Method and kit for detecting genotypes of SNP (Single Nucleotide Polymorphism) loci in target genes by utilizing cascade amplification

Abstract

The invention provides a method and a kit for detecting genotypes of SNP (single nucleotide polymorphism) sites in target genes by cascade amplification, and the method comprises the following steps: designing a first primer, a second primer and a medium probe aiming at the target genes containing to-be-detected SNP sites, and designing an amplification probe according to a medium sequence of the medium probe, and designing a detection probe according to the single-chain report sequence of the amplification probe, and carrying out PCR amplification and amplification product analysis in a reaction system containing the upstream first primer, the second primer, the medium probe, the amplification probe, the detection probe, a nucleic acid sample containing the target gene and DNA polymerase. The method is a non-target-dependent detection method, a melting peak with a melting point of a certain duplex body is detected in melting curve analysis, the target gene SNP typing corresponding to the duplex body can be judged, and by means of the advantages of PCR amplification and two times of PCR index amplification by combining the medium primer and the amplification probe, the SNP typing of the target gene corresponding to the duplex body can be judged. A large amount of target nucleic acid can be enriched, and the detection sensitivity is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and in particular to a method for detecting the genotype of a SNP site in a target gene by using cascade amplification and a kit thereof. Background Art

[0002] Drug-resistant mutation refers to the phenomenon that microorganisms (such as bacteria, viruses, fungi, etc.) undergo genetic mutations after being exposed to antibiotics or other antimicrobial drugs, resulting in reduced or complete loss of sensitivity to drugs. This mutation may be caused by the direct action of drugs on the genetic material of microorganisms (such as DNA), or it may be caused by changes in the microorganism's own repair mechanism or metabolic pathway. Drug-resistant mutation is a common phenomenon in nature and an important mechanism for microorganisms to resist external pressure. However, drug-resistant mutations have brought huge challenges to clinical treatment. At present, drug-resistant mutations have become a public health problem worldwide and are one of the important factors that seriously threaten human health and life safety. Therefore, detecting the mutation status of microbial gene resistance mutation sites is of great significance for guiding clinical medication, reducing unnecessary drug use, promoting precision medicine, and improving treatment effects.

[0003] On the other hand, DNA variation occurring at a single nucleotide at a specific site in the genome, namely single nucleotide polymorphism (SNP), is one of the most common forms of genetic variation in humans. SNP is closely related to genetic diseases and has an important influence on regulating gene expression, determining human phenotypes and regulating metabolic processes. It explains the heritable inter-individual differences in complex phenotypes and the relationship between genes and diseases, and is related to individual differences in disease susceptibility, disease pathogenesis and drug response. Therefore, the detection of SNP can promote the diagnosis, prevention and treatment of early genetic diseases and play a guiding role in clinical diagnosis.

[0004] At present, the main methods for detecting gene polymorphism include direct sequencing of PCR products, polymerase chain reaction restriction fragment length polymorphism (PCR RFLP), reverse membrane hybridization, solid phase gene chip and real-time fluorescence quantitative PCR, amplification blocking PCR, molecular beacon method and high-resolution melting curve method.

[0005] Among them, the direct sequencing of PCR products has the advantages of intuitive and reliable results, ability to analyze unknown DNA sequences and mutation sites, long read capacity of one-way reaction and high accuracy. However, the conventional PCR method used in the direct sequencing of PCR products has low detection sensitivity, which limits its clinical application.

[0006] In addition, the TaqMan probe method is also a common detection method in clinical applications. It has the characteristics of simplicity, high sensitivity, and high accuracy. However, this method only uses the fluorescent signal generated by fluorescence changes for detection and analysis in one dimension. One SNP site requires the design of two specific probes with different fluorescent labels at both ends to identify different alleles. For multiple detection, due to the limitation of fluorescence channels, it can only be used for the analysis of a small number of SNP sites, making it difficult to distinguish multiple targets, and it is more difficult to design probes for SNP sites that are close in distance.

[0007] Multicolor melting curve analysis technology combines melting curve analysis on the basis of multicolor fluorescence, and performs detection and analysis in two dimensions, namely fluorescence change and Tm value, to achieve multiple detection in one channel. This method uses the Tm value generated by the hybridization of the probe with a large number of single-stranded DNA to perform melting curve analysis. Therefore, asymmetric PCR is used to obtain a large number of single-stranded products complementary to the fluorescent probe by adjusting the concentration ratio of upstream and downstream primers, and asymmetric amplification is combined with the melting temperature of DNA to form a characteristic peak of the melting curve. This method circumvents the fluorescence channel limitation of the PCR instrument during multiple detection of other methods, and has the advantages of high throughput, simple operation, low cost, and reliable accuracy. However, the multicolor melting curve analysis technology still faces many challenges. Since asymmetric amplification belongs to linear amplification, non-exponential amplification is prone to low amplification yield and low sensitivity. At the same time, the ratio of upstream and downstream primers is difficult to optimize, and the design is difficult. The molecular beacon used in asymmetric melting curve undergoes the process of hairpin structure, free single strand, hybrid double strand, and free single strand in the reaction. The fluorescence signal changes from weak to strong and from strong to weak, and it is easy to have problems such as uneven baseline and inverted peak.

