WO2020048307A1 - 一种变性泡介导的靶标核酸扩增方法及其专用试剂盒与应用 - Google Patents

一种变性泡介导的靶标核酸扩增方法及其专用试剂盒与应用 Download PDF

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WO2020048307A1
WO2020048307A1 PCT/CN2019/101293 CN2019101293W WO2020048307A1 WO 2020048307 A1 WO2020048307 A1 WO 2020048307A1 CN 2019101293 W CN2019101293 W CN 2019101293W WO 2020048307 A1 WO2020048307 A1 WO 2020048307A1
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nucleic acid
target nucleic
reaction
reaction system
amplification
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PCT/CN2019/101293
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French (fr)
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石超
马翠萍
王雪娇
姬艳丽
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青岛耐德生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the invention relates to the field of biotechnology, in particular to a method for amplifying a target nucleic acid mediated by a denatured vesicle, a special kit and application thereof.
  • Nucleic acids can be divided into deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are the basic elements of all life forms.
  • DNA carries genetic information and is responsible for encoding the basic unit amino acids of proteins.
  • RNA plays an important role in the encoding, decoding, regulation and expression of genes. Therefore, nucleic acid is used as an important biomarker for biological research and medical diagnosis.
  • the advent of nucleic acid amplification technology has provided an important theoretical basis for the detection of pathogenic microorganisms and meat-derived components.
  • a simple, easy-to-operate and Sensitive and rapid nucleic acid detection methods are the main targets in the field of biological detection.
  • PCR Polymerase chain reaction
  • LAMP Loop Mediated Isothermal Amplification
  • HDA Helicase-dependent nucleic acid isothermal amplification technology
  • HAD Helicase-dependent Isothermal Deoxyribonucleic Acid
  • SEA Denaturation bubble-mediated strand exchange amplification technology
  • SEA technology can detect DNA and one-step RNA detection. It has the advantages of simple, fast, and strong specificity. It can complete the chain exchange amplification reaction under isothermal conditions, and it can get rid of the dependence on large and complicated instruments and is suitable for medical institutions. On-site rapid detection, grass-roots unit disease screening, food pathogenic microorganism detection and species identification, inspection and quarantine institutions, handling of large-scale emergency incidents, military field rapid detection and other occasions.
  • the technical problem to be solved by the present invention is how to realize the rapid detection of low-concentration target nucleic acid based on denatured bubble-mediated chain exchange technology (SEA).
  • SEA denatured bubble-mediated chain exchange technology
  • the present invention first provides a method for amplifying a target nucleic acid mediated by a denaturing bubble.
  • the denatured bubble-mediated target nucleic acid amplification method provided by the present invention includes the step of selectively amplifying a nucleic acid sample to be detected under the action of denatured bubbles and DNA polymerase;
  • the amplified reaction system includes one or more pairs of oligonucleotide primers for amplifying the target nucleic acid, one or more DNA polymerases, reaction buffers, dNTPs, and preparations to aid melting;
  • the preparation for assisting melting includes single-chain binding protein and / or polyethylene glycol.
  • the amplification is isothermal amplification.
  • the reaction temperature of the isothermal amplification may be 20-75 ° C.
  • reaction temperature of the isothermal amplification may be 35-65 ° C.
  • reaction temperature of the isothermal amplification may be 55-65 ° C; preferably 59 ° C, 60 ° C, 61 ° C, or 62 ° C.
  • the target nucleic acid may be a single-stranded DNA molecule or a single-stranded RNA molecule, or a double-stranded DNA molecule.
  • the target nucleic acid may be 20-60 bp in length.
  • the target nucleic acid may be 35-50 bp in length.
  • the target nucleic acid may be 38-50 bp in length.
  • the length of the target nucleic acid is specifically 38 bp, 41 bp, 43 bp, or 50 bp.
  • a ratio of the oligonucleotide primer, the DNA polymerase, the dNTPs, the single-chain binding protein, and the polyethylene glycol in the reaction system is 10 -6 mol: (1-30) ⁇ 10 6 U: (0.1-10) ⁇ 10 -3 mol: (0.1-100) ⁇ 10 3 ⁇ g: (0.001-0.2) L.
  • the oligonucleotide primer is a pair of oligonucleotide primers, wherein one primer hybridizes to the 5 ′ end of the target nucleic acid, and the other primer hybridizes to the 3 ′ end of the target nucleic acid.
  • the melting temperature of the oligonucleotide primer is within a range of the reaction temperature of the isothermal amplification ⁇ 5 ° C.
  • the length of the oligonucleotide primer may be 15-30 bp.
  • the GC content of the oligonucleotide primer may be 40-60%.
  • the concentration of the oligonucleotide primer in the reaction system may be 10 -7 -10 -5 M, and specifically may be 10 -6 M.
  • the target nucleic acid refers to a partial sequence in a template, and two ends of the partial sequence are complementary to two primer sequences, respectively, and are a nucleic acid portion between the 5 ′ end of one primer and the 5 ′ end of another primer.
  • the DNA polymerase may be selected from the Klenow fragment and the Bst polymerase large fragment of E. coli DNA polymerase I, or may be a polymerase having 80% or more homology with the Bst polymerase large fragment. Mutants or isomerases can also be complexes of enzymes that bind large fragments of Bst polymerase to other compounds or nucleic acids or proteins and have a hot start effect.
  • the DNA polymerase lacks 5 'to 3' exonuclease activity and has strand displacement activity.
  • the DNA polymerase has reverse transcription activity in addition to polymerase activity.
  • the concentration of the DNA polymerase in the reaction system may be 1-30 U / ⁇ L.
  • the concentration of the DNA polymerase in the reaction system may be 8-24 U / ⁇ L, specifically 8 U / ⁇ L, 16 U / ⁇ L, or 24 U / ⁇ L.
  • the single-chain binding protein includes, but is not limited to, phage T4, 32SSB, E. Coli, SSB, phage T7, 2.5SSB, and phage phi 29SSB, or derivatives thereof.
  • the single-stranded binding protein is an extremely thermostable single-stranded binding protein (ETSSB) derived from E. coli.
  • ETSB extremely thermostable single-stranded binding protein
  • the extremely high thermostable single-stranded binding protein still has a high activity in the reaction temperature range, and can bind single-stranded DNA or RNA, that is, the nucleic acid double-strands that are released due to respiration can be more stable in a single-stranded form. , Thereby increasing the binding efficiency of primers and targets.
  • the highly thermostable single-stranded binding protein can increase the activity of DNA polymerase, thereby speeding up the reaction.
  • the concentration of the single-chain binding protein in the reaction system may be 0.1-100 ⁇ g / mL.
  • the concentration of the single-chain binding protein in the reaction system may be 1.0-50 ⁇ g / mL, specifically 5 ⁇ g / mL, 12.5 ⁇ g / mL, 25 ⁇ g / mL, or 50 ⁇ g / mL, and preferably 5 ⁇ g / mL mL.
  • the polyethylene glycol is selected from the group consisting of polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 2000, and polyethylene glycol 4000.
  • polyethylene glycol is polyethylene glycol 200 (PEG 200).
  • the polyethylene glycol 200 can speed up the hybridization process and increase the binding efficiency of complementary double-stranded DNA, thereby speeding up the reaction speed.
  • the volume fraction of the polyethylene glycol in the reaction system may be 0.1-20%.
  • volume fraction of the polyethylene glycol in the reaction system may be 1.0-10%, specifically 1.0%, 2.5%, 5% or 10%, preferably 2.5%.
  • the concentration of the dNTPs in the reaction system may be 0.1-10 mM.
  • the concentration of the dNTPs in the reaction system may be 0.5-1.5 mM, specifically may be 0.5 mM, 0.8 mM, or 1.0 mM, and preferably 0.8 mM.
  • the reaction buffer may be an Isothermal reaction buffer, and its formula is as follows: the solvent is water, the solute and its concentration are 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , 0.1% Triton X-100; pH 8.8@25°C.
  • reaction system may be 1) or 2) as follows:
  • the reaction system comprises the oligonucleotide primer, the single-stranded binding protein, the polyethylene glycol 200, the DNA polymerase, the dNTPs, the reaction buffer, Template (nucleic acid sample to be detected) and water composition;
  • the reaction system is composed of a Buffer A solution, a Buffer B solution and water;
  • the Buffer A solution is composed of the reaction buffer, the dNTPs, the oligonucleotide primer, and the polyethylene glycol;
  • the Buffer B solution is composed of the reaction buffer, the DNA polymerase, and the single-stranded binding protein.
  • the reaction system in 1) or the Buffer A solution in 2) further includes a fluorescent dye; if the amplification product is detected by a colorimetric method, all The reaction system described in 1) and the reaction system described in 2) further includes an acid-base indicator.
  • the reaction system in 1) is as follows: template (nucleic acid sample to be detected) 1 ⁇ L, a pair of oligonucleotide primers (both final concentrations in the reaction system are 10 -6 M), dNTPs (10 mM ) 0.8 ⁇ L, Isothermal reaction buffer (1 ⁇ ), Evagreen (20 ⁇ ) 0.25 ⁇ L, Bst 2.0 WarmStart TM DNA polymerase (8U / ⁇ L) 0.1 ⁇ L, ET SSB (final concentration in the reaction system is 5 ⁇ g / mL), PEG 200 0.25 ⁇ L, and water to make up the total volume of the system is 10 ⁇ L.
