WO2020168710A1 - 一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法及试剂盒 - Google Patents

一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法及试剂盒 Download PDF

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WO2020168710A1
WO2020168710A1 PCT/CN2019/106693 CN2019106693W WO2020168710A1 WO 2020168710 A1 WO2020168710 A1 WO 2020168710A1 CN 2019106693 W CN2019106693 W CN 2019106693W WO 2020168710 A1 WO2020168710 A1 WO 2020168710A1
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sequence
target
nucleic acid
constant temperature
primer
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French (fr)
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叶邦策
尹斌成
王婷
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华东理工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification

Definitions

  • the invention belongs to the technical field of nucleic acid constant temperature amplification, and specifically relates to a Cas9 nickase-mediated quantitative nucleic acid detection method and kit, which are suitable for constant temperature quantitative detection of genome or double-stranded DNA.
  • the nucleic acid amplification reaction is a very important and valuable technology not only in molecular biology and medicine, but also in forensic medicine, epidemiology and paleontology. Among them, the most widely used is polymerase chain reaction (PCR), a type of in vitro nucleic acid amplification technology, which is used in almost all biological and clinical diagnostic laboratories.
  • PCR polymerase chain reaction
  • the PCR method needs to rely on a thermal cycler with precise temperature control, making PCR-based nucleic acid amplification detection technology not suitable for rapid on-site detection or detection in a remote, poor, and unrelated environment. Since the nucleic acid isothermal amplification technology does not require temperature change operations, it gets rid of the dependence on thermal cycling equipment, and has shown good application prospects in clinical and on-site rapid diagnosis.
  • nucleic acid constant temperature detection methods include autonomous sequence replication system (3SR), nucleic acid sequence dependent amplification technology (NASBA), constant temperature exponential amplification technology (EXPAR), strand displacement amplification technology (SDA), rolling circle nucleic acid amplification (RCA), helicase amplification technology (HAD), loop-mediated isothermal amplification technology (LAMP), recombinase polymerase amplification (RPA), etc.
  • 3SR and NASBA amplify single-stranded RNA through a cascade of reverse transcription, digestion and transcription mediated by reverse transcriptase, RNase H and T7 RNA polymerase, avoiding thermal denaturation reactions.
  • EXPAR and RCA are also more suitable for direct amplification of short single-stranded DNA or ssRNA.
  • SDA requires an initial heating reaction to denature the double-stranded DNA template for primer annealing reaction.
  • LAMP also requires an initial thermal denaturation step, and primer design requirements are high.
  • HAD is restricted by DNA helicase and is only suitable for amplifying small DNA sequences.
  • RPA requires the participation of multiple enzymes, including recombinase (gp32, uxsX, uvsY), single-stranded binding protein, creatine kinase and DNA polymerase, which undoubtedly increases the cost of detection.
  • nucleic acid constant temperature amplification technology solves the thermal cycling reaction that the PCR method depends on, some of them still require initial thermal denaturation operations to separate the nucleic acid double-stranded structure, or a variety of enzyme proteins and special reaction reagents participate in the reaction, or complicated Primer design. Therefore, it is still a challenge to develop a universal isothermal nucleic acid amplification method with high efficiency, easy operation, high sensitivity, strong specificity, and low detection cost.
  • the present invention proposes a constant temperature nucleic acid detection and analysis method based on Cas9 nickase (Cas9n) coupled with DNA polymerase.
  • the specific detection of target nucleic acid under constant temperature conditions includes the following steps:
  • the two sets of Cas9n-sgRNA complexes are respectively adjacent to the protospacer-adjacent motif (PAM, 5′-NGG-3′).
  • PAM protospacer-adjacent motif
  • a single-stranded target DNA sequence is obtained through Cas9n single-mouth cleavage enzyme activity and thermostatic DNA polymerase strand displacement activity, which is compounded between primer 1 and primer 2, DNA polymerase and Cas9n-sgRNA
  • primer hybridization, extension, cutting, strand displacement reactions are repeated under constant temperature conditions, and a large number of target DNA sequences are obtained by amplification.
  • the combination of fluorescent dyes and double-stranded DNA products generates fluorescent signals, and the fluorescence in the reaction system is measured in real time. The signal intensity is compared with the standard working curve to calculate the concentration of the target genome or DNA sequence.
  • the target DNA sequence includes a DNA genome, a plasmid or a double-stranded DNA fragment.
  • DNA genome is the genome of biological samples such as microorganisms, tissues, blood or cells.
  • Cas9 nickase is a domain that inactivates the double-stranded nuclease of Cas9.
  • Cas9 has two catalytic domains, HNH and RuvC.
  • HNH domain cuts complementary DNA sequences
  • RuvC cuts non-complementary DNA sequences.
  • DNA sequence, and Cas9 nickase only contains an active catalytic domain, HNH or RuvC.
  • the sgRNA is a single guide RNA (single guide RNA), and contains a seed sequence of 20 bases that is complementary to the target DNA sequence (probe sequence).
  • the PAM is a protospacer adjacent motif, which is located in a target sequence of 20 base pairs immediately following the complementary pairing with sgRNA, usually a 5'-NGG-3' sequence.
  • the Cas9n-sgRNA complex specifically recognizes a strand of the target DNA sequence in the adjacent region of the PAM through the seed sequence of the sgRNA, and after complementary binding, forms a Cas9n-sgRNA-target DNA binary composite structure to guide the Cas9n nicking
  • the enzyme specifically cleaves the phosphodiester bond between the third and fourth nucleotides in the adjacent front region sequence of the target PAM.
  • isothermal DNA polymerase with strand displacement activity is exo - Klenow fragment, (exo-)DNA polymerase or phi29DNA polymerase.
  • primer 1 and primer 2 are designed to contain a 5'-CCN-3' sequence and a sequence adjacent to and complementary to the two sgRNA seed sequences, respectively.
  • the fluorescent dye is SYBR Green I or EvaGreen.
  • the temperature of the constant temperature reaction is 25-45°C, and the reaction time is 30-200 minutes.
  • the present invention also provides a kit comprising 50-400 nM Cas9n, 100-800 nM sgRNA, 50-500 ⁇ M dNTPs, 200-800 nM primer 1, 200-800 nM primer 2, 0.1-0.6 U/ ⁇ L DNA polymerization Enzyme, fluorescent dye (0.1 ⁇ 1.0 ⁇ SYBR Green I or 0.1 ⁇ 1.0 ⁇ EvaGreen), 0.1 ⁇ 1.0U RNase inhibitor, 50 ⁇ 200mM Tris-HCl, 100 ⁇ 600mM NaCl, 50 ⁇ 400mM MgCl 2 , 50 ⁇ 200 ⁇ g/ mL BSA, 5-20mM dithiothreitol, pH 7.6-8.3, target nucleic acid and DEPC water.
  • a kit comprising 50-400 nM Cas9n, 100-800 nM sgRNA, 50-500 ⁇ M dNTPs, 200-800 nM primer 1, 200-800 nM primer 2, 0.1-0.6 U/ ⁇ L DNA polymerization Enzyme, fluorescent
  • reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction Solution B includes 2 ⁇ L DNA template (DNA genome, plasmid or double-stranded DNA fragment), 0.5 ⁇ L dNTP solution (250 ⁇ M), 0.8 ⁇ L primer 1 (400nM), 0.8 ⁇ L primer 2 (400nM) and 1 ⁇ L buffer (10mM Tris- HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and react at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification curve.
