
  • Figure 1 shows an embodiment of nucleic acid detection using a linear structured fluorophore-quencher double-labeled blocking primer.
  • the detection system includes a nucleic acid sample to be tested, a nucleic acid polymerase, a pair of blocking primers for amplifying a target sequence to generate an amplicon, and a deblocking agent.
  • One blocking primer in the primer pair includes a first nucleotide attached to a fluorophore (R), a second nucleotide attached to a quencher (Q), and a blocking nucleotide (ddN) located at the 3' end of the blocking primer. ).
  • the blocking primer is free to coil in the natural state, the fluorescent energy emitted by the fluorescent group (R) is absorbed by the quencher group (Q), and no fluorescent signal can be detected in the system.
  • the blocking primer is bound to the template DNA, the blocking primer is in a rigid state, the distance between the fluorescent group (R) and the quenching group (Q) is separated, and the fluorescent energy emitted by the fluorescent group (R) cannot be quenched. (Q) absorbed, enabling the detection of fluorescent signals in the system.
  • the deblocking agent cannot remove the blocking nucleotide (ddN) from the blocking primer, and thus the nucleic acid polymerase cannot extend from the blocking primer to generate an amplification product.
  • the fluorescent signal in the system is very weak due to the low content of background DNA in the system.
  • the deblocking agent removes the blocking nucleotide (ddN) from the blocking primer so that the nucleic acid polymerase can extend from the blocking primer to generate an amplification product.
  • more and more blocking primers can bind to the target sequence in the amplification product and assume a rigid state, so that the fluorescent signal in the system increases exponentially.
  • Figure 2 shows an embodiment of nucleic acid detection using a stem-loop fluorophore-quencher double-labeled blocking primer.
  • the detection system comprises nucleic acid samples to be tested, nucleic acid polymerase, a pair of blocking primers for amplifying the target sequence to generate amplicons, and a deblocking agent.
  • One blocking primer in the primer pair includes a first nucleotide attached to a fluorophore (R), a second nucleotide attached to a quencher (Q), and a blocking nucleotide (ddN) located at the 3' end of the blocking primer. ).
  • the blocking primer is in a stem-loop structure in the natural state, the distance between the fluorescent group (R) and the quencher group (Q) is close, and the fluorescent signal of the fluorescent group (R) is quenched by the connection quencher group (Q), the system No fluorescent signal was detected.
  • the blocking primer is bound to the template DNA, the blocking primer is in a rigid state, the distance between the fluorescent group (R) and the quenching group (Q) is separated, and the fluorescent energy emitted by the fluorescent group (R) cannot be quenched. (Q) absorbed, enabling the detection of fluorescent signals in the system.
  • the deblocking agent cannot remove the blocking nucleotide (ddN) from the blocking primer, and thus the nucleic acid polymerase cannot extend from the blocking primer to generate an amplification product.
  • the fluorescent signal in the system is very weak due to the low content of background DNA in the system.
  • the deblocking agent removes the blocking nucleotide (ddN) from the blocking primer so that the nucleic acid polymerase can extend from the blocking primer to generate an amplification product.
  • more and more blocking primers can bind to the target sequence in the amplification product and assume a rigid state, so that the fluorescent signal in the system increases exponentially.
  • the selection and position of the fluorophore and the quenching group of the fluorophore-quencher double-labeled blocking primer need to meet the following conditions: 1) the absorption spectrum of the quenching group is consistent with the emission of the fluorophore The spectra overlap each other; 2) The distance from the quencher group to the fluorescent group is 15-35 nucleotides, and the quencher group of the primer can quench the fluorescent group in the natural state and can emit light when the primer is in a rigid state.
  • the 5' terminal nucleotide of the blocked primer is covalently bonded to a fluorescent group, and a nucleotide modification in the middle has a quenching group; otherwise, the 5' terminal nucleoside of the blocked primer
  • the acid is covalently linked to a quenching group, and a nucleotide modification in the middle carries a fluorescent group.
  • the nucleotide connected to the fluorescent or quencher group is preferably deoxyribonucleotide thymine (dTMP).
  • the sequence between the first nucleotide and the second nucleotide on the blocking primer is partially complementary or not complementary to the target sequence.
  • sequence close to the blocking nucleotide (ddN) on the blocking primer is at least partially complementary to the target sequence.
  • the blocking primer is 8 to 70 nucleotides in length. In some embodiments, the blocking primer is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.
  • blocking primers can be further modified to reduce amplification of unwanted nucleic acids.
  • the modification is the introduction of at least one mismatched nucleotide in the primer that is not complementary to the target sequence when the blocking primer hybridizes to the target sequence.