  • Buffer A and Buffer B are mixed, and the total volume of the system is made up by adding water to 25 ⁇ L.
  • the specific formula of Buffer A is as follows: 1.75 ⁇ L of Isothermal reaction buffer (10 ⁇ ), 2 ⁇ L of dNTPs (10 mM), a pair of oligonucleotide primers (the final concentrations in the reaction system are both 10 -6 M), PEG 200 0.625 ⁇ L, Evagreen (20 ⁇ ) 0.625 ⁇ L;
  • the specific formula of Buffer B is as follows: Isothermal reaction buffer (10 ⁇ ) 0.75 ⁇ L, ET SSB (final concentration in the reaction system is 5 ⁇ g / mL), Bst 2.0 WarmStart TM DNA polymerase (8U / ⁇ L) 0.25 ⁇ L.
  • the above method further includes the step of detecting the amplified product; the method of detecting the amplified product is fluorescent detection, electrophoretic detection, or colorimetric detection.
  • the target nucleic acid is a pathogen from a biological sample, and the pathogen can be detected by amplifying the target nucleic acid.
  • the target nucleic acid is located on a chromosome or a mitochondria or a ribosome, and the method further includes a step of detecting a variation of the target nucleic acid sequence.
  • the method for detecting a target nucleic acid sequence variation may be a conventional method in the prior art.
  • the sequence variation may be a single nucleotide polymorphism.
  • the nucleic acid sample to be detected may be genomic DNA and RNA of a biological sample extracted using a kit, or may be directly mashed the biological sample and mixed with a lysate, and then heated at 95 ° C for 5 minutes and then centrifuged. The collected supernatant containing the target nucleic acid.
  • the method of the present invention is based on a denatured vesicle that opens a double-strand for extended chain exchange reaction (SEA).
  • SEA extended chain exchange reaction
  • primers can form exponential amplification by invading the denatured vesicle structure, extending and replacing the original complementary strand under the action of polymerase.
  • DNA polymerase has strong reverse transcription activity when the target strand length is about 100 bp or less.
  • the strand exchange reaction is designed to detect targets between approximately 35-50bp in length, and the two upstream and downstream primers are designed to bind to the target at the moment of double-strand opening.
  • DNA polymerase can be used to amplify under isothermal conditions to achieve DNA or RNA. One-step detection.
  • the present invention also provides a target nucleic acid amplification kit.
  • the target nucleic acid amplification kit provided by the present invention includes the oligonucleotide primer, the DNA polymerase, the reaction buffer, the dNTPs, and the auxiliary melting preparation.
  • the kit includes a Buffer A solution and a Buffer B solution;
  • the Buffer A solution includes the reaction buffer, the dNTPs, the oligonucleotide primer, and the polyethylene glycol;
  • the Buffer B solution includes the reaction buffer, the DNA polymerase, and the single-stranded binding protein.
  • the kit When the amplification product is detected by a fluorescence method, the kit further includes a fluorescent dye.
  • the kit further includes an acid-base indicator.
  • the kit consists of the oligonucleotide primer, the DNA polymerase, the reaction buffer, the dNTPs, the polyethylene glycol, the single-chain binding protein, and the fluorescence
  • the dye and water consist or the kit consists of a Buffer A solution, a Buffer B solution and water.
  • the Buffer A solution consists of the reaction buffer, the dNTPs, the oligonucleotide primers, the polyethylene glycol, and the fluorescent dye;
  • the Buffer B solution consists of the reaction buffer, the DNA polymerase and the single-stranded binding protein.
  • the present invention also provides new uses of the above method or the above kit.
  • the invention provides the application of the above method or the above kit in the detection of pathogenic microorganisms.
  • the invention also provides the application of the above method or the above kit in species identification.
  • the invention also provides the application of the above method or the above kit in gene detection.
  • the gene detection includes, but is not limited to, detection fields such as defective gene detection, drug resistance gene detection, disease detection, disease risk prediction, or gene physical examination.
  • Figure 1 is a schematic diagram of a chain exchange reaction.
  • the strand exchange reaction requires only two primers upstream and downstream, a DNA polymerase, to achieve rapid amplification and detection of nucleic acids under isothermal conditions.
  • the denatured vesicles produced by the "breathing" of the nucleic acid double strand itself make the nucleic acid appear in a single-stranded state.
  • the upstream or downstream primers bind to the target and extend to the 3 'end in the presence of the polymerase.
  • a cycle of 35-70bp Short-chain target DNA continue to use this as a target cycle to generate a large number of reaction products, to achieve signal amplification.
  • Fig. 2 is a fluorescence diagram of ET SSB which greatly improves the reaction efficiency.
  • - ⁇ - represents the amplification curve when 10 ⁇ L reaction system contains ET SSB with a final concentration of 50 ⁇ g / mL
  • Figure 3 is a graph showing the effect of different concentrations of PEG 200 on the amplification test.
  • - ⁇ - represents the amplification curve when 10 ⁇ L reaction system contains PEG 200 at a final concentration of 1.0%
  • Figure 4 is a graph showing the effect of different concentrations of Bst DNA polymerase on the amplification test.
  • - ⁇ - represents the amplification curve when 10U of the reaction system contains 8U DNA and polymerase
  • Figure 5 is a graph showing the effect of different volumes of dNTPs on the amplification test.
  • - ⁇ - represents the amplification curve when water is used as the target in a 10 ⁇ L reaction system
  • - ⁇ - represents the amplification curve when 0.8 ⁇ L dNTPs is contained in a 10 ⁇ L reaction system
  • FIG. 6 is a diagram of the feasibility verification result of detecting S. aureus by the kit of the present invention.
  • Figure B is a PAGE diagram corresponding to Figure A, where lane M is a 20bp Marker, lanes 1-3 correspond to three positive repeats, and lane 4 is a blank control.
  • Figure C is a colorimetric chart corresponding to Figure A, where a-c corresponds to three positive repeats, and d corresponds to a blank control.
  • FIG. 7 is a diagram of SEA amplification reaction of a target nucleic acid obtained by two different methods. Among them-represents the amplification curve when the duck meat genomic DNA and RNA mixture extracted from the kit is used as a target, - ⁇ -represents the amplification curve when the duck meat genomic DNA and RNA mixture obtained by simple lysis is used as a target,- ⁇ -represents a blank control with water as the target.
  • FIG. 8 is an aging experiment of the SEA reaction system of the present invention.
  • - ⁇ - represents the amplification curve of the reaction Mix just removed from the -20 ° C refrigerator
  • FIG. 9 is a repeated freeze-thaw experiment of the SEA reaction system of the present invention.
  • 1 represents the amplification curve of the positive target when repeatedly freezing and thawing once
  • 2 represents the amplification curve of the positive target when repeatedly freezing and thawing twice
  • 3 represents the amplification curve of the positive target when repeatedly freezing and thawing three times
  • 4 represents four freeze and thaw cycles.
  • 5 represents the amplification curve of the positive target at five freeze-thaw cycles
  • 6 represents the amplification curve of the positive target at six freeze-thaw cycles
  • 7 represents water when the freeze-thaw cycle is repeated once.
  • 8 represents the amplification curve with water as the target when repeatedly freezing and thawing twice
  • 9 represents the amplification curve with water as the target when repeatedly freezing and thawing three times
  • 10 represents water as the target when repeatedly freezing and thawing four times
  • 11 represents the amplification curve with water as the target when repeatedly freezing and thawing five times
  • 12 represents the amplification curve with water as the target when repeatedly freezing and thawing six times.
  • FIG. 10 is the detection limit of the SEA reaction system of the present invention.
  • Increasing curve - ⁇ -represents the amplification curve when the target (S. aureus PCR product) final concentration is 10 -13 M
  • the amplification curve represents the amplification curve when the final concentration of the target (S. aureus PCR product) is 10 -15 M
  • ⁇ represents the blank control with water as the target.
  • genomic DNA and RNA of biological samples were extracted by DNA / RNA Isolation Kit.
  • DNA / RNA Isolation Kit is a product of Tiangen Biochemical Technology (Beijing) Co., Ltd., product number DP422 .
  • the solvent of the Isothermal reaction buffer is water, and the solute and its concentration are as follows: 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgSO 4 , and 0.1% Triton X-100. ; PH8.8@25°C.
  • Example 1 Application of single-chain binding protein in improving SEA reaction efficiency
  • Single-stranded binding protein can accelerate the efficiency of DNA polymerase, stabilize the structure of single-stranded DNA, and greatly promote the amplification reaction.
  • an extremely high thermostable single-stranded binding protein extracted from E. coli is used as an auxiliary melting preparation to perform isothermal amplification, and the optimal concentration of ETSSB is obtained by optimization. Specific steps are as follows:
  • Isothermal amplified target nucleic acid The pine wood nematode (Bursaphelenchus xylophilus) 28S rRNA gene (sequence is 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGCATTGCCG-3 ') (SEQ ID No. 1) is used as the target nucleic acid.
  • reaction system of isothermal amplification is as follows:
  • Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3 '(SEQ ID No. 2)): the final concentration in the SEA reaction system is 10 -6 M;
  • Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3 '(SEQ ID No. 3)): the final concentration in the SEA reaction system is 10 -6 M;
  • dNTPs (10 mM): 0.8 ⁇ L;
  • ETSSB solution 500 ⁇ g / mL
  • different volumes of ETSSB solution 500 ⁇ g / mL (New England Biolabs, # M2401S) were added to the system, so that the final concentration of ETSSB in the reaction system was 0, 5 ⁇ g / mL, 12.5 ⁇ g, respectively.