  • the present invention has the following advantages and good effects:
  • the amplification mechanism is simple and efficient: the specific sequence targeting and single-strand cleavage characteristics of Cas9n-sgRNA complex and the strand displacement activity of thermostatic DNA polymerase are used to specifically obtain the target nucleic acid to be detected, which is induced by primer 1 and primer 2. The reaction of primer hybridization, extension, cleavage, and strand displacement reaction occurs, exponentially amplifying the target to be tested.
  • High sensitivity it can detect single-copy target genome nucleic acid molecules.
  • High-throughput analysis By using eight-strip tubes or 96-well PCR plates, the standard working curve and the detection of the samples to be tested can be produced at the same time, reducing detection errors.
  • the method of the invention has the advantages of simple operation, high sensitivity, strong specificity, wide application range, low cost, etc., and is a simple and practical nucleic acid detection technology.
  • Figure 1 is a schematic diagram of the principle of an analysis method for detecting DNA templates (DNA genome, plasmids or double-stranded DNA fragments) using Cas9n with HNH domain activity.
  • Figure 2 is a comparison of amplification curves and histograms of invA target genes of different lengths in the detection of Salmonella genome.
  • Figure 3 shows the sensitivity amplification curve and linear relationship of the 88bp invA target gene detected in the Salmonella genome.
  • Figure 4 shows the sensitivity amplification curve and linear relationship for detecting the 329bp invA target gene in the Salmonella genome.
  • Figure 5 shows the sensitivity amplification curve and linear relationship for detecting the 859bp invA target gene in the Salmonella genome.
  • Figure 6 shows the sensitivity amplification curve and linear relationship for detecting the 1188bp invA target gene in the Salmonella genome.
  • Figure 7 shows the sensitivity amplification curve and linear relationship for detecting the 1849bp invA target gene in the Salmonella genome.
  • Figure 8 shows the sensitivity amplification curve and linear relationship for detecting uidA target genes in the E. coli genome.
  • Figure 9 shows the sensitivity amplification curve and linear relationship for detecting katG target genes in Mycobacterium tuberculosis genome.
  • Figure 10 shows the sensitivity amplification curve and linear relationship of detecting indA target genes in the genome of Rhododerma.
  • Figure 11 is an investigation of method specificity.
  • Figure 12 is the detection of the G13D point mutation of the KRAS gene in the human genome.
  • the DNA amplification template comes from the genome or plasmid extracted from the kit.
  • the primers are synthesized by a biotechnology company.
  • the sgRNA comes from the T7 transcription kit in vitro transcription DNA template.
  • the 20 bases at the 5′ end of the sgRNA are the seed sequence complementary to the pre-spacer region. .
  • step 1 Cas9n with HNH activity is used, the first cycle (steps 1-3), step 1 consists of two sgRNA sequences complementary to the target DNA sequence (20 bases) and Cas9n to form two sets of Cas9n-sgRNA After the complex is combined with the adjacent sequence adjacent motif (protospacer-adjacent motif, PAM, 5′-NGG-3′) of the matching target DNA, after the complex is combined with the sequence b and sequence e, through step 2, the Cas9n single-port cleavage enzyme activity With constant temperature DNA polymerase strand displacement activity, a single-stranded target DNA sequence is obtained.
  • step 2 the first cycle (steps 1-3) consists of two sgRNA sequences complementary to the target DNA sequence (20 bases) and Cas9n to form two sets of Cas9n-sgRNA
  • the adjacent sequence adjacent motif protospacer-adjacent motif, PAM, 5′-NGG-3′
  • This sequence enters the second cycle under the joint action of primer 1 and primer 2, DNA polymerase and Cas9n-sgRNA complex (step 4-11 ), repeat primer hybridization, extension, cleavage, strand displacement reactions under constant temperature conditions, and amplify to obtain a large number of target DNA sequences.
  • Fluorescent dyes and double-stranded DNA products combine to generate fluorescent signals, and the fluorescence signal intensity in the reaction system is measured in real time. Comparing standard working curves, calculate the concentration of the target genome or DNA sequence to be tested.
  • the amplification template sequence is the invA target gene sequence in the Salmonella genome
  • the primer 1 sequence used in the 88bp amplification template is:
  • primer 2 5'-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3', the sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the primer 1 sequence used for the 329bp amplification template is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the primer 1 sequence used in the 859bp amplification template is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the primer 1 sequence used in the 1188bp amplification template is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the sequence of primer 1 used in the 1849bp amplification template is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the invA target gene sequence in the Salmonella genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTACTCCCCTAATTTGATGGATCTCATT-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the invA target gene sequence in the Salmonella genome
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the invA target gene sequence in the Salmonella genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGA-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the invA target gene sequence in the Salmonella genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the invA target gene sequence in the Salmonella genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the uidA target gene sequence in the E. coli genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the katG target gene sequence in the Mycobacterium tuberculosis genome, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the indA target gene sequence in the genome of Rhododerma, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is to construct a plasmid with the indA target gene sequence in the Rhododendula genome with a single base mutation.
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the amplified template sequence is the wild-type KRAS gene and the mutant KRAS G13D gene sequence, and the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to specifically detect invA target genes of different lengths in the Salmonella genome under constant temperature conditions.
  • the detection principle is shown in Figure 1.
  • Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the sequence of the target gene to be detected invA is as follows, the PAM complementary sequence is marked by the box, the sgRNA recognition sequence is underlined, and the Cas9n-sgRNA complex cleavage site is marked with a black triangle symbol:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • Reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction Solution B includes 2 ⁇ L Salmonella genome sample or water (as blank sample), 0.5 ⁇ L dNTP solution (250 ⁇ M), 0.8 ⁇ L primer 1 (400nM), 0.8 ⁇ L primer 2 (400nM) and 1 ⁇ L buffer (10mM Tris-HCl, 50mM) NaCl, 10mM MgCl 2, 1mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and incubate at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification curve.
  • Working curve preparation Prepare Salmonella genome standard solution and blank sample of known concentration respectively, follow the above steps to measure the amplification curve of each reaction solution, and obtain the final state fluorescence value (F) of the target gene and the final state of the blank sample Fluorescence value (F 0 ).
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to specifically detect the uidA target gene in the E. coli genome under constant temperature conditions.
  • the detection principle is shown in Figure 1.
  • Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the uidA sequence of the target gene to be detected is as follows, the PAM complementary sequence is marked by the box, the sgRNA recognition sequence is underlined, and the Cas9n-sgRNA complex cleavage site is marked with a black triangle symbol:
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the specific implementation of the detection of the uidA target gene in the E. coli genome will further illustrate the present invention.
  • the experimental methods without specific conditions are usually in accordance with the conventional conditions or the conditions recommended by the manufacturer.
  • the specific operation steps are as follows: Prepare reaction solutions A and B on ice.
  • Reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • Reaction Solution B includes 2 ⁇ L of E.