[0008] For the detection of genetic polymorphism, the asymmetric melting curve method currently widely used designs mutation sites on the probe and uses the difference in the Tm value of a fluorescent probe to distinguish between the wild type and the mutant type. The Tm value difference for the ATGC>GC mutation is easy to distinguish, but for the A>T mutation, the Tm difference of the probe binding is small, and the genotype is difficult to distinguish. In addition, due to the limitations of probe temperature and two peaks in a single channel, a maximum of 2-3 SNP sites can be designed for one fluorescent channel. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides a method for detecting the genotype of a SNP site in a target gene by using cascade amplification, the method comprising the following steps: 1. A method for detecting the genotype of a SNP site in a target gene using cascade amplification, characterized in that the method comprises the following steps: Step 1: Design the first primer, the second primer and the intermediate probe for the target gene containing the SNP site to be detected. 1.1. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be detected; 1.2. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence in order from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementary bind to the target gene, and along the 5' end to the 3' end, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents extension; Step 2: Design an amplification probe according to the mediator sequence of the mediator probe in step 1, wherein the amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded mediator sequence-specific binding sequence, wherein the single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during PCR amplification; the mediator sequence-specific binding sequence specifically and complementary binds to the mediator sequence in the mediator probe, and the double-stranded chain formed after the mediator sequence binds to the mediator sequence-specific binding sequence is flush with the double-stranded chain of the auxiliary sequence, that is, there is no intervening base between the two double-stranded chains; the end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents extension; Step 3: Designing a detection probe according to the single-stranded reporter sequence of the amplification probe, wherein the detection probe sequentially comprises a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5' end of the auxiliary sequence, and an extension sequence, wherein the first base of the extension sequence along the direction from the 3' end to the 5' end cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end; Step 4: Perform PCR amplification and analysis of amplification products in a reaction system containing the first primer, the second primer, the intermediate probe, the amplification probe, the detection probe, the nucleic acid sample containing the target gene, and a DNA polymerase: 4.1. When the first base of the target gene specific binding sequence is complementary to the wild-type base of the target gene SNP site: 4.1.1. If the SNP site of the target gene is wild-type, then the first base of the target gene-specific binding sequence in the mediator probe complementarily binds to the wild-type target gene to form a SNP site base pair, and the first primer and the second primer are extended and amplified. When extending from the 5' end to the 3' end to the last base of the mediator sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the SNP site base pair and the first adjacent base pair, and the cut sequence fragment contains the mediator sequence and the first base. This fragment is called the first mediator primer, and the mediator primer is connected to the single-stranded end of the amplification probe. The mediator sequence-specific binding binds sequence-specifically and forms an invasion structure of a single base. After the structure is formed, the DNA polymerase cuts off the single-stranded reporter sequence of the amplification probe together with the first invaded base. The cut sequence fragment contains the single-stranded reporter sequence together with the first invaded base, and the sequence fragment is called the first reporter primer; the first reporter primer is completely complementary to the detection probe and extends along the extension sequence portion of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type; 4.1.2. If the SNP site of the target gene is a mutant type, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the mutant target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. The first primer and the second primer are both extended and amplified. When extending from the 5' end to the 3' end to the first base of the target gene specific binding sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the first base and the second base in the target gene specific binding sequence that are paired with the target gene, and the cut sequence fragment includes the mediator sequence, the first base of the target gene specific binding sequence, and The first base that pairs with the target gene is called the second mediator primer; the second mediator primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the structure is formed, the DNA polymerase cuts off the reporter sequence of the amplification probe together with the first and second invaded bases, and the sequence fragment is called the second reporter primer; when the second reporter primer complementarily binds to the detection probe, the last base at the 3' end of the second reporter primer cannot complementarily bind to the detection probe, and the second reporter primer cannot extend along the detection probe, thereby obtaining a second double-stranded product. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is a mutant type; 4.1.3. If the SNP site of the target gene is heterozygous, the first double-stranded product and the second double-stranded product will be generated at the same time. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed at the same time, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous; 4.2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the target gene SNP site: 4.2.1. If the SNP site of the target gene is a wild type or a synonymous mutation, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. As in the above 4.1.2, only the formation of the second double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a wild type or a synonymous mutation; 4.2.2. If the SNP site of the target gene is a missense mutation, at this time, the first base of the target gene specific binding sequence in the intermediate probe complementarily binds to the missense mutation target gene to form a SNP site base pair. As in the above 4.1.1, only the formation of the first double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a missense mutation; 4.2.3. As in 4.1.3 above, when it is detected that the first double-stranded product and the second double-stranded product are formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

[0010] In one embodiment, when the target gene contains multiple SNP sites, corresponding mediator probes, amplification probes and detection probes are designed for each SNP site, so that each SNP site forms a corresponding first double-stranded product and a second double-stranded product.