  • NTC target nucleic acid
  • Isothermal amplification reaction conditions Isothermal amplification was performed at 62 ° C using a Bole CFX96 TM real-time quantitative PCR instrument, and the fluorescence signal was scanned every minute. The results are shown in FIG. 2.
  • polyethylene glycol can increase the binding efficiency of complementary nucleic acid strands, based on patent application number 201610101384.5, isothermal amplification is performed using polyethylene glycol as a preparation for auxiliary melting, to further optimize the concentration of polyethylene glycol . Specific steps are as follows:
  • Isothermal amplified target nucleic acid The pine wood nematode (Bursaphelenchus xylophilus) 28S rRNA gene (sequence is 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGCATTGCCG-3 ') (SEQ ID No. 1) is used as the target nucleic acid.
  • reaction system of isothermal amplification is as follows:
  • Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3 '(SEQ ID No. 2)): the final concentration in the SEA reaction system is 10 -6 M;
  • Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3 '(SEQ ID No. 3)): the final concentration in the SEA reaction system is 10 -6 M;
  • dNTPs (10 mM): 0.8 ⁇ L;
  • ETSSB 500 ⁇ g / mL: 0.1 ⁇ L
  • polyethylene glycol 200 (PEG 200, Sigma, Aldrioh, 1002300576, P3015-250G) with different concentrations was added to the system, so that their volume fractions in the SEA reaction system were 1%, 2.5%, 5 % And 10%.
  • Isothermal amplification reaction conditions The isothermal amplification was performed with a Bole CFX96 TM real-time quantitative PCR instrument at 62 ° C, and the fluorescence signal was scanned every minute. The results are shown in Figure 3.
  • Isothermal amplified target nucleic acid The pine wood nematode (Bursaphelenchus xylophilus) 28S rRNA gene (sequence is 5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGCATTGCCG-3 ') (SEQ ID No. 1) is used as the target nucleic acid.
  • reaction system of isothermal amplification is as follows:
  • Primer P1 (sequence is 5'-AGCCTTCTGGGCGCGT-3 '(SEQ ID No. 2)): the final concentration in the SEA reaction system is 10 -6 M;
  • Primer P2 (sequence is 5'-CGGCAATGCACAAACACCA-3 '(SEQ ID No. 3)): the final concentration in the SEA reaction system is 10 -6 M;
  • dNTPs (10 mM): 0.8 ⁇ L;
  • ETSSB 500 ⁇ g / mL: 0.1 ⁇ L
  • 0.1 ⁇ L of Bst 2.0 WarmStart TM DNA polymerase at a concentration of 8 U / ⁇ L, 16 U / ⁇ L, and 24 U / ⁇ L were added to the system, and the rest was used to make up the total volume of the system to 10 ⁇ L.
  • Isothermal amplification reaction conditions Isothermal amplification was performed using a Bole CFX96 TM real-time quantitative PCR instrument at 62 ° C, and the fluorescence signal was scanned every minute. The results are shown in Figure 4.
  • Isothermal amplified target nucleic acid Listeria monocytogenes 16S rRNA gene (sequence 5'-GTCATTGGAAACTGGAAGACTGGAGTGCAGAAGAGGAGAGTGG-3 '(SEQ ID No. 4)) was used as the target nucleic acid.
  • reaction system of isothermal amplification is as follows:
  • Primer P1 (sequence 5'-GTCATTGGAAACTGGAAGACTG-3 '(SEQ ID NO. 5)): the final concentration in the SEA reaction system is 10 -6 M;
  • Primer P2 (sequence is 5'-CCACTCTCCTCTTCTGCAC-3 '(SEQ ID NO.6)): the final concentration in the SEA reaction system is 10 -6 M;
  • ETSSB 500 ⁇ g / mL: 0.1 ⁇ L
  • dNTPs 10 mM
  • NTC target nucleic acid
  • Isothermal amplification reaction conditions Isothermal amplification was performed with a Bole CFX96 TM real-time quantitative PCR instrument at 62 ° C, and the fluorescence signal was scanned every minute. The results are shown in Figure 5.
  • Example 3 Preparation of a kit for target nucleic acid detection and its feasibility verification
  • the kit for target nucleic acid detection of the present invention includes Buffer A and Buffer B.
  • the formulas of Buffer A and Buffer B are as follows:
  • Buffer A formula is as follows:
  • Primer P1 the final concentration in the total reaction system is 10 -6 M;
  • Primer P2 the final concentration in the total reaction system is 10 -6 M;
  • Buffer B formula is as follows:
  • ETSSB 500 ⁇ g / mL: 0.25 ⁇ L
  • the total volume of the reaction system is 25 ⁇ L, and the total volume is made up by adding water to 25 ⁇ L.
  • the 10 ⁇ L system in Examples 1 and 2 can also be used for SEA amplification.
  • Evagreen fluorescent dye is not added to Buffer A, and an acid-base indicator with a final concentration of 10% is added to the reaction system to achieve reaction monitoring.
  • Isothermal amplified target nucleic acid Staphylococcus aureus 16S rRNA gene (sequence 5'-GGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCGCGC-3 '(SEQ ID ID No. 7)) was used as the target nucleic acid.
  • the primer sequences for amplifying the target nucleic acid of S. aureus are as follows: 5'-GGTTCAAAAGTGAAAGACGGTCTTG-3 '(SEQ ID No. 8) and 5'-GCGCGGATCCATCTATAAGTGAC-3' (SEQ ID No. 9).
  • Isothermal amplification reaction system Buffer A, Buffer B and 1 ⁇ L of Staphylococcus aureus genomic DNA and RNA mixture (10 -10 M) were mixed to obtain the SEA reaction system. The experiment was set up in triplicate.
  • NTC target nucleic acid
  • Isothermal amplification reaction conditions Isothermal amplification was performed at 61 ° C using Bole CFX96 TM real-time quantitative PCR instrument.
  • the fluorescence signal was scanned every minute using a Bole CFX96 TM real-time quantitative PCR instrument at 61 ° C. The results are shown in FIG. 6A.
  • the amplified products were subjected to PAGE electrophoresis at 10-18%, and the voltage was first applied at 180 V for 1 min, and then applied at 135 V for 55 min. The results are shown in Figure 6B.
  • lanes 1-3 have a clear product band at 41 bp, which is consistent with the designed length, which proves that there is a product. There is no product band in lane 4, only unreacted primer bands are around 20bp.
  • An acid-base indicator (purchased from Qingdao Naide Biotechnology Co., Ltd., K3005) capable of indicating acidity and alkalinity is added to the reaction system so that the final concentration thereof is 10%. Observe the color change of the reaction system before and after the reaction. If the reaction system changes from yellow to red before and after the reaction, it means that the test sample contains the target nucleic acid. If the reaction system does not change color before and after the reaction, it means that the test sample does not contain the target nucleic acid. The results are shown in Figure 6C.
  • the duck targets obtained in two different ways are used for isothermal amplification.
  • the specific steps are as follows:
  • Kit extraction TIAN DNA / RNA Isolation Kit was used to extract genomic DNA and RNA from duck meat of Anas platyrhynchos to obtain duck genomic DNA and RNA mixture (pork1).
  • a mallard duck mitochondrial gene (sequence 5'-CGCATAACCCTCCTAGTCCAAGCCGGACGGACTCGTATCCC-3 '(SEQ ID No. 10)) was used as a target nucleic acid.
  • the primer sequences for amplifying the mallard duck mitochondrial target nucleic acid are as follows: 5'-CGCATAACCCTCCTAGTCCAAG-3 '(SEQ ID No. 11) and 5'-CCCTCTGCTCAGGCAGGC-3' (SEQ ID No. 12).
  • the target nucleic acid was detected using the kit in Example 3.
  • NTC target nucleic acid
  • the fluorescence signal was scanned every minute using a Bole CFX96 TM real-time quantitative PCR instrument at 61 ° C. The results are shown in FIG. 7.
  • the fluorescence value of duck genomic DNA and RNA mixture (pork 1) extracted from the kit started to peak at 17 minutes, and the fluorescence value of mashed duck genomic DNA and RNA mixture (pork 2) was directly broken at 24 minutes.
  • Significant changes indicate that the amplification system of the present invention can not only detect genomic targets quickly, but also detect samples that have not undergone complex pre-processing, providing a theoretical basis for field detection.
  • Isolate the duck target isothermally by using bufferA and bufferB stored under different conditions for different times.
  • the specific steps are as follows: Using the duck genomic DNA and RNA mixture (pork 1) obtained in step 1 as a template, the kit in Example 3 was used. Detection of the target nucleic acid. According to the storage time and storage conditions of bufferA and bufferB, they are divided into the following groups:
  • BufferA and bufferB removed from the refrigerator at -20 °C for target nucleic acid detection
  • NTC target nucleic acid
  • the fluorescence signal was scanned every minute using a Bole CFX96 TM real-time quantitative PCR instrument at 61 ° C. The results are shown in FIG. 8.
  • reaction solution stored at room temperature for 4 months still has good reaction efficiency.
  • the reaction solution stored at room temperature for 8 months has a reaction time 13 minutes longer than the reaction solution stored at room temperature for 4 months, but the detection ability It has not weakened. It shows that the kit of the present invention has good stability, can be stored for a long time and is not easy to fail.