  • Working curve preparation Prepare the E. coli genome standard solution and blank sample of known concentration respectively, follow the above steps to measure the amplification curve of each reaction system, and obtain the final state fluorescence value (F) of the target gene and the final blank sample State fluorescence value (F 0 ). Then, according to the ratio (F/F 0 ) of the negative logarithm of the content of the E. coli genome standard solution and the final state fluorescence value (F) of the target gene and the final state fluorescence value (F 0 ) of the blank sample (F/F 0 ), as shown in Figure 8 ( As shown in B), the ratio (F/F 0 ) of the negative logarithm of the content of the E.
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to specifically detect the katG target gene in the genome of Mycobacterium tuberculosis under constant temperature conditions.
  • the detection principle is shown in Figure 1.
  • Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the katG sequence of the target gene to be detected is as follows, the PAM complementary sequence is marked by the box, the sgRNA recognition sequence is underlined, and the Cas9n-sgRNA complex cleavage site is marked with a black triangle symbol:
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTTATCCCCACGGATGCGTCGCAGGAAC-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction Solution B includes 2 ⁇ L of Mycobacterium tuberculosis genome sample or water (as a blank sample), 0.5 ⁇ L of dNTP solution (250 ⁇ M), 0.8 ⁇ L of primer 1 (400nM), 0.8 ⁇ L of primer 2 (400nM) and 1 ⁇ L of buffer (10mM Tris- HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and incubate at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification The curve is shown in Figure 9(A).
  • Preparation of working curve Prepare a standard solution of Mycobacterium tuberculosis genome and a blank sample of known concentration, follow the above steps to measure the amplification curve of each reaction system, and obtain the final state fluorescence value (F) and blank of the target gene The final fluorescence value of the sample (F 0 ).
  • the ratio (F/F 0 ) of the negative logarithm of the content of the Mycobacterium tuberculosis genome standard solution to the final fluorescence value (F) of the target gene and the final fluorescence value (F 0 ) of the blank sample (F/F 0 ) is 2 copies
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to specifically detect indA target genes in the genome of Rhododendron under constant temperature conditions.
  • the detection principle is shown in Figure 1.
  • Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the target gene sequence is as follows, the PAM complementary sequence is marked by a box, the sgRNA recognition sequence is underlined, and the Cas9n-sgRNA complex cleavage site is marked with a black triangle symbol:
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is: 5'-GACTAGTTATCCCGCGCGATCGACGCCCCCAGC-3',
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • Reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction Solution B includes 2 ⁇ L of Rhododendrum genome sample or water (as a blank sample), 0.5 ⁇ L of dNTP solution (250 ⁇ M), 0.8 ⁇ L of primer 1 (400nM), 0.8 ⁇ L of primer 2 (400nM) and 1 ⁇ L of buffer (10mM Tris-HCl, It consists of 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and incubate at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification The curve is shown in Figure 10(A).
  • Preparation of working curve Prepare a standard solution of the Rhododendron genome of known concentration and a blank sample (does not contain the Rhodophyte genome, that is, the concentration of the Rhododerma genome is 0), follow the above steps to determine the amplification curve of each reaction system, The final fluorescence value (F) of the target gene and the final fluorescence value (F 0 ) of the blank sample are obtained.
  • Figure 11 shows the specific experiment of the method of the present invention.
  • the amplified template sequence is used to construct a plasmid with a single-base mutation of the Rhododerma indA target gene sequence.
  • Md is the single base mutation sequence adjacent to the motif PAM of the sequence before the sequence e. All single base mutations are marked in bold.
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to conduct the specific investigation of the invention under constant temperature conditions.
  • the detection principle is shown in Figure 1. Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the indA target gene sequence of the Rhododerma spp. genome with single-base mutation was amplified from the plasmid with the single-base mutation indA target gene sequence of Rhododerma spp., and the indA target gene standard with known concentration of single-base mutation was prepared respectively.
  • Solution and blank sample without single-base mutant indA target gene, that is, single-base mutant indA target gene content is 0
  • single-base mutation of indA target gene of Rhododendron was detected (Figure 11).
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • Reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction solution B includes 2 ⁇ L of single-base mutation indA target gene sample or water (as a blank sample), 0.5 ⁇ L dNTP solution (250 ⁇ M), 0.8 ⁇ L primer 1 (400nM), 0.8 ⁇ L primer 2 (400nM) and 1 ⁇ L buffer ( It consists of 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and incubate at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification Curve to obtain the final fluorescence value (F) of the target gene and the final fluorescence value (F 0 ) of the blank sample.
  • the ratio (F/F 0 ) of the final fluorescence value of the target gene (F) and the final fluorescence value (F 0 ) of the blank sample (F/F 0 ) is used for specific analysis, as shown in Figure 11 (B), the single-base mutation indA target gene
  • the ratio of the final fluorescence value (F) and the final fluorescence value of the blank sample (F 0 ) (F/F 0 ) to the final fluorescence value (F) of the wild-type indA target gene and the final fluorescence value of the blank sample (F 0 ) (F/F 0 ) has a great difference, and the experimental results show that the analysis method has a good single-base recognition ability.
  • the constant temperature nucleic acid detection and analysis method of Cas9 nickase coupled with DNA polymerase is used to specifically detect the G13D point mutation (G ⁇ A) of the KRAS gene in the human genome under constant temperature conditions.
  • the detection principle is shown in Figure 1.
  • Cas9n with HNH domain activity, exo - Klenow fragment DNA polymerase and double-stranded DNA binding dye SYBR Green I are used.
  • the KRAS G13D point mutation sequence of the target gene to be detected is as follows, the PAM sequence is marked by the box, the sgRNA recognition sequence is underlined, the Cas9n-sgRNA complex cleavage site is marked with a black triangle symbol, and the G13D single-base mutation site is capitalized Bold base A.
  • the primer 1 sequence used is:
  • primer 2 The sequence of primer 2 is:
  • sequence of sgRNA1 is:
  • sequence of sgRNA2 is:
  • the specific implementation of the G13D point mutation detection of the KRAS gene in the human genome will further illustrate the present invention.
  • the experimental methods without specific conditions are usually in accordance with the conventional conditions or the conditions recommended by the manufacturer.
  • the specific operation steps are as follows: Prepare reaction solutions A and B on ice.
  • Reaction solution A includes 1.9 ⁇ L Cas9n-sgRNA complex, 0.8 ⁇ L exo-Klenow polymerase (0.2U/ ⁇ L), 2.5 ⁇ L RNase inhibitor (0.1U), 1 ⁇ L reaction buffer (10mM Tris-HCl, 50mM NaCl, 10mM MgCl 2 , 1mM dithiothreitol, pH 7.9), 0.4 ⁇ L fluorescent dye (0.4 ⁇ SYBR Green I or 0.4 ⁇ EvaGreen), 3.4 ⁇ L DEPC water composition.
  • Cas9n-sgRNA complex 0.5 ⁇ L Cas9n (200nM) and 0.4 ⁇ L sgRNA (for example: sgRNA1 and sgRNA2, 200nM) dissolved in 1 ⁇ L buffer (10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA) , PH 7.9), incubate at 37°C for 20 minutes.