[0011] In one embodiment, whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

[0012] In one embodiment, the single-stranded reporter sequence of the amplification probe is labeled with a fluorescent group; the double-stranded auxiliary sequence of the amplification probe is labeled with a quencher group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quencher group corresponding to the fluorescent group labeled in the single-stranded reporter sequence of the amplification probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the melting curve method.

[0013] In one embodiment, the detection probe is labeled with a fluorescent group and a corresponding quenching group, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

[0014] In one embodiment, the intermediary probe sequence, the amplification probe and / or the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0015] In one embodiment, the DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

[0016] In one embodiment, a kit for detecting the genotype of a SNP site in a target gene is provided, the kit comprising: a DNA polymerase, a first primer designed for a target gene containing a SNP site to be detected, a second primer and an intermediate probe, an amplification probe and a detection probe, a. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be tested; b. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence in order from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementary bind to the target gene, and along the 5' end to the 3' end, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents extension; c. Design an amplification probe according to the media sequence of the media probe, wherein the amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded media sequence-specific binding sequence, wherein the single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during PCR amplification; the media sequence-specific binding sequence specifically and complementary binds to the media sequence in the media probe, and the double-stranded chain formed after the media sequence binds to the media sequence-specific binding sequence is flush with the double-stranded chain of the auxiliary sequence, that is, there is no intervening base between the two double-stranded chains; the end of the single-stranded media sequence-specific binding sequence is labeled with a group that prevents extension; d. Designing a detection probe according to the single-stranded reporter sequence of the amplification probe, the detection probe sequentially comprises a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5' end of the auxiliary sequence, and an extension sequence, wherein the first base of the extension sequence along the direction from the 3' end to the 5' end cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end.

[0017] Compared with the prior art, the present invention has the following advantages: 1. The method of the present invention includes two amplification cycles. The first amplification cycle is PCR amplification. As the PCR amplification reaction proceeds, the mediator primer is continuously generated; the second amplification is the combination of the mediator primer and the amplification probe, cutting and releasing the reporter primer, and then the reporter primer continuously combines with the detection probe to form a double-stranded product. In the second amplification cycle, the mediator primer will not be consumed, but will act as a catalyst, and as the amplification reaction proceeds, it will continuously combine with the amplification probe, continuously cut and release the reporter primer. However, the two amplification cycles are interconnected and independent of each other. Since the mediator primer will not be consumed, even if the PCR amplification cycle stops, it will not affect the continuous occurrence of the second amplification cycle, thereby realizing cascade amplification of the target gene detection and further improving the detection sensitivity; 2. The method of the present invention is a non-target-dependent detection method, that is, the sequence of each reporter primer and detection probe is artificially designed, known or predetermined, so the first double-stranded product and the second-stranded product can be determined. In the melting curve detection, the melting point (Tm value) of each duplex can be calculated in advance. Therefore, by detecting the melting peak with the melting point (Tm value) of a certain duplex in the melting curve analysis, the target gene SNP typing corresponding to the duplex can be determined, which solves the problem of melting curve peak offset and easy misjudgment caused by the easy mutation of the target gene sequence to be tested, and well solves the problem that the melting curve analysis method is not suitable for RNA samples; 3. The asymmetric melting curve method, which is widely used at present, designs the mutation site on the probe, and uses the difference in the Tm value of a fluorescent probe to distinguish the wild type and the mutant type. The difference in the Tm value of the ATGC>GC mutation is easy to distinguish, but the difference in the Tm value of the probe binding to the A>T mutation is small, and the genotype is difficult to distinguish. At present, the asymmetric melting curve is widely used in SNP genotyping. This method designs the mutation site on the probe. Since the probe has no selectivity, only one probe is designed for one SNP site to distinguish the two genotypes, and the genotype is distinguished by the single-base Tm value of the probe binding to the single strand in the same fluorescent channel. The difference in the Tm value of the ATGC>GC mutation is easy to distinguish, but the difference in the Tm value of the probe binding to the A>T mutation is small, and the genotype is difficult to distinguish. In addition, due to the limitations of the probe temperature and the two peaks of a single channel, a maximum of 2-3 SNP sites can be designed for one fluorescent channel. The method of the present invention designs the first base of the sequence specifically binding the media probe to the target gene as a SNP site, and uses whether the SNP site of the target gene to be tested is complementary to the media probe to generate two different media primers by cutting. Due to the difference in the 3' terminal sequence, the different media primers will form invasion structures with different numbers of bases after binding to the amplification probe. The different invasion structures will generate two different reporter primers. Due to the difference in the 3' terminal sequence of the two reporter primers, after binding to the detection probe, only one of the reporter primers can extend along the detection probe, while the other reporter primer can only bind to the detection probe but cannot extend. Therefore, the melting point (Tm value) of the double helix can be arbitrarily constructed, and the melting curve peak of the SNP site can be customized, thereby solving the problem of indistinguishable Tm values ​​of the wild type and mutant type of the same SNP site, reducing the design difficulty, and enabling the detection of the target site to achieve specific distinction of A>T base mutations. For different SNP sites, the Tm value is distinguished by controlling the amplicon length and GC content between the reporter primer and the detection probe, and different SNP sites are distinguished by different melting curve Tm values, thereby increasing the number of detectable SNP sites.