  • Freeze and thaw twice (repeatedly freeze and thaw twice);
  • Freeze and thaw three times three times of repeated freeze-thaw cycles
  • Freeze and thaw 6 times (repeated freeze-thaw 6 times).
  • the above six groups used water as the target as a blank control.
  • the fluorescence signal was scanned every minute using a Bole CFX96 TM real-time quantitative PCR instrument at 61 ° C. The results are shown in FIG. 9.
  • Example 5 DNA detection limit of the kit of the present invention
  • This embodiment uses different concentrations of target nucleic acids as templates to perform isothermal amplification.
  • the specific steps are as follows: Staphylococcus aureus PCR products with different concentrations PCR products obtained by PCR amplification of the primers shown in SEQ ID No. 13 and SEQ ID No. 14), and the target nucleic acid is detected using the kit in Example 3, and equal volumes are added to each reaction tube separately Isothermal amplification of S. aureus PCR products at different concentrations.
  • the different final concentrations of S. aureus PCR products in the reaction system they are divided into the following groups: S. aureus PCR products have a final concentration of 10 -11 M; S.
  • aureus PCR products have a final concentration of 10 -12 M; gold The final concentration of S. aureus PCR products is 10 -13 M; the final concentration of S. aureus PCR products is 10 -14 M; the final concentration of S. aureus PCR products is 10 -15 M. At the same time, water was used as a blank control.
  • the fluorescence signal was scanned every minute using a Bole CFX96 TM real-time PCR instrument at 61 ° C. The results are shown in FIG. 10.
  • the detection limit of the SEA reaction system of the present invention reaches 10 -14 M.
  • the sensitivity is increased by 1000 times, which is more conducive to low.
  • the detection of concentration targets provides advantages for rapid detection in the field.
  • the invention provides a method for amplifying a target nucleic acid mediated by denaturation bubbles and a kit used for the method.
  • the experiment proves that the kit of the present invention has higher sensitivity, can detect the target of 10 -14 M, and improves the sensitivity of SEA technology by 1000 times. It is suitable for genetic detection fields such as pathogenic microorganisms and species identification, and is stable. Good performance and long-term storage advantages.

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Abstract

一种变性泡介导的靶标核酸扩增方法及其专用试剂盒与应用。该试剂盒包括一对或多对用于扩增靶标核酸的寡核苷酸引物、一种或多种DNA聚合酶、反应缓冲液、dNTPs和辅助解链的制剂。该辅助解链的制剂包括单链结合蛋白和/或聚乙二醇。该试剂盒具有更高的灵敏度，能够实现10 -14 M靶标的检测，将SEA技术的灵敏度提高了1000倍，适合多种病原微生物以及物种鉴定等基因快速检测，且具有稳定性好，可长期存储的优点。

Description

一种变性泡介导的靶标核酸扩增方法及其专用试剂盒与应用 技术领域
本发明涉及生物技术领域，具体涉及一种变性泡介导的靶标核酸扩增方法及其专用试剂盒与应用。
背景技术
核酸可分为脱氧核糖核酸(DNA)和核糖核酸(RNA)，是所有生命形式的基本要素。DNA携带遗传信息并负责编码蛋白质的基本单元氨基酸。RNA在基因的编码、解码、调节和表达中起到重要的作用。因此，核酸被用作重要的生物标志物用于生物研究和医学诊断，而核酸扩增技术的出现为病原微生物以及肉源成分的检测提供了重要的理论依据，建立一种简单、易于操作和灵敏快速的核酸检测方法是生物检测领域中的主要目标。
聚合酶链式扩增反应(Polymerase Chain Reaction，PCR)技术作为最早出现的核酸扩增技术，直至目前仍然应用广泛。该技术以长片段核酸作为靶标，设计上下游两条引物，高温退火解开双链，低温结合引物，适温时在聚合酶的存在下延伸，经反复循环，实现靶标片段的指数性扩增。传统的PCR技术扩增结果需要琼脂糖凝胶电泳进行验证，后来发展的实时荧光定量PCR技术通过加入荧光染料实现了荧光信号输出实验结果。但是由于三步扩增导致PCR技术总是离不开精密的控温仪器，限制了该技术的研究发展及基层应用。
自20世纪90年代初以来，各种等温扩增技术被开发成为PCR的替代品，如环介导等温扩增(Loop Mediated Isothermal Amplification，LAMP)、依赖解旋酶的核酸等温扩增技术(Helicase-dependent Isothermal Deoxyribonucleic Acid，HDA)等技术。LAMP技术因为灵敏度高、特异性好而被大家熟知，但是该技术易污染，且引物设计困难，无法实现高突变物种靶标的检测。而HAD技术的反应体系中需要两个酶，双酶体系容易引起非特异扩增，影响实验结果的判断。这些缺点在一定程度上限制了这些技术的推广使用。
1953年沃森和克里克提出了DNA双螺旋结构。在生理条件下，DNA是处在动态平衡中，由于波动，一些碱基氢键之间会发生断裂，双链DNA会自发的变性，并导致双螺旋结构发生变化，该现象被称为DNA的呼吸作用，解开的单链区域叫做DNA变性泡。
基于变性泡介导的链交换扩增技术(Strand Exchange Amplification，SEA)是一种基于DNA呼吸作用引起的变性泡介导的等温核酸扩增技术。在基于SEA技术检测靶核酸时，仅需设计上下游两条引物，该引物可通过侵入变性泡结构，在聚合酶的作用下延伸并置换下原有的互补链来形成指数式扩增。SEA技术可以检测DNA，也可以实现RNA的一步法检测，具有简单、快速、特异性强等优点，可在等温条件下完成链交换扩增反应，摆脱了对大型复杂仪器的依赖，适合医疗机构的现场快速检测，基层单位的疾病筛查，食品致病微生物检测及物种鉴 定、检验检疫机构、处理大型应急事件，军队野外快速检测等场合。
但是，目前报道的变性泡介导的链交换技术，最低检测限均较高，对于低浓度靶标的检出能力较弱。
发明公开
本发明要解决的技术问题是如何基于变性泡介导的链交换技术(SEA)实现低浓度靶标核酸的快速检测。
为了解决上述技术问题，本发明首先提供了一种变性泡介导的靶标核酸扩增方法。
本发明提供的变性泡介导的靶标核酸扩增方法包括将待检测核酸样品在变性泡和DNA聚合酶的作用下进行选择性扩增的步骤；
所述扩增的反应体系包括一对或多对用于扩增所述靶标核酸的寡核苷酸引物、一种或多种DNA聚合酶、反应缓冲液、dNTPs和辅助解链的制剂；
所述辅助解链的制剂包括单链结合蛋白和/或聚乙二醇。
上述方法中，所述扩增为等温扩增。所述等温扩增的反应温度可为20-75℃。