  • 1 ⁇ L buffer 10mM NaCl, 5mM Tris-HCl, 1mM MgCl 2 , 10 ⁇ g/mL BSA
  • Reaction Solution B includes 2 ⁇ L of human genome sample or water (as a blank sample), 0.5 ⁇ L of dNTP solution (250 ⁇ M), 0.8 ⁇ L of primer 1 (400nM), 0.8 ⁇ L of primer 2 (400nM) and 1 ⁇ L of buffer (10mM Tris-HCl, It consists of 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, pH 7.9) and 4.9 ⁇ L DEPC water. Mix the reaction solution A and B on ice. The total volume of the mixed reaction solution is 20 ⁇ L. Place the above reaction solution in a fluorescent quantitative PCR machine and incubate at 37°C for 60 minutes. The fluorescence signal is detected every 2.5 minutes to obtain amplification The curve is shown in Figure 12(A).
  • the G13D point mutation detection results of the KRAS gene in the human genome were analyzed by t-test, and a mixed sample and a blank sample of the wild-type KRAS gene and the mutant KRAS G13D gene of known concentrations were prepared respectively (without the human genome, that is, the human genome content is 0).
  • the present invention utilizes the specific sequence targeting and single-stranded cleavage characteristics of Cas9n-sgRNA complex and the strand displacement activity of thermostatic DNA polymerase to specifically cut a specific single-stranded nucleic acid sequence from the target nucleic acid, and then introduce a Cas9n-sgRNA complex recognition and cleavage sequence primer 1 and primer 2 pair single-stranded nucleic acid sequence under constant temperature conditions, repeated primer hybridization, extension, cleavage, strand displacement reaction to achieve exponential amplification of target nucleic acid molecules; using fluorescence
  • the dye combines with the double-stranded DNA product to generate a fluorescent signal.
  • the intensity of the fluorescent signal in the reaction system is measured in real time and compared with the standard working curve to calculate the target nucleic acid concentration.
  • the kit of the present invention contains Cas9n, two sgRNA sequences complementary to 20 bases of target nucleic acid, thermostatic DNA polymerase, primer pairs complementary to two sgRNA sequences, dNTP solution, and embedded fluorescent dye.
  • the method of the invention has the advantages of high-efficiency amplification mechanism, high sensitivity, strong specificity, simple operation, low detection cost, etc., and can be applied to genome detection in biological samples such as microorganisms, tissues, blood and cells, and is especially suitable for immediate nucleic acid detection.

Abstract

本发明公开了一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，是在恒温条件下特异性检测靶标核酸；包括以下步骤：以两条分别与靶DNA序列互补的sgRNA序列，两组Cas9n-sgRNA复合物分别与前间区序列邻近基序毗邻的匹配靶DNA序列结合后，通过Cas9n单口切割酶活性和恒温DNA聚合酶链置换活性，获得一条单链靶DNA序列，该序列在引物1和引物2，DNA聚合酶及Cas9n-sgRNA复合物的共同作用下，在恒温条件下反复进行引物杂交，延伸，切割，链置换反应，扩增获得大量靶DNA序列，荧光染料与双链DNA产物结合产生荧光信号，实时测定反应体系中的荧光信号强度，与标准工作曲线对比，计算出靶标基因组或DNA序列的浓度。本发明适用于核酸即时检测。

Description

一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法及试剂盒 技术领域
本发明属于核酸恒温扩增技术领域，具体涉及一种基于Cas9切口酶介导的定量核酸检测方法及试剂盒，适合对基因组或双链DNA的恒温定量检测。
背景技术
核酸扩增反应不仅在分子生物学和医学，而且在法医学、流行病学和古生物学中都是一种非常重要而有价值的技术。其中应用最为广泛的是聚合酶链式反应(PCR)，一类核酸体外扩增技术，几乎应用于所有的生物及临床诊断实验室。然而PCR方法需要依赖控温精确的热循环仪，使得以PCR为基础的核酸扩增检测技术不适用于现场快速检测或是偏远、贫穷、没有相关仪器的环境进行检测。核酸等温扩增技术由于不需要温度变化操作，摆脱了对热循环仪器的依赖，在临床和现场快速诊断中展示了良好的应用前景。目前，常用的核酸恒温检测方法包括自主序列复制系统(3SR)、核酸序列依赖扩增技术(NASBA)、恒温指数扩增技术(EXPAR)、链置换扩增技术(SDA)、滚环核酸扩增(RCA)、解旋酶扩增技术(HAD)、环介导等温扩增技术(LAMP)、重组酶聚合酶扩增(RPA)等。3SR和NASBA通过采用逆转录酶、RNase H和T7RNA聚合酶共同介导的逆转录、消化和转录的级联反应扩增单链RNA，避免了热变性反应。同样地，EXPAR和RCA也更适用于短单链DNA或ssRNA的直接扩增反应。对于长双链DNA或基因组DNA扩增，SDA需要初始加热反应使双链DNA模板变性，用于引物退火反应。LAMP同样也需要初始热变性步骤，而且引物设计要求高。HAD受DNA解旋酶限制，只适用于扩增小片段的DNA序列。RPA需要多种酶参与，包括重组酶(gp32，uxsX，uvsY)，单链结合蛋白，肌酸激酶和DNA聚合酶，无疑增加了检测成本。现有的核酸恒温扩增技术虽然解决了PCR方法依赖的热循环反应，但它们有些仍需要初始热变性操作来分离核酸双链结构，或多种酶蛋白、特殊反应试剂参与反应，或复杂的引物设计。因此，开发扩增机制高效、操作简便、灵敏度高、特异性强、检测成本低的通用等温核酸扩增方法仍然是一个挑战。
发明内容
针对现有技术的不足，本发明提出一种基于Cas9切口酶(Cas9n)偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测靶标核酸，包括以下步骤：
以两条分别与靶DNA序列(20个碱基)互补的sgRNA序列，两组Cas9n-sgRNA复合物分别与前间区序列邻近基序(protospacer-adjacent motif，PAM，5′-NGG-3′)毗邻的匹配靶DNA序列结合后，通过Cas9n单口切割酶活性和恒温DNA聚合酶链置换活性，获得一条单链靶DNA序列，该序列在引物1和引物2，DNA聚合酶及Cas9n-sgRNA复合物的共同作用下，在恒温条件下反复进行引物杂交，延伸，切割，链置换反应，扩增获得大量靶DNA序列，荧光染料与双链DNA产物结合产生荧光信号，实时测定反应体系中的荧光信号强度，与标准工作曲线对比，计算出靶标基因组或DNA序列的浓度。
进一步的，所述靶DNA序列包括DNA基因组、质粒或双链DNA片段。
进一步的，所述DNA基因组为微生物、组织、血液或细胞等生物样本的基因组。
进一步的，所述Cas9切口酶(Cas9n)是失活了Cas9双链核酸酶的一个结构域，Cas9具有HNH和RuvC两个催化结构域，HNH结构域切割互补的DNA序列，RuvC切割非互补的DNA序列，而Cas9切口酶仅包含一个具有活性的催化结构域，HNH或RuvC。