  • the mismatched nucleotide is on the 5' side with a blocking nucleotide.
  • the distance between the mismatched nucleotide and the blocking nucleotide is 2-18 nucleotides.
  • template nucleic acid comprising a target sequence can be detected by a fluorescent signal in the methods of the present disclosure.
  • the fluorescent signal detected in the reaction system may be the background or "noise" of the reaction.
  • the amplified signal can be distinguished from the background by a baseline, ie, the level of fluorescent signal at the initial cycling period, and a threshold, ie, a level of fluorescent signal significantly above the baseline signal.
  • the methods of the present disclosure further comprise the step of detecting the amplification Ct value of the amplicon.
  • Ct Threshold cycle, threshold cycle number
  • the target nucleic acid can be quantitatively detected by fluorescent PCR to specifically amplify the fluorescent amplification signal of the target nucleic acid or detect the Ct value. Accordingly, detection methods of the present disclosure may be qualitative or quantitative.
  • the methods provided by the present disclosure can simultaneously detect multiple target sequences in a sample. Accordingly, the present disclosure provides, in another aspect, multiplexed nucleic acid detection methods.
  • the multiple detection method of the present disclosure can simultaneously detect multiple target sequences through at least two approaches.
  • different target sequences can be distinguished by different fluorescent signals.
  • the blocking primers in the reaction system of the present disclosure may include multiple blocking primers with different fluorophore-quencher combinations. Among them, blocking primers with different fluorescent groups can be designed for different target sequences in the same reaction system (or detection system), so as to distinguish amplification products of multiple target sequences in one reaction; The same universal quencher or different quenchers can be selected depending on the fluorophore. In some embodiments, 2, 3, 4, or 5 amplification products can be distinguished in one reaction using different fluorescent signals.
  • different target sequences can be distinguished by the melting curve or melting temperature of the amplification products.
  • the dissolution curve refers to the curve of the degree of degradation of the double helix structure of DNA as the temperature increases.
  • Melting temperature (Tm) refers to the temperature at which half of the entire DNA double helix structure is degraded.
  • Tm melting temperature
  • Melt curve analysis can be used to identify various reaction products, including nonspecific products.
  • the melting curve is generated by gradually increasing the temperature while monitoring the fluorescent signal at each step.
  • the fluorescent dye or fluorescent probe returns to the free state, resulting in a decrease in the fluorescent signal.
  • the negative first derivative of the fluorescence signal change is plotted against the temperature, and there is a characteristic peak (Tm value) at the melting temperature of the amplified product, and the position of the characteristic peak can specifically distinguish different amplified products.
  • the DNA melting curve can distinguish between 2, 3, 4, or 5 amplification products.
  • different fluorescent signals and melting curves can be combined to distinguish different target sequences.
  • multiple blocking primers are designed, which have the same or different fluorescent groups for different templates; on this basis, PCR melting curve analysis is also performed on the detection system.
  • up to 4 fluorophores that can emit different fluorescent signals can be selected according to needs, and up to 5 Tm characteristic peaks can be distinguished in each fluorescent channel according to the PCR melting curve, Thereby, the purpose of simultaneously detecting 20 target sequences in one reaction is achieved.
  • different target sequences can be distinguished by the size of the amplicons.
  • Techniques for sizing amplicons are well known in the art and include, but are not limited to, electrophoresis (eg, gel electrophoresis, capillary electrophoresis, etc.), mass spectrometry, and the like.
  • different fluorescent signals and amplicon sizes can be combined to distinguish different target sequences.
  • multiple blocking primers are designed, which have the same or different fluorescent groups for different templates; on this basis, the size of the amplicon is also detected.
  • up to 4 fluorophores that can emit different fluorescent signals can be selected according to needs, and at least 2, 3, 4 fluorophores can be distinguished in each fluorescent channel according to the size of the amplicon. , 5, 5, 6, 7, 8, 9, 10, 20 or 30 amplicons, so as to simultaneously detect at least 20, 30, 40, 50, 60, 70, 80, 90, 100 in one reaction, 200 or more target sequences.
  • the present disclosure provides a novel fluorescent PCR method for nucleic acid detection, which not only ensures the specificity of fluorescent PCR amplification, but also maintains the efficiency of fluorescent PCR amplification by combining primers to activate polymerization reactions and specific fluorescent marker-probes.
  • the present disclosure also provides a linear fluorophore-quencher dual-labeled blocking primer and a stem-loop fluorophore-quencher dual-labeled blocking primer, two different structural types of fluorophore-quencher double labeling
  • Blocking primers simplifies probe design, combining blocking primers and probes into one, reducing the difficulty of primer design and optimization as well as the cost of use.