进一步地，所述等温扩增的反应温度可为35-65℃。
更进一步地，所述等温扩增的反应温度可为55-65℃；优选为59℃、60℃、61℃或62℃。
上述方法中，所述靶标核酸可以为单链DNA分子或单链RNA分子，也可以为双链DNA分子。
上述方法中，所述靶标核酸的长度可为20-60bp。
进一步地，所述靶标核酸的长度可为35-50bp。
更进一步地，所述靶标核酸的长度可为38-50bp。在本发明中，所述靶标核酸的长度具体为38bp、41bp、43bp或50bp。
上述方法中，所述反应体系中的所述寡核苷酸引物、所述DNA聚合酶、所述dNTPs、所述单链结合蛋白和所述聚乙二醇的配比为10 -6mol：(1-30)×10 6U：(0.1-10)×10 -3mol：(0.1-100)×10 3μg：(0.001-0.2)L。
上述方法中，所述寡核苷酸引物为一对寡核苷酸引物，其中一条引物与所述靶标核酸的5'端杂交，另一条引物与所述靶标核酸的3'端杂交。
进一步地，所述寡核苷酸引物的熔解温度在所述等温扩增的反应温度±5℃的范围内。
所述寡核苷酸引物的长度可为15-30bp。
所述寡核苷酸引物的GC含量可为40-60％。
所述寡核苷酸引物在所述反应体系中的浓度可为10 -7-10 -5M，具体可为10 -6M。
在本发明中，所述靶标核酸是指模板中的部分序列，该部分序列的两端分别与两条引物序列互补，为一条引物5'端到另一条引物5'端之间的核酸部分。
上述方法中，所述DNA聚合酶可选自大肠杆菌DNA聚合酶I的Klenow片段和 Bst聚合酶大片段，也可为与Bst聚合酶大片段具有80％或80％以上同源性的聚合酶突变体或异构酶，还可为Bst聚合酶大片段与其他化合物或核酸或蛋白质结合且具有热启动效果的酶的复合物。所述DNA聚合酶缺少5'到3'外切核酸酶活性，具有链置换活性。
当靶标核酸为RNA分子时，所述DNA聚合酶除了具有聚合酶活性外还具有反转录活性。
进一步地，所述DNA聚合酶在所述反应体系中的浓度可为1-30U/μL。
更进一步地，所述DNA聚合酶在所述反应体系中的浓度可为8-24U/μL，具体可为8U/μL、16U/μL或24U/μL。
上述方法中，所述单链结合蛋白包括但不限于噬菌体T4 32SSB、E.Coli SSB、噬菌体T7 2.5SSB和噬菌体phi 29SSB，或它们的衍生物。
进一步地，所述单链结合蛋白为来源于大肠杆菌的极高热稳定性单链结合蛋白(ET SSB)。所述极高热稳定性单链结合蛋白在反应温度范围内仍然具有较高活性，可结合单链的DNA或者RNA，即能够使得由于呼吸作用解开的核酸双链更加稳定的以单链形式存在，从而增加引物和靶标的结合效率。除此之外，极高热稳定性单链结合蛋白还能够提高DNA聚合酶的活性，从而加快反应速度。
所述单链结合蛋白在所述反应体系中的浓度可为0.1-100μg/mL。
更进一步地，所述单链结合蛋白在所述反应体系中的浓度可为1.0-50μg/mL，具体可为5μg/mL、12.5μg/mL、25μg/mL或50μg/mL，优选为5μg/mL。
上述方法中，所述聚乙二醇选自聚乙二醇200、聚乙二醇400、聚乙二醇2000和聚乙二醇4000。
进一步地，所述聚乙二醇为聚乙二醇200(PEG 200)。所述聚乙二醇200可加快杂交进程，增加互补双链DNA的结合效率，从而加快反应速度。
所述聚乙二醇在所述反应体系中的体积分数可为0.1-20％。
更进一步地，所述聚乙二醇在所述反应体系中的体积分数可为1.0-10％，具体可为1.0％、2.5％、5％或10％，优选为2.5％。
上述方法中，所述dNTPs在所述反应体系中的浓度可为0.1-10mM。
进一步地，所述dNTPs在所述反应体系中的浓度可为0.5-1.5mM，具体可为0.5mM、0.8mM或1.0mM，优选为0.8mM。
上述方法中，所述反应缓冲液可为Isothermal reaction buffer，其配方如下：溶剂为水，溶质及其浓度分别为20mM Tris-HCl，10mM KCl，10mM(NH 4) 2SO 4，2mM MgSO 4，0.1％Triton X-100；pH8.8@25℃。
上述方法中，所述反应体系可为如下1)或2)：
所述1)中，所述反应体系由所述寡核苷酸引物、所述单链结合蛋白、所述聚乙二醇200、所述DNA聚合酶、所述dNTPs、所述反应缓冲液、模板(待检测核酸样品)和水组成；
所述2)中，所述反应体系由Buffer A溶液、Buffer B溶液和水组成；
所述Buffer A溶液由所述反应缓冲液、所述dNTPs、所述寡核苷酸引物和所述聚乙二醇组成；
所述Buffer B溶液由所述反应缓冲液、所述DNA聚合酶和所述单链结合蛋白组成。
进一步地，若采用荧光法检测所述扩增产物时，所述1)中反应体系或所述2)中Buffer A溶液还包括荧光染料；若采用比色法检测所述扩增产物时，所述1)中的反应体系和所述2)中反应体系还包括酸碱指示剂。
更进一步地，所述1)中的反应体系具体如下：模板(待检测核酸样品)1μL、一对寡核苷酸引物(在反应体系中的终浓度均为10 -6M)、dNTPs(10mM)0.8μL、Isothermal reaction buffer反应缓冲液(1×)、Evagreen(20×)0.25μL、Bst 2.0 WarmStart TM DNA polymerase(8U/μL)0.1μL、ET SSB(在反应体系中的终浓度为5μg/mL)、PEG 200 0.25μL，加水补足体系总体积为10μL。
所述2)中的反应体系是将Buffer A和Buffer B混匀，加水补足体系总体积为25μL。所述Buffer A配方具体如下：Isothermal reaction buffer(10×)1.75μL、dNTPs(10mM)2μL、一对寡核苷酸引物(在反应体系中的终浓度均为10 -6M)、PEG 200 0.625μL、Evagreen(20×)0.625μL；所述Buffer B配方具体如下：Isothermal reaction buffer(10×)0.75μL、ET SSB(在反应体系中的终浓度为5μg/mL)、Bst 2.0 WarmStart TM DNA polymerase(8U/μL)0.25μL。
上述方法还包括检测扩增产物的步骤；所述检测扩增产物的方法为荧光检测或电泳检测或比色法检测。
上述方法中，所述靶标核酸为来自生物样品中的病原体，可通过扩增所述靶标核酸来检测所述病原体。
上述方法中，所述靶标核酸位于染色体或线粒体或核糖体，所述方法还包括检测所述靶标核酸序列变异情况的步骤。所述检测靶标核酸序列变异的方法可为现有技术中的常规方法。所述序列变异可为单核苷酸多态性。
上述方法中，所述待检测核酸样品可为利用试剂盒提取的生物样品的基因组DNA和RNA，也可为将生物样品直接捣碎后混合裂解液，然后在95℃条件下加热5min，再离心收集的含有靶标核酸的上清液。
本发明的方法基于变性泡打开双链进行延伸的链交换反应(SEA)。等温过程中引物可通过侵入变性泡结构，在聚合酶的作用下延伸并置换下原有的互补链来形成指数式扩增。另外，DNA聚合酶在靶标链长度约100bp及以下时具有较强的反转录活性。而链交换反应设计检测全长约35-50bp之间的靶标，设计上下游两条引物在双链打开瞬间与靶标结合，通过DNA聚合酶的作用在等温条件下扩增，可实现DNA或者RNA的一步法检测。
为了解决上述技术问题，本发明还提供了靶标核酸扩增试剂盒。
本发明提供的靶标核酸扩增试剂盒包括上述寡核苷酸引物、上述DNA聚合酶、上述反应缓冲液、上述dNTPs和上述辅助解链的制剂。
进一步地，所述试剂盒包括Buffer A溶液和Buffer B溶液；
所述Buffer A溶液包括所述反应缓冲液、所述dNTPs、所述寡核苷酸引物和所述聚乙二醇；
所述Buffer B溶液包括所述反应缓冲液、所述DNA聚合酶和所述单链结合蛋白。
若采用荧光法检测所述扩增产物时，所述试剂盒中还包括荧光染料。
若采用比色法检测所述扩增产物时，所述试剂盒中还包括酸碱指示剂。
更进一步地，所述试剂盒由所述寡核苷酸引物、所述DNA聚合酶、所述反应缓冲液、所述dNTPs、所述聚乙二醇、所述单链结合蛋白、所述荧光染料和水组成或所述试剂盒由Buffer A溶液、Buffer B溶液和水组成。所述Buffer A溶液由所述反应缓冲液、所述dNTPs、所述寡核苷酸引物、所述聚乙二醇和所述荧光染料组成；所述Buffer B溶液由所述反应缓冲液、所述DNA聚合酶和所述单链结合蛋白组成。
为了解决上述技术问题，本发明还提供了上述方法或上述试剂盒的新用途。
本发明提供了上述方法或上述试剂盒在病原微生物检测中的应用。
本发明还提供了上述方法或上述试剂盒在物种鉴定中的应用。
本发明还提供了上述方法或上述试剂盒在基因检测中的应用。
上述应用中，所述基因检测包括但不限于缺陷型基因检测、耐药基因检测、疾病检测、疾病风险预测或基因体检等检测领域。
附图说明
图1为链交换反应原理图。链交换反应仅需上下游两条引物，一种DNA聚合酶，在等温条件下实现核酸快速扩增检测。核酸双链自身的“呼吸”作用产生的变性泡使得核酸呈现单链状态，此时的上或下游引物与靶标结合，在聚合酶的存在下向3'端延伸，一个循环后形成35-70bp的短链靶DNA，继续以此为靶标循环产生大量的反应产物，实现信号放大。
图2为ET SSB大幅提高反应效率的荧光图。其中－▆－代表10μL反应体系中含有终浓度为50μg/mL的ET SSB时的扩增曲线，－●－代表10μL反应体系中含有终浓度为25μg/mL的ET SSB时的扩增曲线，－▲－代表10μL反应体系中含有终浓度为12.5μg/mL的ET SSB时的扩增曲线，－▼－代表10μL反应体系含有终浓度为5.0μg/mL的ET SSB时的扩增曲线，－◆－代表10μL反应体系不添加ET SSB时的扩增曲线；
Figure PCTCN2019101293-appb-000001
代表10μL以水为靶标的反应体系中含有终浓度为50μg/mL的ET SSB时的扩增曲线，
Figure PCTCN2019101293-appb-000002