进一步的，所述sgRNA是单向导RNA(single guide RNA)，含有与靶DNA序列(前间区序列)互补的20个碱基的种子序列。
进一步的，所述PAM是前间区序列邻近基序(protospacer adjacent motif)，位于紧随着与sgRNA互补配对的20个碱基对的靶序列，通常为5′-NGG-3′序列。
进一步的，所述Cas9n-sgRNA复合物通过sgRNA的种子序列特异性地识别PAM相邻区域的靶DNA序列的一条链，互补结合后，形成Cas9n-sgRNA-靶DNA二元复合结构，引导Cas9n切口酶特异性切割靶PAM毗邻的前间区序列中第三和第四个核苷酸之间的磷酸二酯键。
进一步的，所述具有链置换活性的恒温DNA聚合酶为exo -Klenow fragment，
Figure PCTCN2019106693-appb-000001
(exo-)DNA聚合酶或phi29DNA聚合酶。
进一步的，所述引物1和引物2分别设计含有5'-CCN-3'序列和毗邻与两条sgRNA种子序列互补的序列。
进一步的，所述荧光染料为SYBR Green I或EvaGreen。
进一步的，所述恒温反应的温度为25～45℃，反应时间为30～200分钟。
本发明还提出了一种试剂盒，所述试剂盒包括50～400nM Cas9n，100～800nM sgRNA，50～500μM dNTPs，200～800nM引物1，200～800nM引物2，0.1～0.6U/μL DNA聚合酶，荧光染料(0.1～1.0×SYBR Green I或0.1～1.0×EvaGreen)，0.1～1.0U RNase抑制剂，50～200mM Tris-HCl，100～600mM NaCl，50～400mM MgCl 2，50～200μg/mL BSA，5～20mM二硫苏糖醇，pH 7.6～8.3，靶核酸和DEPC水。
为了实现上述目的，本发明的技术方案如下：
本发明恒温核酸检测分析方法的具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL DNA模板(DNA基因组、质粒或双链DNA片段)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃反应60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线。
本发明与现有技术相比，具有以下优点和良好效果：
1、扩增机制简单而高效：利用Cas9n-sgRNA复合物特异性序列靶向性和单链切割特性及恒温DNA聚合酶的链置换活性特异性获得待检测靶标核酸，通过引物1和引物2诱导进行引物杂交，延伸，切割，链置换反应的反应发生，指数扩增待测靶标。
2、灵敏度高：可实现单拷贝靶基因组核酸分子的检测。
3、特异性强：可实现靶标单碱基突变检测(如单核苷酸多态性检测)，例如人基因组中结肠癌相关的KRAS G13D点突变检测，最低可以检测出0.5％KRAS G13D点突变。
4、普适性广：靶标核酸待测序列长度没有限制，引物设计简单，可迅速应用于不同核酸序列特异性检测。
5、高通量分析：通过采用八联管或96孔PCR板，可以实现同时制作标准工作曲线和待测样本的检测，减少检测误差。
6、操作简便：整个操作过程只有简单的试剂添加过程。
7、检测成本低：反应组分简单、Cas9n-sgRNA复合物、恒温DNA聚合酶、普通引物和荧光染料。
8、无污染：整个检测过程不需要用到有机溶剂或有毒试剂。
本发明方法具有操作简便、灵敏度高、特异性强、适用范围广、成本低等优点，是一种简便实用的核酸检测技术。
附图说明
图1是采用HNH结构域活性的Cas9n检测DNA模板(DNA基因组、质粒或双链 DNA片段)分析方法的原理示意图。
图2是检测沙门氏菌基因组中不同长度invA靶基因扩增曲线及柱状图比较。
图3是检测沙门氏菌基因组中88bp invA靶基因灵敏度扩增曲线及线性关系。
图4是检测沙门氏菌基因组中329bp invA靶基因灵敏度扩增曲线及线性关系。
图5是检测沙门氏菌基因组中859bp invA靶基因灵敏度扩增曲线及线性关系。
图6是检测沙门氏菌基因组中1188bp invA靶基因灵敏度扩增曲线及线性关系。
图7是检测沙门氏菌基因组中1849bp invA靶基因灵敏度扩增曲线及线性关系。
图8是检测大肠杆菌基因组中uidA靶基因灵敏度扩增曲线及线性关系。
图9是检测结核分枝杆菌基因组中katG靶基因灵敏度扩增曲线及线性关系。
图10是检测红霉菌基因组中indA靶基因灵敏度扩增曲线及线性关系。
图11是方法特异性考察。
图12是检测人基因组中KRAS基因的G13D点突变。
具体实施方式
下面结合实施例对本发明作进一步详细的说明。其中DNA扩增模板来自试剂盒提取的基因组或质粒，引物委托生物技术公司合成，sgRNA来自T7转录试剂盒体外转录DNA模板，sgRNA的5′端20个碱基为与前间隔区互补的种子序列。
符号说明：
图1中，采用具有HNH活性的Cas9n，第一个循环(步骤1-3)，步骤1为两条分别与靶DNA序列(20个碱基)互补的sgRNA序列与Cas9n组成两组Cas9n-sgRNA复合物分别与前间区序列邻近基序(protospacer-adjacent motif，PAM，5′-NGG-3′)毗邻的匹配靶DNA的序列b和序列e结合后，通过步骤2，Cas9n单口切割酶活性和恒温DNA聚合酶链置换活性，获得一条单链靶DNA序列，该序列在引物1和引物2，DNA聚合酶及Cas9n-sgRNA复合物的共同作用下，进入第二个循环(步骤4-11)，在恒温条件下反复进行引物杂交，延伸，切割，链置换反应，扩增获得大量靶DNA序列，荧光染料与双链DNA产物结合产生荧光信号，实时测定反应体系中的荧光信号强度，与标准工作曲线对比，计算出待测靶标基因组或DNA序列的浓度。
图2中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，88bp扩增模板采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，引物2序列为：
5′-GACTAGTACTCCCCTAATTTGATGGATCTCATT-3′，
sgRNA1序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
329bp扩增模板采用的引物1序列为：
5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGAGAGCGGCTGCTCGCCTT-3′，
引物2序列为：5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
859bp扩增模板采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：
5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGA-3′，
sgRNA1序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
1188bp扩增模板采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
1849bp扩增模板采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，
引物2序列为：
5′-GACTAGTTATCCCATCTGGTTGATTTCCTGATC-3′，
sgRNA1序列为：
5′- AUCUGGUUGAUUUCCUGAUCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图3中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，
引物2序列为：5′-GACTAGTACTCCCCTAATTTGATGGATCTCATT-3′，
sgRNA1序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区 序列互补。
图4中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGAGAGCGGCTGCTCGCCTT-3′，
引物2序列为：
5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图5中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGA-3′，
sgRNA1序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图6中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：
5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图7中，扩增模板序列为沙门氏菌基因组中invA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，
引物2序列为：
5′-GACTAGTTATCCCATCTGGTTGATTTCCTGATC-3′，
sgRNA1序列为：
5′- AUCUGGUUGAUUUCCUGAUCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图8中，扩增模板序列为大肠杆菌基因组中uidA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCGGTGAAGGTTATCTCTATGAACCCGGTTGCCAGAGGT-3′，
引物2序列为：
5′-GACTAGTTATCCCCCGGGAATGGTGATTACCGA-3′，
sgRNA1序列为：
5′- CCGGGAAUGGUGAUUACCGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GGUGAAGGUUAUCUCUAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图9中，扩增模板序列为结核分枝杆菌基因组中katG靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCCGTGGACCTGGTCTTCGGGTACGCGGCTGCCGGTCCA-3′，
引物2序列为：
5′-GACTAGTTATCCCCACGGATGCGTCGCAGGAAC-3′，
sgRNA1序列为：
5′- GUUCCUGCGACGCAUCCGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- ACCCGAAGACCAGGUCCACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图10中，扩增模板序列为红霉菌基因组中indA靶基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCCCGGGATGTTCAGCGTCTCCCGGTGGTCGGCTACCGG-3′，
引物2序列为：
5′-GACTAGTTATCCCGCGCGATCGACGCCCCCAGC-3′，
sgRNA1序列为：
5′- GGAGACGCUGAACAUCCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GCUGGGGGCGUCGAUCGCGCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图11中，方法的特异性考察，扩增模板序列为构建带有单碱基突变的红霉菌基因组中indA靶基因序列的质粒，采用的引物1序列为：
5′-GACTAGTTATCCCCCGGGATGTTCAGCGTCTCCCGGTGGTCGGCTACCGG-3′，
引物2序列为：
5′-GACTAGTTATCCCGCGCGATCGACGCCCCCAGC-3′，
sgRNA1序列为：
5′- GGAGACGCUGAACAUCCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GCUGGGGGCGUCGAUCGCGCUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
图12中，扩增模板序列为野生型KRAS基因与突变型KRAS G13D基因序列，采用的引物1序列为：
5′-GACTAGTTATCCCACGTCACCAGCTCCAACTACCGTAGGCAAGAGTGCCT-3′，
引物2序列为：
5′-GACTAGTTATCCCTGTCTTGTCTTTGCTGATGT-3′，
sgRNA1序列为：
5′- GUAGUUGGAGCUGGUGACGUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- ACAUCAGCAAAGACAAGACAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
通过下述实施例将有助于理解本发明，但是不能限制本发明的内容。
实施例1
利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测沙门氏菌基因组中不同长度invA靶基因，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。待检测靶基因invA序列如下，PAM互补序列由方框标出，sgRNA识别序列为下划线部分，Cas9n-sgRNA复合物切割位点采用黑色三角符号标出：
Figure PCTCN2019106693-appb-000002
检测沙门氏菌基因组中不同长度invA靶基因扩增曲线及柱状图比较如图2所示，分别制备已知浓度的沙门氏菌基因组标准溶液，加入反应液中靶基因终含量依次为20拷贝，100拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含沙门氏菌基因组，即沙门氏菌基因组含量为0)，对88bp扩增模板进行灵敏度检测，获得扩增曲线，如附图3(A)所示，采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，
引物2序列为：
5′-GACTAGTACTCCCCTAATTTGATGGATCTCATT-3′，
sgRNA1序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列为与靶标互补序列；
分别制备已知浓度的沙门氏菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷 贝以及空白样本(不含沙门氏菌基因组，即沙门氏菌基因组含量为0)，对329bp扩增模板进行灵敏度检测，获得扩增曲线，如附图4(A)所示，采用的引物1序列为：
5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGAGAGCGGCTGCTCGCCTT-3′，
引物2序列为：
5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列为与靶标互补序列；
分别制备已知浓度的沙门氏菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含沙门氏菌基因组，即沙门氏菌基因组含量为0)，对859bp扩增模板进行灵敏度检测，获得扩增曲线，如附图5(A)所示，采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：
5′-GACTAGTTATCCCCTCAGTATTGAGGAAAAAGA-3′，
sgRNA1序列为：
5′- CUCAGUAUUGAGGAAAAAGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列为与靶标互补序列；
分别制备已知浓度的沙门氏菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含沙门氏菌基因组，即沙门氏菌基因组含量为0)，对1188bp扩增模板进行灵敏度检测，获得扩增曲线，如附图6(A)所示，采用的引物1序列为：
5′-GACTAGTTATCCCCTAATTTGATGGATCTCATTTAGCGGAGGCTTCCGGG-3′，
引物2序列为：
5′-GACTAGTTATCCCCCCTGTCTACTTATACCATG-3′，
sgRNA1序列为：
5′- CCCUGUCUACUUAUACCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- CUAAUUUGAUGGAUCUCAUUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列为与靶标互补序列；
分别制备已知浓度的沙门氏菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含沙门氏菌基因组，即沙门氏菌基因组含量为0)，对1849bp扩增模板进行灵敏度检测，获得扩增曲线，如附图7(A)所示，采用的引物1序列为：
5′-GACTAGTTATCCCTCGATTTATTAAGAAAATGACGACGGACATCGACAGA-3′，
引物2序列为：
5′-GACTAGTTATCCCATCTGGTTGATTTCCTGATC-3′，
sgRNA1序列为：
5′- AUCUGGUUGAUUUCCUGAUCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- UCGAUUUAUUAAGAAAAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
下面对沙门氏菌基因组中不同长度invA靶基因检测的具体实施来进一步说明本发明。其中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA 复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL沙门氏菌基因组样本或水(作为空白样本)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线。
工作曲线的制作：分别制备已知浓度的沙门氏菌基因组标准溶液和空白样本，按照上述步骤进行操作，测定各个反应液的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)。然后根据沙门氏菌基因组标准溶液含量的负对数值和靶基因终态荧光值(F)与空白样本终态荧光值(F 0)比值(F/F 0)制作标准工作曲线，如附图3(B)所示为沙门氏菌基因组88bp扩增模板标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在20拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝0.69×lg[S.typhimurium]-0.05。如附图4(B)所示为沙门氏菌基因组329bp扩增模板标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝1.08×lg[S.typhimurium]+0.05。如附图5(B)所示为沙门氏菌基因组859bp扩增模板标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝1.49×lg[S.typhimurium]+0.49。如附图6(B)所示为沙门氏菌基因组1188bp扩增模板标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝3.11×lg[S.typhimurium]+2.24。如附图7(B)所示为沙门氏菌基因组1849bp扩增模板标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝2.88×lg[S.typhimurium]+0.83。