[0018] 4. In the method of the present invention, the design of the double-stranded auxiliary sequence of the amplification probe cleverly forms a large steric hindrance, preventing excessive amplification probe from combining with the detection probe to produce false positive background peaks, thereby avoiding the problem of false positive background peaks interfering with the accuracy of interpretation; 5. In the method of the present invention, when the melting curve method is used to detect the first double-stranded product and the second-stranded product, a larger proportion of the fluorescence signal fluctuations of the reporter primer and the detection probe are reflected in the melting curve analysis process, a flatter baseline is obtained, the appearance of non-specific peaks is reduced, and the correctness and accuracy of the detection method are improved. At the same time, this method is not limited to the number of fluorescent channels detected, and different target genes are distinguished according to the melting point and fluorescent color, the number of targets detected in a single well is increased, and the detection throughput is improved. The method of the present invention can be used for multiple target detection, the baseline of the melting peak can be kept flat, the peak value is higher, there is no non-specific peak, and it has a higher sensitivity; at the same time, the detection range is wider, and the melting curve detection T m The value range is: 40~80℃; 6. When using the melting curve method for detection, the method of the present invention for detecting target nucleic acid is a melting curve method of symmetrical amplification. Compared with the common melting curve method of asymmetrical amplification (a linear amplification method), this method utilizes the advantages of PCR exponential amplification, can enrich a large amount of target nucleic acid, and improve the sensitivity of detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 is a schematic diagram of the amplification probe used in the method of the present invention; Figure 2 It is a schematic diagram of the basic principle of the method of the present invention; Figure 3 Melting curve analysis of MTHFR (677C>T) genotype is CC wild type; Figure 4 Melting curve analysis of the MTHFR (677C>T) genotype being the TT homozygous mutant type; Figure 5 Melting curve analysis of MTHFR (677C>T) genotype is CT heterozygous mutation type; Figure 6 Melting curve analysis of MTHFR (1298A>C) genotype is AA wild type; Figure 7 Melting curve analysis of the MTHFR (1298A>C) genotype as CC homozygous mutant type; Figure 8 Melting curve analysis of MTHFR (1298A>C) genotype AC heterozygous mutant type; Fig. 9 Melting curve analysis diagram of both SNP sites being mutant; Fig.10 Melting curve analysis of both SNP sites being wild type; Fig.11 Melting curve analysis of MTHFR (677C>T) as wild type and MTHFR (1298A>C) as mutant type; Fig.12 MTHFR (677C>T) is the melting curve analysis diagram of wild type MTHFR (1298A>C) is the heterozygous type; Fig.13 Melting curve analysis of MTHFR (677C>T) as heterozygous and MTHFR (1298A>C) as mutant; Fig.14 Melting curve analysis diagram of MTHFR (677C>T) as heterozygous and MTHFR (1298A>C) as heterozygous. DETAILED DESCRIPTION