代表10μL以水为靶标的反应体系中含有终浓度为25μg/mL的ET SSB时的扩增曲线，
Figure PCTCN2019101293-appb-000003
代表10μL以水为靶标的反应体系中含有终浓度为12.5μg/mLET SSB时的扩增曲线，－★－代表10μL以水为靶标的反应体系含有终浓度为5μg/mL的ET SSB 时的扩增曲线，
Figure PCTCN2019101293-appb-000004
代表10μL以水为靶标的反应体系不添加ET SSB时的扩增曲线。
图3为不同浓度的PEG 200对扩增试验的影响结果图。其中－▆－代表10μL反应体系中含有终浓度为1.0％的PEG 200时的扩增曲线，－●－代表10μL反应体系中含有终浓度为2.5％的PEG 200时的扩增曲线，－▲－代表10μL反应体系中含有终浓度为5.0％的PEG 200时的扩增曲线，－▼－代表10μL反应体系中含有终浓度为10.0％的PEG 200时的扩增曲线，－◆－代表10μL反应体系不添加PEG 200时的扩增曲线；
Figure PCTCN2019101293-appb-000005
代表10μL以水为靶标的反应体系中含有终浓度为1.0％的PEG 200时的扩增曲线，
Figure PCTCN2019101293-appb-000006
代表10μL以水为靶标的反应体系中含有终浓度为2.5％的PEG 200时的扩增曲线，
Figure PCTCN2019101293-appb-000007
代表10μL以水为靶标的反应体系中含有终浓度为5.0％的PEG 200时的扩增曲线，－★－代表10μL以水为靶标的反应体系中含有终浓度为10.0％的PEG 200的扩增曲线，
Figure PCTCN2019101293-appb-000008
代表10μL以水为靶标的反应体系中不添加PEG 200时的扩增曲线。
图4为不同浓度的Bst DNA polymerase对扩增试验的影响结果图。其中－▆－代表10μL反应体系中含有8U Bst DNA polymerase时的扩增曲线，－●－代表10μL反应体系中含有16U Bst DNA polymerase时的扩增曲线，－▲－代表10μL反应体系中含有24U Bst DNA polymerase时的扩增曲线，－▼－代表10μL反应体系中含有24U Bst DNA polymerase但没有靶标核酸时的扩增曲线。
图5为不同体积的dNTPs对扩增试验的影响结果图。其中－▆－代表10μL反应体系中以水为靶标时的扩增曲线，－●－代表10μL反应体系中含有0.8μL dNTPs时的扩增曲线，－▲－代表10μL dNTPs反应体系中含有1μL dNTPs时的扩增曲线，－▼－代表10μL反应体系中含有0.5μL dNTPs时的扩增曲线。
图6为本发明试剂盒检测金黄色葡萄球菌可行性验证结果图。其中图A中－▼－代表以水为靶标的空白对照。其余三条荧光曲线是同一靶标浓度的三个重复性验证。图B为与图A对应的PAGE图，其中泳道M为20bp的Marker，泳道1-3对应三个阳性重复，泳道4为空白对照。图C为与图A对应的比色图，其中a-c对应三个阳性重复，d对应空白对照。
图7为两种不同方法得到的靶标核酸的SEA扩增反应图。其中－●－代表由试剂盒提取的鸭肉基因组DNA和RNA混合物作为靶标时的扩增曲线，－▆－代表由简单裂解得到的鸭肉基因组DNA和RNA混合物作为靶标时的扩增曲线，－▲－代表以水为靶标的空白对照。
图8为本发明SEA反应体系的老化实验。其中－▆－代表刚从-20℃冰箱中取出的反应Mix的扩增曲线，－●－代表室温储存1个月的反应mix的扩增曲线，－▲－代表室温储存4个月的反应mix的扩增曲线，－▼－代表室温储存8个月的反应mix的扩增曲线，－◆－代表以水为靶标的空白对照。
图9为本发明SEA反应体系的反复冻融实验。其中1代表反复冻融一次时 阳性靶标的扩增曲线，2代表反复冻融两次时阳性靶标的扩增曲线，3代表反复冻融三次时阳性靶标的扩增曲线，4代表反复冻融四次时阳性靶标的扩增曲线，5代表反复冻融五次时阳性靶标的扩增曲线，6代表反复冻融六次时阳性靶标的扩增曲线，7代表反复冻融一次时以水为靶标的扩增曲线，8代表反复冻融两次时以水为靶标的扩增曲线，9代表反复冻融三次时以水为靶标的扩增曲线，10代表反复冻融四次时以水为靶标的扩增曲线，11代表反复冻融五次时以水为靶标的扩增曲线，12代表反复冻融六次时以水为靶标的扩增曲线。
图10为本发明的SEA反应体系的检测限。其中－▆－代表靶标(金黄色葡萄球菌PCR产物)终浓度为10 -11M时的扩增曲线，－●－代表靶标(金黄色葡萄球菌PCR产物)终浓度为10 -12M时的扩增曲线，－▲－代表靶标(金黄色葡萄球菌PCR产物)终浓度为10 -13M时的扩增曲线，－▼－代表靶标(金黄色葡萄球菌PCR产物)终浓度为10 -14M时的扩增曲线，－◆－代表靶标(金黄色葡萄球菌PCR产物)终浓度为10 -15M时的扩增曲线，－◣－代表以水为靶标的空白对照。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中生物样品的基因组DNA和RNA(基因组DNA和RNA混合物)均通过DNA/RNA Isolation Kit提取获得，DNA/RNA Isolation Kit是天根生化科技(北京)有限公司的产品，货号为DP422。
下述实施例中Isothermal reaction buffer反应缓冲液的溶剂为水，溶质及其浓度分别如下：20mM Tris-HCl，10mM KCl，10mM(NH 4) 2SO 4，2mM MgSO 4，0.1％Triton X-100；pH8.8@25℃。
实施例1：单链结合蛋白在提高SEA反应效率中的应用
由于单链结合蛋白(SSB)能够加速DNA聚合酶的作用效率，稳定单链DNA的结构，极大促进扩增反应。本实施例将从大肠杆菌提取的极高热稳定性单链结合蛋白(ET SSB)作为辅助解链的制剂进行等温扩增，并优化得到ET SSB最佳浓度。具体步骤如下：
1、等温扩增的靶标核酸：以松材线虫(Bursaphelenchus xylophilus)28S rRNA基因(序列为5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3')(SEQ ID No.1)为靶标核酸。
2、等温扩增的反应体系(SEA反应体系)如下：
引物P1(序列为5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2))：在SEA反应体系中的终浓度为10 -6M；
引物P2(序列为5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3))：在SEA 反应体系中的终浓度为10 -6M；
dNTPs(10mM)：0.8μL；
Isothermal reaction buffer反应缓冲液(1×)；
Evagreen(20×)(Bridgen，D013-1)：0.25μL；
Bst 2.0 WarmStart TM DNA polymerase(8U/μL)(New England Biolabs，M0537L)：0.1μL；
Genomic DNA and RNA Mix of B.Xylophilus(松材线虫基因组DNA和RNA混合物)(10 -10M)：1μL；
除以上成分之外，向体系中加入不同体积的ET SSB溶液(500μg/mL)(New England Biolabs，#M2401S)，使ET SSB在反应体系中的终浓度分别为0、5μg/mL、12.5μg/mL、25μg/mL、50μg/mL，其余用水补足体系总体积至10μL。
同时在含有不同浓度ET SSB的SEA反应体系中均以未加入靶标核酸(NTC)作为对照，其余用水补足体系总体积至10μL。
3、等温扩增的反应条件：在62℃下利用伯乐CFX96 TM实时荧光定量PCR仪进行等温扩增，且每分钟对荧光信号进行扫描，结果如图2。
从图中可以看出，加入不同浓度的ET SSB对SEA链交换反应具有明显的促进作用。和不加ET SSB相比较，在SEA反应体系中加入适当浓度的ET SSB可以大幅度缩短反应时间，提高反应速率。其中，ET SSB在SEA反应体系中的终浓度为5μg/mL时，起峰时间最早。
实施例2：SEA反应体系的优化
一、聚乙二醇浓度优化实验
由于聚乙二醇能够增加互补核酸链的结合效率，在专利申请号为201610101384.5的基础上，将聚乙二醇作为辅助解链的制剂进行等温扩增，以进一步对聚乙二醇浓度进行优化。具体步骤如下：
1、等温扩增的靶标核酸：以松材线虫(Bursaphelenchus xylophilus)28S rRNA基因(序列为5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3')(SEQ ID No.1)为靶标核酸。
2、等温扩增的反应体系如下：
引物P1(序列为5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2))：在SEA反应体系中的终浓度为10 -6M；
引物P2(序列为5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3))：在SEA反应体系中的终浓度为10 -6M；
dNTPs(10mM)：0.8μL；
Isothermal reaction buffer反应缓冲液(1×)；
Evagreen(20×)：0.25μL；
ET SSB(500μg/mL)：0.1μL；
Bst 2.0 WarmStart TM DNA polymerase(8U/μL)：0.1μL；
Genomic DNA and RNA Mix of B.Xylophilus(松材线虫基因组DNA和RNA混合物)(10 -10M)：1μL；
除以上成分之外，向体系中加入不同浓度的聚乙二醇200(PEG 200，Sigma Aldrioh，1002300576 P3015-250G)，使其在SEA反应体系中的体积分数分别为1％、2.5％、5％和10％。