实施例2
利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测大肠杆菌基因组中uidA靶基因，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。待检测靶基因uidA序列如下，PAM互补序列由方框标出，sgRNA识别序列为下划线部分，Cas9n-sgRNA 复合物切割位点采用黑色三角符号标出：
Figure PCTCN2019106693-appb-000003
分别制备已知浓度的大肠杆菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含大肠杆菌基因组，即大肠杆菌基因组含量为0)，对大肠杆菌基因组中uidA靶基因进行灵敏度检测(图8)。
采用的引物1序列为：
5′-GACTAGTTATCCCGGTGAAGGTTATCTCTATGAACCCGGTTGCCAGAGGT-3′，
引物2序列为：
5′-GACTAGTTATCCCCCGGGAATGGTGATTACCGA-3′，
sgRNA1序列为：
5′- CCGGGAAUGGUGAUUACCGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GGUGAAGGUUAUCUCUAUGAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
下面对大肠杆菌基因组中uidA靶基因检测的具体实施来进一步说明本发明。其中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL大肠杆菌基因组样本或水(作为空白样本)， 0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线，如附图8(A)所示。
工作曲线的制作：分别制备已知浓度的大肠杆菌基因组标准溶液和空白样本，按照上述步骤进行操作，测定各个反应体系的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)。然后根据大肠杆菌基因组标准溶液含量的负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)制作标准工作曲线，如附图8(B)所示，大肠杆菌基因组标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝1.13×lg[E.coli]-0.04。
实施例3
利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测结核分枝杆菌基因组中katG靶基因，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。待检测靶基因katG序列如下，PAM互补序列由方框标出，sgRNA识别序列为下划线部分，Cas9n-sgRNA复合物切割位点采用黑色三角符号标出：
Figure PCTCN2019106693-appb-000004
分别制备已知浓度的结核分枝杆菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含结核分枝杆菌基因组，即结核分枝杆菌基因组含量为0)，对结 核分枝杆菌基因组中katG靶基因进行灵敏度检测(图9)。
采用的引物1序列为：
5′-GACTAGTTATCCCCGTGGACCTGGTCTTCGGGTACGCGGCTGCCGGTCCA-3′，
引物2序列为：5′-GACTAGTTATCCCCACGGATGCGTCGCAGGAAC-3′，
sgRNA1序列为：
5′- GUUCCUGCGACGCAUCCGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- ACCCGAAGACCAGGUCCACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
下面对结核分枝杆菌基因组中katG靶基因检测的具体实施来进一步说明本发明。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL结核分枝杆菌基因组样本或水(作为空白样本)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线，如附图9(A)所示。
工作曲线的制作：分别制备已知浓度的结核分枝杆菌基因组标准溶液和空白样本，按照上述步骤进行操作，测定各个反应体系的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)。然后根据结核分枝杆菌基因组标准溶液含量的负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)制作标准工作曲线，如附图9(B)所示，结核分枝杆菌基因组标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝1.64×lg[M.tuberculosis]+0.25。
实施例4
利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测红霉菌基因组中indA靶基因，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。靶基因序列如下，PAM互补序列由方框标出，sgRNA识别序列为下划线部分，Cas9n-sgRNA复合物切割位点采用黑色三角符号标出：
Figure PCTCN2019106693-appb-000005
分别制备已知浓度的红霉菌基因组标准溶液，加入反应液中靶基因终含量依次为2拷贝，10拷贝，20拷贝，200拷贝，2×10 3拷贝，2×10 4拷贝，2×10 5拷贝和2×10 6拷贝以及空白样本(不含红霉菌基因组，即红霉菌基因组含量为0)，对红霉菌基因组中indA靶基因进行灵敏度检测(图10)。
采用的引物1序列为：
5′-GACTAGTTATCCCCCGGGATGTTCAGCGTCTCCCGGTGGTCGGCTACCGG-3′，
引物2序列为：5′-GACTAGTTATCCCGCGCGATCGACGCCCCCAGC-3′，
sgRNA1序列为：
5′- GGAGACGCUGAACAUCCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GCUGGGGGCGUCGAUCGCGCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区 序列互补。
下面对红霉菌基因组中indA靶基因检测的具体实施来进一步说明本发明。其中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL红霉菌基因组样本或水(作为空白样本)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线，如附图10(A)所示。
工作曲线的制作：分别制备已知浓度的红霉菌基因组标准溶液和空白样本(不含有红霉菌基因组，即红霉菌基因组浓度为0)，按照上述步骤进行操作，测定各个反应体系的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)。根据红霉菌基因组标准溶液含量的负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)制作标准工作曲线，如附图10(B)所示，红霉菌基因组标准溶液的含量负对数值与靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)在2拷贝～2×10 6拷贝范围内呈线性关系，线性方程为F/F 0＝1.33×lg[S.erythraea]+1.41。
实施例5
附图11所示为本发明方法的特异性实验，扩增模板序列为构建带有单碱基突变的红霉菌indA靶基因序列的质粒，如附图11(A)所示，质粒模板的前间区序列单碱基突变：Mb1-Mb20，Me1-Me20(其中数字1-20代表突变位点的位置)，WTb和WTe是无单碱基突变的野生型，PAM1，PAM2为靶DNA的序列b和序列e前间区序列邻近基序突变型，Md为序列e前间区序列邻近基序PAM毗邻的单碱基突变序列，所有单碱基突变由大写加粗标出。利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下进行该发明的特异性考察，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。
从带有单碱基突变的红霉菌indA靶基因序列的质粒上扩增得到带有单碱基突变的红 霉菌基因组indA靶基因序列，分别制备已知浓度的单碱基突变的indA靶基因标准溶液和空白样本(不含单碱基突变indA靶基因，即单碱基突变indA靶基因含量为0)，对红霉菌indA靶基因单碱基突变进行检测(图11)。
采用的引物1序列为：
5′-GACTAGTTATCCCCCGGGATGTTCAGCGTCTCCCGGTGGTCGGCTACCGG-3′，
引物2序列为：
5′-GACTAGTTATCCCGCGCGATCGACGCCCCCAGC-3′，
sgRNA1序列为：
5′- GGAGACGCUGAACAUCCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- GCUGGGGGCGUCGAUCGCGCUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
下面对红霉菌基因组中单碱基突变的indA靶基因检测的具体实施来进一步说明本发明。其中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL单碱基突变的indA靶基因样本或水(作为空白样本)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)。