[0021] In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without making creative work should belong to the scope of protection of the present application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0022] Embodiment 1 Basic principle of the present invention like Figure 1 and Figure 2 As shown, the present invention provides a method for detecting the genotype of a SNP site in a target gene by using cascade amplification, and the method comprises the following steps: Step 1: Design the first primer, the second primer and the intermediate probe for the target gene containing the SNP site to be detected. 1.1. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be detected; 1.2. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence in order from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementary bind to the target gene, and along the 5' end to the 3' end, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents extension; Step 2: Design an amplification probe based on the mediator sequence of the mediator probe described in step 1, such as Figure 1 As shown in a, the amplification probe of the present invention can be designed into various forms, and the amplification probe can be composed of one chain, or two chains or more chains; the amplification probe sequentially includes a single-chain reporter sequence, a double-chain auxiliary sequence and a single-chain mediator sequence-specific binding sequence, the single-chain reporter sequence does not bind to the target gene, and the single-chain reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-chain structure of the auxiliary sequence remains stable during PCR amplification; the mediator sequence-specific binding sequence specifically and complementary binds to the mediator sequence in the mediator probe, and the double-chain formed after the mediator sequence binds to the mediator sequence-specific binding sequence is exactly flush with the double-chain of the auxiliary sequence, that is, there is no spacing base between the above two double-chains (such as Figure 1 b); the end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents extension; Step 3: Designing a detection probe according to the single-stranded reporter sequence of the amplification probe, wherein the detection probe sequentially comprises a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5' end of the auxiliary sequence, and an extension sequence, wherein the first base of the extension sequence along the direction from the 3' end to the 5' end cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end; Step 4: Perform PCR amplification and analysis of amplification products in a reaction system containing the first primer, the second primer, the intermediate probe, the amplification probe, the detection probe, the nucleic acid sample containing the target gene, and a DNA polymerase: 4.1. When the first base of the target gene specific binding sequence is complementary to the wild-type base of the target gene SNP site: 4.1.1. If the SNP site of the target gene is wild-type, at this time, the first base of the target gene-specific binding sequence in the mediator probe is complementary to the wild-type target gene to form a SNP site base pair, and the first primer and the second primer are both extended and amplified. When extending from the 5' end to the 3' end to the last base of the mediator sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the SNP site base pair and the first adjacent base pair, and the cut sequence fragment contains the mediator sequence and the first base. This fragment is called the first mediator primer. The mediator primer specifically binds to the single-stranded mediator sequence-specific binding sequence of the amplification probe and forms an invasion structure of a single base (such as Figure 1 c) After the structure is formed, the DNA polymerase cuts off the single-stranded reporter sequence of the amplification probe together with the first invaded base, and the cut sequence fragment contains the single-stranded reporter sequence together with the first invaded base, and the sequence fragment is called the first reporter primer; the first reporter primer is completely complementary to the detection probe, and extends along the extended sequence portion of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type; 4.1.2. If the SNP site of the target gene is a mutant type, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the mutant target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. The first primer and the second primer are both extended and amplified. When extending from the 5' end to the 3' end to the first base of the target gene specific binding sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the first base and the second base in the target gene specific binding sequence that are paired with the target gene, and the cut sequence fragment includes the mediator sequence, the first base of the target gene specific binding sequence, and the second base of the target gene specific binding sequence. and the first base that pairs with the target gene, and this fragment is called the second mediator primer; the second mediator primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the structure is formed, the DNA polymerase cuts off the reporter sequence of the amplification probe together with the invaded first and second bases, and this sequence fragment is called the second reporter primer; when the second reporter primer complementarily binds to the detection probe, the last base at the 3' end of the second reporter primer cannot complementarily bind to the detection probe, and the second reporter primer cannot extend along the detection probe, thereby obtaining a second double-stranded product. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is a mutant type; 4.1.3. If the SNP site of the target gene is heterozygous, the first double-stranded product and the second double-stranded product will be generated at the same time. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed at the same time, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous; 4.2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the target gene SNP site: 4.2.1. If the SNP site of the target gene is a wild type or a synonymous mutation, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. As in the above 4.1.2, only the formation of the second double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a wild type or a synonymous mutation; 4.2.2. If the SNP site of the target gene is a missense mutation, at this time, the first base of the target gene specific binding sequence in the intermediate probe complementarily binds to the missense mutation target gene to form a SNP site base pair. As in the above 4.1.1, only the formation of the first double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a missense mutation; 4.2.3. As in 4.1.3 above, when it is detected that the first double-stranded product and the second double-stranded product are formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

[0023] In the method of the present invention, the single-stranded reporter sequence of the amplification probe is labeled with a fluorescent group; the double-stranded auxiliary sequence of the amplification probe is labeled with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence of the amplification probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the melting curve method.

[0024] In the method of the present invention, the detection probe is labeled with a fluorescent group and a corresponding quenching group, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

[0025] In the method of the present invention, the amplification reaction system may contain not only DNA polymerase but also endonuclease and / or exonuclease.

[0026] In the method of the present invention, the intruder base may be complementary to the sense strand or antisense strand of part of the double-stranded auxiliary sequence of the amplification probe, or may not be complementary to any strand of the double-stranded auxiliary sequence of the amplification probe.

[0027] In the present invention, since the base sequence and length of the detection probe can be pre-designed, the first double-stranded product obtained by extending the first reporter primer and the detection probe has a predictable length and structure, and accordingly, the first double-stranded product has a predictable melting point (T m1 Similarly, the second double-stranded product obtained by the combination of the second reporter primer and the detection probe also has a predictable length and structure. Accordingly, the second double-stranded product has a predictable melting point (T m2 value), and because the first double-stranded product has a longer base sequence and structure than the second double-stranded product, T m1 >T m2 The double-stranded product obtained in step 4 is subjected to melting curve analysis and detection. By detecting the peak position of the melting curve of the double-stranded product, the corresponding genotype can be determined.