  • the fluorophore-quencher double-labeled blocking primer design provided in the present disclosure can be used for upstream primers or downstream primers or both upstream and downstream primers.
  • a single primer or upstream and downstream primers with different fluorescent groups there are multiple (>5,>10,>15) fluorescent group combinations, one fluorescent combination corresponds to one template, breaking through the detection of fluorescence by current fluorescent PCR instruments The bottleneck limit of the channel.
  • the detection of the target nucleic acid can be confirmed by the melting curve of the fluorescent PCR specific amplification of the target nucleic acid, and each pair of fluorophore-quencher double labels can correspond to amplification products with different melting curves. Further expand the multiplicity of detection.
  • the fluorescent PCR method of the present disclosure can carry out a single reaction, and can also use multiple fluorophore-quencher double-labeled blocking primers with different fluorophores, so as to distinguish multiple templates in one reaction to achieve a single tube
  • Multiplex fluorescent PCR reaction overcomes the problem that dye-based SYBR fluorescent PCR cannot perform multiple reactions and has poor specificity, simplifies operation and reduces reagent consumption.
  • the present disclosure combines PAP technology with fluorescent PCR technology, which not only inherits the characteristics of high sensitivity, high selectivity, and high specificity of PAP technology, but also has the function of detecting nucleic acid quantitatively.
  • One tube of multiple PAP fluorescent PCR reactions will not produce cross-reactions, which solves the problem of non-specific reactions in multiple PCR reactions and avoids false positive results.
  • the disclosed method is simple and convenient to operate, and the results can be observed in real time without tube opening and PCR post-treatment, and PCR product pollution will not occur.
  • Two mutant nucleic acid sequences of the human deafness gene SLC26A4 were obtained from the COSMIC database: IVS15+5G>A, 2162C>T, and the mutant nucleic acid sequences of IVS15+5G>A, 2162C>T and their corresponding wild-type genes were chemically synthesized
  • the fragments were respectively inserted into the pUC57 vector to construct mutant and wild-type recombinant plasmid DNA.
  • the extracted recombinant plasmid DNA was quantified by ultraviolet absorption at 260nm. Each plasmid was diluted with TE buffer to a concentration of 10000 copies/ ⁇ l.
  • Each reaction mixture contained 50mM Tris PH8.0, 0.2mM dNTP, 3mM MgCl2, 90nM pyrophosphate, 2 units of KlenTaq-S DNA polymerase, each blocking primer concentration was 0.5 ⁇ M, wild-type genomic DNA and / Or plasmid DNA, add DNase/RNase-free water to a final volume of 20 ⁇ L, use ABI 7500 fluorescent quantitative PCR instrument or Hongshi SLAN 96S to amplify, run the following program: 95°C for 2 minutes; 95°C for 15 seconds, 60°C °C for 120 seconds, 40 cycles, 60 °C to collect FAM or/and HEX fluorescence signals.
  • Each reaction mixture contained 50mM Tris PH8.0, 0.2mM dNTP, 3mM MgCl2, 90nM pyrophosphate, 2 units of KlenTaq-S DNA polymerase, each blocking primer concentration was 0.5 ⁇ M, wild-type genomic DNA and / Or plasmid DNA, add DNase/RNase-free water to a final volume of 20 ⁇ L, use ABI 7500 fluorescent quantitative PCR instrument or Hongshi SLAN 96S to amplify, run the following program: 95°C for 2 minutes; 95°C for 15 seconds, 60°C 120 seconds at °C, 40 cycles; 5 minutes at 95°C ⁇ 2 minutes at 55°C ⁇ 2 minutes at 55°C ⁇ 90°C, among which, the melting curve analysis is carried out at a heating rate of 0.03°C/s at 55°C ⁇ 90°C, and at 55°C ⁇ 90°C Fluorescence signal is collected in the stage; instrument fluorescence channel selection: FAM or/and HEX.
  • Example 2 Stem-loop A-type and B-type fluorophore-quencher double-labeled blocking primers for single and multiplex PAP fluorescent PCR detection of deafness gene mutations
  • a single-plex PAP fluorescent PCR reaction detects the IVS15+5G>A mutation using a downstream stem-loop A-type fluorophore-quencher double-labeled blocking primer (SEQ ID NO: 2) and an upstream blocking primer (SEQ ID NO :1) (Table 1)
  • another single-plex PAP fluorescent PCR reaction detects IVS15+5G>A mutation using a downstream stem-loop B-type fluorophore-quencher double-labeled blocking primer (SEQ ID NO:3) and an upstream blocking primer (SEQ ID NO: 1) (Table 1).