同时以SEA反应体系中未加入靶标核酸且含有体积分数为10％的PEG 200(NTC)作为对照。
3、等温扩增的反应条件：在62℃下利用伯乐CFX96 TM实时荧光定量PCR仪进行等温扩增，且每分钟对荧光信号进行扫描，结果如图3。
从图中可以看出：加入不同浓度的PEG 200对SEA链交换反应具有明显的促进作用。和不加PEG 200相比较，在SEA反应体系中加入适当浓度的PEG 200可以大幅度缩短反应时间，提高反应速率。其中，PEG 200在SEA反应体系中的体积分数为2.5％时，起峰时间最早。
二、Bst DNA polymerase浓度优化实验
采用不同浓度的Bst DNA polymerase进行等温扩增，研究Bst DNA polymerase对扩增反应的影响。具体步骤如下：
1、等温扩增的靶标核酸：以松材线虫(Bursaphelenchus xylophilus)28S rRNA基因(序列为5'-AGCCTTCTGGGCGCGTGATTGGTGTTTGTGCATTGCCG-3')(SEQ ID No.1)为靶标核酸。
2、等温扩增的反应体系如下：
引物P1(序列为5'-AGCCTTCTGGGCGCGT-3'(SEQ ID No.2))：在SEA反应体系中的终浓度为10 -6M；
引物P2(序列为5'-CGGCAATGCACAAACACCA-3'(SEQ ID No.3))：在SEA反应体系中的终浓度为10 -6M；
dNTPs(10mM)：0.8μL；
Isothermal reaction buffer反应缓冲液(1×)；
Evagreen(20×)：0.25μL；
ET SSB(500μg/mL)：0.1μL；
PEG 200(100％)：0.25μL；
Genomic DNA and RNA Mix of B.Xylophilus(松材线虫基因组DNA和RNA混合物)(10 -10M)：1μL；
除以上成分之外，向体系中分别加入0.1μL浓度为8U/μL、16U/μL和24U/μL的Bst 2.0 WarmStart TM DNA polymerase，其余用水补足体系总体积至10μL。
同时以SEA反应体系中未加入靶标核酸且含有24U Bst DNA polymerase(NTC)作为对照。
3、等温扩增的反应条件：在62℃下利用伯乐CFX96 TM实时荧光定量PCR仪 进行等温扩增，且每分钟对荧光信号进行扫描，结果如图4。
从图中可以看出：3种不同浓度的Bst DNA polymerase下，阳性结果之间没有明显差别。
三、dNTPs浓度优化实验
采用不同体积的dNTPs进行等温扩增，研究dNTPs对扩增反应的影响。具体步骤如下：
1、等温扩增的靶标核酸：以单增李斯特菌(Listeria monocytogenes)16S rRNA基因(序列为5'-GTCATTGGAAACTGGAAGACTGGAGTGCAGAAGAGGAGAGTGG-3'(SEQ ID No.4))为靶标核酸。
2、等温扩增的反应体系如下：
引物P1(序列为5'-GTCATTGGAAACTGGAAGACTG-3'(SEQ ID NO.5))：在SEA反应体系中的终浓度为10 -6M；
引物P2(序列为5'-CCACTCTCCTCTTCTGCAC-3'(SEQ ID NO.6))：在SEA反应体系中的终浓度为10 -6M；
Isothermal reaction buffer反应缓冲液(1×)；
Evagreen(20×)：0.25μL；
ET SSB(500μg/mL)：0.1μL；
PEG 200：0.25μL；
Bst 2.0WarmStart TM DNA polymerase(8U/μL)：0.1μL；
Genomic DNA and RNA Mix of L.monocytogenes(单增李斯特菌基因组DNA和RNA混合物)(10 -10M)：1μL；
除以上成分之外，分别向体系中加入0.5μL、0.8μL和1.0μL的dNTPs(10mM)，使其在体系中的终浓度分别为0.5mM、0.8mM和1.0mM，其余用水补足体系总体积至10μL。
同时以水作为靶标核酸(NTC)且含有1.0μL的dNTPs作为对照。
3、等温扩增的反应条件：在62℃下利用伯乐CFX96 TM实时荧光定量PCR仪进行等温扩增，且每分钟对荧光信号进行扫描，结果如图5。
从图中可以看出：不同浓度的dNTPs，对SEA反应表现出不同的促进作用，加入反应体系的dNTPs体积为0.8μL时(dNTPs在反应体系中的终浓度为0.8mM)，阳性结果起峰时间更短，反应效率更高。
实施例3：用于靶标核酸检测的试剂盒的制备及其可行性验证
一、用于靶标核酸检测的试剂盒的制备
本发明的用于靶标核酸检测的试剂盒包括Buffer A和Buffer B。Buffer A和Buffer B的配方分别如下：
Buffer A配方如下：
Isothermal reaction buffer(10×)：1.75μL；
dNTPs(10mM)：2μL；
引物P1：在总反应体系中的终浓度为10 -6M；
引物P2：在总反应体系中的终浓度为10 -6M；
PEG 200：0.625μL；
Evagreen(20×)：0.625μL；
Buffer B配方如下：
Isothermal reaction buffer(10×)：0.75μL；
ET SSB(500μg/mL)：0.25μL；
Bst 2.0 WarmStart TM DNA polymerase(8U/μL)：0.25μL。
当需要对靶标核酸进行检测时，将Buffer A和Buffer B混匀，然后加入待检测核酸样品，等温条件下实现SEA扩增检测。实际应用中，反应体系的总体积为25μL，加水补足总体积为25μL。也可以按照实施例1和2中的10μL体系进行SEA扩增。
采用比色法进行结果检测时，Buffer A中不添加Evagreen荧光染料，在反应体系中额外加入终浓度为10％的酸碱指示剂，实现反应监控。
二、用于靶标核酸检测的试剂盒的可行性检测
1、等温扩增的靶标核酸：以金黄色葡萄球菌(Staphylococcus aureus)16S rRNA基因(序列为5'-GGTTCAAAAGTGAAAGACGGTCTTGCTGTCACTTATAGATGGATCCG CGC-3'(SEQ ID No.7))为靶标核酸。
扩增金黄色葡萄球菌靶标核酸的引物序列如下：5'-GGTTCAAAAGTGAAAGACGGTCTTG-3'(SEQ ID No.8)和5'-GCGCGGATCCATCTATAAGTGAC-3'(SEQ ID No.9)。
2、等温扩增的反应体系：将Buffer A、Buffer B和1μL金黄色葡萄球菌基因组DNA和RNA混合物(10 -10M)混匀得到SEA反应体系。实验设置三个重复。
同时以水作为靶标核酸(NTC)作为空白对照。
3、等温扩增的反应条件：在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪进行等温扩增。
4、结果检测
(1)荧光法实现检测
在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪每分钟对荧光信号进行扫描，结果如图6A。
从图中可以看出：三个重复样在一分钟误差范围内同时起峰，空白对照没有荧光值变化。说明优化后的体系重复性好，稳定性强。
(2)PAGE胶电泳法实现检测
将扩增产物进行10-18％的PAGE电泳，先180V电压1min，再135V电压55min。结果如图6B。
从图中可以看出，泳道1-3在41bp有明显的产物条带，与设计长度一致，证明有产物。泳道4无任何产物条带，仅在20bp左右有未反应的引物带。
(3)酸碱指示剂指示的比色法实现检测
将能够指示酸碱性的酸碱指示剂(购自青岛耐德生物技术有限公司，货号为K3005)加入反应体系中，使其终浓度为10％。观察反应体系在反应前后的颜色变化情况。若反应体系在反应前后由黄色变为红色，则表明待测样品含有靶标核酸，若反应体系在反应前后没有发生颜色变化，则表明待测样品不含有靶标核酸。结果如图6C。
从图中可以看出，a-c在反应结束后颜色由黄色变为红色，空白对照没有颜色变化。
实施例4：本发明试剂盒的性能检测
一、不同方法获取的靶标核酸的检测效果比较
采用两种不同方式得到的鸭靶标进行等温扩增，具体步骤如下：
1、靶标核酸的获取
1)试剂盒提取：采用TIAN DNA/RNA Isolation Kit提取绿头鸭(Anas platyrhynchos)鸭肉基因组DNA和RNA，得到鸭基因组DNA和RNA混合物(pork1)。
2)直接捣碎裂解：将绿头鸭(Anas platyrhynchos)鸭肉直接捣碎后，与PBS混匀，然后在95℃下加热5min，再用小型离心机离心，得到鸭基因组DNA和RNA混合物(pork 2)。
2、靶标核酸的检测
以绿头鸭线粒体基因(序列为5'-CGCATAACCCTCCTAGTCCAAGCCGGACGGACTCGTATCCC-3'(SEQ ID No.10))为靶标核酸。
扩增绿头鸭线粒体基因靶标核酸的引物序列如下：5'-CGCATAACCCTCCTAGTCCAAG-3'(SEQ ID No.11)和5'-CCCTCTGCTCAGGCAGGC-3'(SEQ ID No.12)。
分别以步骤1获得的鸭基因组DNA和RNA混合物(pork 1)和鸭基因组DNA和RNA混合物(pork 2)为模板，采用实施例3中的试剂盒进行靶标核酸的检测。
两种鸭基因组DNA和RNA混合物分别加入2.5μL。
同时以水作为靶标核酸(NTC)作为空白对照。
在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪每分钟对荧光信号进行扫描，结果如图7。