[0028] In the present invention, the fluorescent group includes various commonly used fluorescent markers, but not limited to these fluorescent markers, such as Pacific Blue, Oregon Green, Bodipy FL-X, FAM, VIC, TET, Bodipy R6G-X, JOE, HEX, Cy3, Cy3B, Rhodamine Red, TAMRA, Texas Red-X, ROX, Cy3.5, Cy5, etc.; the quenching group includes various commonly used quenchers, but not limited to these quenchers, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0029] Example 2 Genotyping of MTHFR C677T locus This example takes the detection of the MTHFR C677T site as an example, and uses the method of the present invention to detect the MTHFR gene. The specific method includes the following steps.

[0030] 1. Primer and probe sequence information The first primer, the second primer, the intermediate probe, the amplification probe and the detection probe are designed according to the target nucleic acid site to be detected, wherein the intermediate probe is designed according to the first base sequence of the target gene specific binding sequence being complementary to the missense mutant base of the SNP site of the target gene, and the sequence information is shown in Table 1 below.

[0031] Table 1

[0032] Note: The bold part of the media probe is the media sequence, the italic part is the SNP site, and the lowercase letters are the target gene-specific sequence; the bold part of the amplifier probe is the reporter sequence, the uppercase letters and underlined part are the double-stranded auxiliary sequence, and the italic part is the media sequence-specific binding sequence.

[0033] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, and the PCR reaction system includes: 1× PCR buffer, 2.5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM intermediate probe, 100 nM amplification probe, 100 nM detection probe 1, 5 μL nucleic acid template.

[0034] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, 40-80°C for melting curve analysis, and detecting fluorescence signals every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0035] 3. Melting Curve Analysis The results of the melting curve analysis are shown in Table 2 below.

[0036] Table 2

[0037] Targeting the MTHFR C677T mutation site, the VIC channel is m The peak appears at 54.5±1℃, which is considered to be wild type. Figure 3 ;VIC channel at T m The peak value is 67.1±1℃, which is judged as homozygous mutant. Figure 4 ;VIC channel at T m The peaks at 54.6±1℃ and 67.3±1℃ appear at the same time, which is considered to be heterozygous. Figure 5 .

[0038] Example 3 Genotyping of MTHFR A1298C locus This example takes the detection of the MTHFR A1298C mutation site as an example, and uses the method of the present invention to perform mutation detection on the MTHFR gene. The specific method includes the following steps.

[0039] 1. Primer and probe sequence information The first primer, the second primer, the intermediate probe, the amplification probe and the detection probe are designed according to the target nucleic acid site to be detected, wherein the intermediate probe is designed according to the first base sequence of the target gene specific binding sequence being complementary to the missense mutant base of the SNP site of the target gene, and the sequence information is shown in Table 3 below.

[0040] Table 3

[0041] Note: The bold part of the media probe is the media sequence, the italic part is the SNP site, and the lowercase part is the target gene specific sequence; the bold part of the amplifier probe is the reporter sequence, the uppercase and underlined part is the double-stranded auxiliary sequence, and the italic part is the media sequence-specific binding sequence.

[0042] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, and the PCR reaction system includes: 1× PCR buffer, 2.5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.1 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM intermediate probe, 100 nM amplification probe, 100 nM detection probe 2, 5 μL nucleic acid template.

[0043] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, 40-80°C for melting curve analysis, and detecting fluorescence signals every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0044] 3. Melting Curve Analysis The results of the melting curve analysis are shown in Table 4 below.

[0045] Table 4

[0046] Targeting the MTHFR A1298C mutation site, ROX pathway is expressed in T m The peak appears at 48.0±1℃, which is considered to be wild type. Figure 6 ;ROX channel in T m The peak value is 65.3±1℃, which is judged as homozygous mutant. Figure 7 ;ROX channel in T m The peaks at 48.1±1℃ and 65.2±1℃ appear at the same time, which is considered to be heterozygous. Figure 8 .

[0047] Example 3 MTHFR C677T and A1298C dual-locus genotyping This example takes the detection of the double mutation sites of MTHFR C677T and MTHFR A1298C as an example, and uses the method of the present invention to perform mutation detection on the MTHFR gene. The specific method includes the following steps: 1. Primer and probe sequence information The first primer, the second primer, the intermediate probe, the amplification probe and the detection probe are designed respectively according to the target nucleic acid site to be detected, wherein the intermediate probe is designed according to the first base sequence of the target gene specific binding sequence being complementary to the missense mutant base of the SNP site of the target gene, and the sequence information is shown in Table 5 below.