  • Stem Loop Type A Fluorophore-Quencher Double Labeled Blocking Primer (SEQ ID NO: 2) stem loop (5' stem arm and 3' stem arm nucleotide labels are underlined) 5' terminal nucleotides are The HEX fluorescent group is covalently linked, and the BHQ1 quencher is covalently linked to the dTMP at the 3' end of the stem arm; the stem-loop B-type fluorescent group-quencher double-labeled blocking primer (SEQ ID NO:3)
  • the stem loop (5' stem arm and 3' stem arm nucleotide markers are underlined) the 5' terminal nucleotide is covalently bonded to the BHQ1 quencher group, and the 3' end dTMP of the stem arm is covalently bonded to HEX Fluorophore.
  • a single-plex PAP fluorescent PCR detection of the 2162C>T mutation uses a downstream stem-loop A-type fluorophore-quencher double-labeled blocking primer (SEQ ID NO:5) and an upstream blocking primer (SEQ ID NO:4) (Table 1), another single-plex PAP fluorescent PCR detection 2162C>T mutation used a downstream stem-loop B-type fluorophore-quencher double-labeled blocking primer (SEQ ID NO:6) and an upstream blocking primer ( SEQ ID NO:4) (Table 1).
  • Stem-loop A-type fluorophore-quencher double-labeled blocking primer (SEQ ID NO: 5) stem-loop (5' stem arm and 3' stem arm nucleotide labels are underlined) 5' terminal nucleotides are The FAM fluorescent group is covalently bonded, and the BHQ1 quencher is covalently bonded to the dTMP at the 3' end of the stem arm; the stem-loop B-type fluorescent group-quencher double-labeled blocking primer (SEQ ID NO:6) The stem loop (5' stem arm and 3' stem arm nucleotide markers are underlined) the 5' terminal nucleotide is covalently bonded to the BHQ1 quencher group, and the 3' end dTMP of the stem arm is covalently bonded to FAM Fluorophore.
  • the stem-loop B-type fluorescent group-quencher double-labeled blocking primer pair The detection Ct is lower than that of type A, showing that the amplification efficiency of type B fluorophore-quencher double-labeled blocking primer pair is higher, and the fluorescence amplification signal is also stronger.
  • Example 3 Stem-loop A-type and B-type fluorophore-quencher double-labeled blocking primers for multiplex PAP fluorescent PCR detection of deafness gene mutations
  • a dual PAP fluorescent PCR simultaneously detects IVS15+5G>A, 2162C>T mutations using two downstream stem-loop A-type fluorophore-quencher double-labeled blocking primers (SEQ ID NO: 2 and SEQ ID NO: 5 ) and two upstream blocking primers (SEQ ID NO:1 and SEQ ID NO:4) (Table 1), another dual fluorescent PCR detection of IVS15+5G>A, 2162C>T mutations used two downstream stem-loop B-type Fluorophore-quencher dual-labeled blocking primers (SEQ ID NO:3 and SEQ ID NO:6) and two upstream blocking primers (SEQ ID NO:1 and SEQ ID NO:4) (Table 2).
  • CT40 means that after 40 cycles of amplification reaction, no amplification product is still detected.
  • Fluorophore-quencher double-labeled blocking primers detect deafness gene mutations by multiple PAP fluorescent PCR melting curves
  • a dual PAP fluorescent PCR melting curve simultaneously detects IVS15+5G>A, 2162C>T mutations using two downstream stem-loop A-type fluorophore-quencher double-labeled blocking primers (SEQ ID NO: 2 and SEQ ID NO :5) and two upstream blocking primers (SEQ ID NO: 1 and SEQ ID NO: 4) (Table 1).
  • the IVS15+5G>A, 2162C>T mutation gene fragments were simultaneously amplified and analyzed by melting curves, resulting in HEX and FAM Melting peaks of target gene products with different Tm values of fluorescent signals; when using 20,000 copies of IVS15+5G>A wild-type plasmid and 20,000 copies of 2162C>T wild-type plasmid as templates, the melting curve analysis was performed without HEX and Target gene product melting peaks of FAM fluorescence signal.

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Abstract

Procédé de PCR par fluorescence pour la détection d'un acide nucléique. Le procédé utilise une amorce fermée reliée à un groupe fluorescent et à un groupe d'extinction, afin qu'un acide nucléique cible puisse être détecté avec une grande sélectivité et une grande spécificité.
PCT/CN2022/124790 2021-10-27 2022-10-12 Procédé de pcr par fluorescence pour la détection d'acides nucléiques WO2023071788A1 (fr)

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