从图中可以看出，试剂盒提取的鸭基因组DNA和RNA混合物(pork 1)在17min荧光值开始起峰，直接捣碎裂解的鸭基因组DNA和RNA混合物(pork 2) 在24min处荧光值发生明显变化，说明本发明的扩增体系既能够快速检测基因组靶标，同时也可以检测未经过复杂前处理的样本，为现场检测提供了理论依据。
二、本发明试剂盒的老化实验
采用在不同条件下存放不同时间的bufferA和bufferB对鸭靶标进行等温扩增，具体步骤如下：以步骤一获得的鸭基因组DNA和RNA混合物(pork 1)为模板，采用实施例3中的试剂盒进行靶标核酸的检测。根据bufferA和bufferB的存放时间和存放条件不同分为如下各组：
-20℃：从-20℃冰箱取出的bufferA和bufferB进行靶标核酸的检测；
a month：用室温储存1个月的bufferA和bufferB进行靶标核酸的检测；
four months：用室温储存4个月的bufferA和bufferB进行靶标核酸的检测；
eight months：用室温储存8个月的bufferA和bufferB进行靶标核酸的检测；
同时以水作为靶标核酸(NTC)作为空白对照。
在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪每分钟对荧光信号进行扫描，结果如图8。
从图中可以看出：室温储存4个月的反应液仍然具有较好的反应效率，室温储存8个月的反应液比起室温储存4个月的反应液的反应时间长13min，但是检测能力并没有减弱。说明本发明试剂盒稳定性好，可以长时间储存且不易失效。
三、本发明试剂盒的反复冻融实验
采用自然冷冻以及融化不同次数的SEA反应体系对鸭靶标进行等温扩增，具体步骤如下：以步骤一获得的鸭基因组DNA和RNA混合物(pork 1)为模板，采用实施例3中的试剂盒进行靶标核酸的检测。将同一批次bufferA和bufferB混合得到的SEA反应体系分装到六个Eppendorf管中，根据SEA反应体系自然冷冻以及融化次数的不同分为如下各组：
自然冷冻及融化1次(反复冻融1次)；
自然冷冻及融化2次(反复冻融2次)；
自然冷冻及融化3次(反复冻融3次)；
自然冷冻及融化4次(反复冻融4次)；
自然冷冻及融化5次(反复冻融5次)；
自然冷冻及融化6次(反复冻融6次)。
同时上述六组分别以水为靶标作为空白对照。
在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪每分钟对荧光信号进行扫描，结果如图9。
从图中可以看出，反复冻融6次时的buffer溶液除起峰时间相对靠后外， 仍然可以达到很好的检测效果。
实施例5：本发明试剂盒的DNA检测限
本实施例以不同浓度的靶标核酸为模板进行等温扩增，具体步骤如下：以不同浓度的金黄色葡萄球菌PCR产物(金黄色葡萄球菌PCR产物是以金黄色葡萄球菌的基因组DNA为模板，采用SEQ ID No.13和SEQ ID No.14所示的引物进行PCR扩增得到的PCR产物)模板，采用实施例3中的试剂盒进行靶标核酸的检测，分别向每个反应管中加入同等体积不同浓度的金黄色葡萄球菌PCR产物进行等温扩增。根据金黄色葡萄球菌PCR产物在反应体系中的终浓度不同分为如下各组：金黄色葡萄球菌PCR产物终浓度为10 -11M；金黄色葡萄球菌PCR产物终浓度为10 -12M；金黄色葡萄球菌PCR产物终浓度为10 -13M；金黄色葡萄球菌PCR产物终浓度为10 -14M；金黄色葡萄球菌PCR产物终浓度为10 -15M。同时以水为靶标作为空白对照。
在61℃下利用伯乐CFX96 TM实时荧光定量PCR仪每分钟对荧光信号进行扫描，结果如图10。
从图中可以看出，本发明的SEA反应体系的检测限达到10 -14M，与专利申请号为201610101384.5中的未经过优化的SEA反应体系相比，灵敏度提高了1000倍，更加有利于低浓度靶标的检测，为现场快速检测提供优势。
工业应用
本发明提供了一种变性泡介导的靶标核酸扩增方法及其所用试剂盒。通过实验证明：本发明的试剂盒具有更高的灵敏度，能够实现10 -14M靶标的检测，提高了SEA技术1000倍的灵敏度，适合多种病原微生物以及物种鉴定等基因检测领域，且具有稳定性好，可长期存储的优点。

Claims (36)

  1. 一种变性泡介导的靶标核酸扩增方法，包括将待检测核酸样品在变性泡和DNA聚合酶的作用下进行选择性扩增的步骤；
    所述扩增的反应体系包括一对或多对用于扩增所述靶标核酸的寡核苷酸引物、一种或多种DNA聚合酶、反应缓冲液、dNTPs和辅助解链的制剂；
    所述辅助解链的制剂包括单链结合蛋白和/或聚乙二醇。
  2. 根据权利要求1所述的方法，其特征在于：所述扩增为等温扩增。
  3. 根据权利要求2所述的方法，其特征在于：所述等温扩增的反应温度为20-75℃。
  4. 根据权利要求3所述的方法，其特征在于：所述等温扩增的反应温度为37-65℃。
  5. 根据权利要求4所述的方法，其特征在于：所述等温扩增的反应温度为55-65℃。
  6. 根据权利要求1所述的方法，其特征在于：所述靶标核酸为DNA分子或RNA分子。
  7. 根据权利要求1或6所述的方法，其特征在于：所述靶标核酸的长度为20-60bp。
  8. 根据权利要求7所述的方法，其特征在于：所述靶标核酸的长度为35-50bp。
  9. 根据权利要求1所述的方法，其特征在于：所述寡核苷酸引物为一对寡核苷酸引物，其中一条引物与所述靶标核酸的5'端杂交，另一条引物与所述靶标核酸的3'端杂交。
  10. 根据权利要求1-5或9任一所述的方法，其特征在于：所述寡核苷酸引物的熔解温度在所述等温扩增的反应温度±5℃的范围内。
  11. 根据权利要求1所述的方法，其特征在于：所述寡核苷酸引物的长度为15-30bp。
  12. 根据权利要求1所述的方法，其特征在于：所述寡核苷酸引物的GC含量为40-60％。
  13. 根据权利要求1所述的方法，其特征在于：所述DNA聚合酶选自大肠杆菌DNA聚合酶I的Klenow片段和Bst聚合酶大片段。
  14. 根据权利要求1所述的方法，其特征在于：所述DNA聚合酶为与Bst聚合酶大片段具有80％或80％以上同源性的聚合酶突变体或异构酶。
  15. 根据权利要求1所述的方法，其特征在于：所述DNA聚合酶为Bst聚合酶大片段与其他化合物或核酸或蛋白质结合且具有热启动效果的酶的复合物。
  16. 根据权利要求1或13-15任一所述的方法，其特征在于：所述DNA聚 合酶缺少5’到3’外切核酸酶活性。
  17. 根据权利要求1或13-15任一所述的方法，其特征在于：所述DNA聚合酶具有链置换活性。
  18. 根据权利要求1所述的方法，其特征在于：所述单链结合蛋白选自噬菌体T4 32 SSB、E.Coli SSB、噬菌体T7 2.5 SSB和噬菌体phi 29 SSB，或它们的衍生物。
  19. 根据权利要求18所述的方法，其特征在于：所述单链结合蛋白为来源于大肠杆菌的极高热稳定性单链结合蛋白。
  20. 根据权利要求1所述的方法，其特征在于：所述聚乙二醇选自聚乙二醇200、聚乙二醇400、聚乙二醇2000和聚乙二醇4000。
  21. 根据权利要求20所述的方法，其特征在于：所述聚乙二醇为聚乙二醇200。
  22. 根据权利要求1所述的方法，其特征在于：所述反应体系中的所述寡核苷酸引物、所述DNA聚合酶、所述dNTPs、所述单链结合蛋白和所述聚乙二醇的配比为10 -6mol：(1-30)×10 6U：(0.1-10)×10 -3mol：(0.1-100)×10 3μg：(0.001-0.2)L。
  23. 根据权利要求1或18或19或22任一所述的方法，其特征在于：所述单链结合蛋白在所述反应体系中的浓度为0.1-100μg/mL。
  24. 根据权利要求1或20-22任一所述的方法，其特征在于：所述聚乙二醇在所述反应体系中的体积分数为0.1-20％。
  25. 根据权利要求1或13-15或22任一所述的方法，其特征在于：所述DNA聚合酶在所述反应体系中的浓度为1-30U/μL。
  26. 根据权利要求1或22所述的方法，其特征在于：所述dNTPs在所述反应体系中的浓度为0.1-10mM。
  27. 根据权利要求1所述的方法，其特征在于：所述反应体系中还包括荧光染料或酸碱指示剂。
  28. 根据权利要求1所述的方法，其特征在于：所述方法还包括检测扩增产物的步骤；所述检测扩增产物的方法为荧光检测或电泳检测或比色法检测。
  29. 根据权利要求1所述的方法，其特征在于：所述靶标核酸来自生物样品中的病原体，可通过扩增所述靶标核酸来检测所述病原体。
  30. 根据权利要求1的方法，其特征在于：所述靶标核酸位于染色体或线粒体或核糖体，所述方法还包括检测所述靶标核酸序列变异情况的步骤。
  31. 根据权利要求30的方法，其特征在于：所述序列变异为单核苷酸多态性。
  32. 靶标核酸扩增试剂盒，其包括权利要求1-31中任一所述的寡核苷酸引物、权利要求1-31中任一所述的DNA聚合酶、权利要求1-31中任一所述的反应缓冲液、权利要求1-31中任一所述的dNTPs和权利要求1-31中任一所述的辅助解链 的制剂。
  33. 根据权利要求32所述的试剂盒，其特征在于：所述试剂盒包括Buffer A溶液和Buffer B溶液；
    所述Buffer A溶液包括所述反应缓冲液、所述dNTPs、所述寡核苷酸引物和所述聚乙二醇；
    所述Buffer B溶液包括所述反应缓冲液、所述DNA聚合酶和所述单链结合蛋白。
  34. 根据权利要求32或33所述的试剂盒，其特征在于：所述试剂盒还包括荧光染料或酸碱指示剂。
  35. 权利要求1-31任一所述的方法或权利要求32-34任一所述的试剂盒在如下1)-3)任一种中的应用：
    1)病原微生物检测；
    2)物种鉴定；
    3)基因检测。
  36. 根据权利要求35所述的应用，其特征在于：所述基因检测包括缺陷型基因检测、耐药基因检测、疾病检测、疾病风险预测和基因体检。
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