[0048] Table 5

[0049] Note: The bold part of the media probe is the media sequence, the italic part is the SNP site, and the lowercase part is the target gene specific sequence; the bold part of the amplifier probe is the reporter sequence, the uppercase and underlined part is the double-stranded auxiliary sequence, and the italic part is the media sequence-specific binding sequence.

[0050] 2. PCR Amplification Reaction System and Procedure The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis, and the PCR reaction system includes: 1× PCR buffer, 5 U BioZues ® HS Multiplex Taq DNA Polymerase, 0.2 mM dNTPs, 40 nM first primer, 40 nM second primer, 20 nM intermediate probe, 100 nM amplification probe, 100 nM detection probe 1 and 100 nM detection probe 2, 5 μL nucleic acid template.

[0051] The fluorescent PCR reaction program of the present invention is 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 60°C annealing and extension for 45 s, collecting fluorescence at the same time, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, 40-80°C for melting curve analysis, and detecting fluorescence signals every 0.04°C. The instrument used in this embodiment is SLAN 96 real-time fluorescent PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0052] For the dual-site one-tube test of MTHFR C677T and MTHFR A1298C, the results of the random template melting curve are shown in the attached Figures 9 to 14 ; a. If the VIC channel peaks only at 67.3±1℃, and the ROX channel peaks only at 65.2±1℃, then both sites are homozygous mutants ( Fig. 9 ); b. If the VIC channel peaks only at 54.6±1℃, and the ROX channel peaks only at 48.1±1℃, then both sites are wild type ( Fig.10 ); c. If the VIC channel peaks only at 54.6±1℃, and the ROX channel peaks only at 65.2±1℃, then C677T is wild type, and the A1298C site is a homozygous mutant ( Fig.11 ); d. If the VIC channel peaks only at 54.6±1℃, and the ROX channel peaks at both 48.1±1℃ and 65.2±1℃, then C677T is wild type, and the A1298C site is a heterozygous mutant ( Fig.12 ); e. If the VIC channel peaks at both 54.6±1℃ and 67.3±1℃, and the ROX channel peaks only at 65.2±1℃, then C677T is a heterozygous mutation and A1298C is a homozygous mutation ( Fig.13 ); f. If the VIC channel peaks at 54.6±1℃ and 67.3±1℃ simultaneously, and the ROX channel peaks at 48.1±1℃ and 65.2±1℃ simultaneously, then the C677T and A1298C sites are both heterozygous mutations ( Fig.14 ).

[0053] 3. Melting Curve Analysis The results of the melting curve analysis are shown in Table 6 below.

[0054] Table 6

[0055] It should be understood that the disclosed invention is not limited only to the specific method, scheme and material of description, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to the appended claims.

[0056] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for detecting the genotype of a SNP site in a target gene using cascade amplification, characterized in that the method comprises the following steps: Step 1: Design the first primer, the second primer and the intermediate probe for the target gene containing the SNP site to be detected. 1.

1. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be detected; 1.

2. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence in order from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementary bind to the target gene, and along the 5' end to the 3' end, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents extension; Step 2: Design an amplification probe according to the mediator sequence of the mediator probe in step 1, wherein the amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded mediator sequence-specific binding sequence, wherein the single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during PCR amplification; the mediator sequence-specific binding sequence specifically and complementary binds to the mediator sequence in the mediator probe, and the double-stranded chain formed after the mediator sequence binds to the mediator sequence-specific binding sequence is flush with the double-stranded chain of the auxiliary sequence, that is, there is no intervening base between the two double-stranded chains; the end of the single-stranded mediator sequence-specific binding sequence is labeled with a group that prevents extension; Step 3: Designing a detection probe according to the single-stranded reporter sequence of the amplification probe, wherein the detection probe sequentially comprises a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5' end of the auxiliary sequence, and an extension sequence, wherein the first base of the extension sequence along the direction from the 3' end to the 5' end cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end; Step 4: Perform PCR amplification and analysis of amplification products in a reaction system containing the first primer, the second primer, the intermediate probe, the amplification probe, the detection probe, the nucleic acid sample containing the target gene, and a DNA polymerase: 4.

1. When the first base of the target gene specific binding sequence is complementary to the wild-type base of the target gene SNP site: 4.1.

1. If the SNP site of the target gene is wild-type, then the first base of the target gene-specific binding sequence in the mediator probe complementarily binds to the wild-type target gene to form a SNP site base pair, and the first primer and the second primer are extended and amplified. When extending from the 5' end to the 3' end to the last base of the mediator sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the SNP site base pair and the first adjacent base pair, and the cut sequence fragment contains the mediator sequence and the first base. This fragment is called the first mediator primer, and the mediator primer is connected to the single-stranded end of the amplification probe. The mediator sequence-specific binding binds sequence-specifically and forms an invasion structure of a single base. After the structure is formed, the DNA polymerase cuts off the single-stranded reporter sequence of the amplification probe together with the first invaded base. The cut sequence fragment contains the single-stranded reporter sequence together with the first invaded base, and the sequence fragment is called the first reporter primer; the first reporter primer is completely complementary to the detection probe and extends along the extension sequence portion of the detection probe to form a first double-stranded product. When only the formation of the first double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type; 4.1.