特异性分析：分别制备已知浓度的单碱基突变indA靶基因标准溶液以及空白样本(不含单碱基突变indA靶基因，即单碱基突变indA靶基因含量为0)，按照上述步骤进行操作，测定各个反应体系的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值 (F 0)。采取靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)进行特异性分析，如附图11(B)所示，单碱基突变indA靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)与野生型indA靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)具有很大差异，实验结果表明该分析方法具有很好的单碱基识别能力。
实施例6
利用Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，在恒温条件下特异性检测人基因组中KRAS基因的G13D点突变(G→A)，检测原理如图1所示。采用HNH结构域活性的Cas9n，exo -Klenow fragment DNA聚合酶和双链DNA结合染料SYBR Green I。待检测靶基因KRAS G13D点突变序列如下，PAM序列由方框标出，sgRNA识别序列为下划线部分，Cas9n-sgRNA复合物切割位点采用黑色三角符号标出，G13D单碱基突变位点为大写加粗碱基A。
Figure PCTCN2019106693-appb-000006
制备人基因组中野生型KRAS基因与突变型KRAS G13D基因的混合样本及空白样本(不含人基因组，即人基因组含量为0)，突变型KRAS G13D基因添加比例分别为0％，0.5％，1％，5％，10％，50％，100％，对人基因组中KRAS基因的G13D点突变进行检测(图12)。
采用的引物1序列为：
5′-GACTAGTTATCCCACGTCACCAGCTCCAACTACCGTAGGCAAGAGTGCCT-3′，
引物2序列为：
5′-GACTAGTTATCCCTGTCTTGTCTTTGCTGATGT-3′，
sgRNA1序列为：
5′- GUAGUUGGAGCUGGUGACGUUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补，
sgRNA2序列为：
5′- ACAUCAGCAAAGACAAGACAUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUG-3′，其中下划线序列与靶标的前间区序列互补。
下面对人基因组中KRAS基因的G13D点突变检测的具体实施来进一步说明本发明。其中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件。具体操作步骤如下：在冰上制备反应液A和B。反应液A包括1.9μL Cas9n-sgRNA复合物，0.8μL exo-Klenow聚合酶(0.2U/μL)，2.5μL RNase抑制剂(0.1U)，1μL反应缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)，0.4μL荧光染料(0.4×SYBR Green I或0.4×EvaGreen)，3.4μL DEPC水组成。其中Cas9n-sgRNA复合物的制备：0.5μL Cas9n(200nM)和0.4μL sgRNA(例如：sgRNA1和sgRNA2，200nM)溶解于1μL缓冲液(10mM NaCl，5mM Tris-HCl，1mM MgCl 2，10μg/mL BSA，pH 7.9)，37℃下孵育20分钟。反应液B包括2μL人基因组组样本或水(作为空白样本)，0.5μL dNTP溶液(250μM)，0.8μL引物1(400nM)，0.8μL引物2(400nM)和1μL缓冲液(10mM Tris-HCl，50mM NaCl，10mM MgCl 2，1mM二硫苏糖醇，pH 7.9)和4.9μL DEPC水组成。将反应液A和B在冰上混合，混合后的反应液总体积为20μL，将上述反应液置于荧光定量PCR仪，于37℃孵育60分钟，每隔2.5分钟检测荧光信号，获得扩增曲线，如附图12(A)所示。
对人基因组中KRAS基因的G13D点突变检测结果进行t检验分析，分别制备已知浓度的野生型KRAS基因与突变型KRAS G13D基因的混合样本及空白样本(不含人基因组，即人基因组含量为0)，按照上述步骤进行操作，测定各个反应体系的扩增曲线，得出靶基因终态荧光值(F)和空白样本终态荧光值(F 0)，采取靶基因终态荧光值(F)和空白样本终态荧光值(F 0)比值(F/F 0)进行t检验分析，如附图12(B)所示，MT(％)表示突变型KRAS G13D基因比例，WT(％)表示野生型KRAS基因比例，将0.5％，1％，5％，10％，50％，100％的MT(％)测试样本分别与100％的WT(％)进行t检验分析，得出P值结果，*P<0.05，**P<0.01，***P<0.001，****P<0.0001，误差分析为3次平行实验的平均值分析，实验结果表明该试剂盒具有很强的特异性，可在混合样本中检测出0.5％的突变样本。
本发明利用Cas9n-sgRNA复合物特异性序列靶向性和单链切割特性及恒温DNA聚合酶的链置换活性，从靶标核酸中特异性切割获取特异性的一段单链核酸序列，然后通过引入含有Cas9n-sgRNA复合物识别和切割序列的引物1和引物2对单链核酸序列在恒温条件下，反复进行引物杂交，延伸，切割，链置换反应，实现对靶核酸分子的指数扩增；采用荧光染料与双链DNA产物结合产生荧光信号，实时测定反应体系中的荧光信号强度， 与标准工作曲线对比，计算出靶标核酸的浓度。
本发明试剂盒中包含Cas9n，两条分别与靶标核酸20个碱基互补的sgRNA序列，恒温DNA聚合酶，与两条sgRNA序列互补的引物对，dNTP溶液，嵌入式荧光染料。
本发明方法具有高效的扩增机制、灵敏度高、特异性强、操作简便、检测成本低等优点，可应用于微生物、组织、血液和细胞等生物样本中基因组检测，尤其适用于核酸即时检测。

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  1. 一种基于Cas9切口酶偶联DNA聚合酶的恒温核酸检测分析方法，是在恒温条件下特异性检测靶标核酸；其特征在于，包括以下步骤：
    以两条分别与靶DNA序列互补的sgRNA序列，两组Cas9n-sgRNA复合物分别与前间区序列邻近基序毗邻的匹配靶DNA序列结合后，通过Cas9n单口切割酶活性和恒温DNA聚合酶链置换活性，获得一条单链靶DNA序列，该序列在引物1和引物2，DNA聚合酶及Cas9n-sgRNA复合物的共同作用下，在恒温条件下反复进行引物杂交，延伸，切割，链置换反应，扩增获得大量靶DNA序列，荧光染料与双链DNA产物结合产生荧光信号，实时测定反应体系中的荧光信号强度，与标准工作曲线对比，计算出靶标基因组或DNA序列的浓度。
  2. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述靶DNA序列包括DNA基因组、质粒和/或双链DNA片段。
  3. 根据权利要求2所述的核酸恒温检测方法，其特征在于，所述DNA基因组为微生物、组织、血液和/或细胞生物样本的基因组。
  4. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述Cas9切口酶是失活了Cas9双链核酸酶的一个结构域，Cas9具有HNH和RuvC两个催化结构域，HNH结构域切割互补的DNA序列，RuvC切割非互补的DNA序列，而Cas9切口酶仅包含一个具有活性的催化结构域，HNH或RuvC。
  5. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述sgRNA是单向导RNA，含有与靶DNA序列的前间区序列互补的20个碱基的种子序列。
  6. 根据权利要求2所述的核酸恒温检测方法，其特征在于，所述PAM是前间区序列邻近基序，位于紧随着与sgRNA互补配对的20个碱基对的靶序列。
  7. 根据权利要求6所述的核酸恒温检测方法，其特征在于，所述靶序列是5′-NGG-3′序列。
  8. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述Cas9n-sgRNA复合物通过sgRNA的种子序列特异性地识别PAM相邻区域的靶DNA序列的一条链，互补结合后，形成Cas9n-sgRNA-靶DNA二元复合结构，引导Cas9n切口酶特异性切割靶PAM毗邻的前间区序列中第三和第四个核苷酸之间的磷酸二酯键。
  9. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述具有链置换活性的恒温DNA聚合酶为exo -Klenow fragment，VentR(exo-)DNA聚合酶或phi29DNA聚合酶。
  10. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述引物1和引物2 分别设计含有5'-CCN-3'序列和毗邻与两条sgRNA种子序列互补的序列。
  11. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述荧光染料为SYBR Green I或EvaGreen。
  12. 根据权利要求1所述的核酸恒温检测方法，其特征在于，所述恒温反应的温度为25～45℃，反应时间为30～200分钟。
  13. 一种试剂盒，其特征在于，所述试剂盒包括50～400nM Cas9n，100～800nM sgRNA，50～500μM dNTPs，200～800nM引物1，200～800nM引物2，0.1～0.6U/μL DNA聚合酶，荧光染料(0.1～1.0×SYBR Green I或0.1～1.0×EvaGreen)，0.1～1.0U RNase抑制剂，50～200mM Tris-HCl，100～600mM NaCl，50～400mM MgCl 2，50～200μg/mL BSA，5～20mM二硫苏糖醇，pH 7.6～8.3，靶核酸和DEPC水。
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