2. If the SNP site of the target gene is a mutant type, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the mutant target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. The first primer and the second primer are both extended and amplified. When extending from the 5' end to the 3' end to the first base of the target gene specific binding sequence in the mediator probe, the DNA polymerase will cut the phosphodiester bond between the first base and the second base in the target gene specific binding sequence that are paired with the target gene. The cut sequence fragment contains the mediator sequence, the first base of the target gene specific binding sequence and the first base paired with the target gene. This fragment is called the second mediator primer; The second mediator primer specifically binds to the amplification probe and forms an invasion structure of two bases. After the structure is formed, the DNA polymerase cuts off the reporter sequence of the amplification probe together with the first and second invaded bases, and the sequence fragment is called the second reporter primer; when the second reporter primer complementarily binds to the detection probe, the last base at the 3' end of the second reporter primer cannot complementarily bind to the detection probe, and the second reporter primer cannot extend along the detection probe, thereby obtaining a second double-stranded product. When only the formation of the second double-stranded product is detected, it can be determined that the SNP site in the nucleic acid sample containing the target gene is a mutant type; 4.1.

3. If the SNP site of the target gene is heterozygous, the first double-stranded product and the second double-stranded product will be generated at the same time. Therefore, when the first double-stranded product and the second double-stranded product are detected to be formed at the same time, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous; 4.

2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the target gene SNP site: 4.2.

1. If the SNP site of the target gene is a wild type or a synonymous mutation, at this time, the first base of the target gene specific binding sequence in the mediator probe does not complementarily bind to the target gene. At this time, the first base of the target gene specific binding sequence and the mediator sequence do not complementarily bind to the target gene. As in the above 4.1.2, only the formation of the second double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a wild type or a synonymous mutation; 4.2.

2. If the SNP site of the target gene is a missense mutation, at this time, the first base of the target gene specific binding sequence in the intermediate probe complementarily binds to the missense mutation target gene to form a SNP site base pair. As in the above 4.1.1, only the formation of the first double-stranded product is detected, and the SNP site in the nucleic acid sample containing the target gene can be determined to be a missense mutation; 4.2.

3. As in 4.1.3 above, when it is detected that the first double-stranded product and the second double-stranded product are formed simultaneously, it can be determined that the SNP site in the nucleic acid sample containing the target gene is heterozygous.

2. The method according to claim 1, characterized in that When the target gene contains multiple SNP sites, corresponding intermediate probes, amplification probes and detection probes are designed for each SNP site, so that each SNP site forms a corresponding first double-stranded product and a second double-stranded product.

3. The method according to claim 2, characterized in that Whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

4. The method according to claim 3, characterized in that The single-stranded reporter sequence of the amplification probe is labeled with a fluorescent group; the double-stranded auxiliary sequence of the amplification probe is labeled with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence of the amplification probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

5. The method according to claim 3, characterized in that: The detection probe is labeled with a fluorescent group and a corresponding quenching group, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

6. The method according to claim 1, characterized in that The intermediary probe sequence, the amplification probe and / or the detection probe sequence comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

7. The method according to claim 1, characterized in that The DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

8. A kit for detecting the genotype of a SNP site in a target gene using cascade amplification, characterized in that: The kit comprises: DNA polymerase, a first primer designed for a target gene containing a SNP site to be detected, a second primer and a mediator probe, an amplification probe and a detection probe, a. The first primer and the second primer specifically bind to the target gene and are used to amplify the target gene containing the SNP site to be tested; b. The mediator probe comprises a mediator sequence and a target gene-specific binding sequence in order from the 5' end to the 3' end, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementary bind to the target gene, and along the 5' end to the 3' end, the first base sequence of the target gene-specific binding sequence corresponds to the SNP site of the target gene, and the first base sequence is complementary to the wild-type base or mutant base of the SNP site of the target gene; the 3' end of the mediator probe is labeled with a group that prevents extension; c. Design an amplification probe according to the media sequence of the media probe, wherein the amplification probe sequentially includes a single-stranded reporter sequence, a double-stranded auxiliary sequence and a single-stranded media sequence-specific binding sequence, wherein the single-stranded reporter sequence does not bind to the target gene, and the single-stranded reporter sequence does not complementarily pair with other sequences in the amplification probe; the double-stranded structure of the auxiliary sequence remains stable during PCR amplification; the media sequence-specific binding sequence specifically and complementary binds to the media sequence in the media probe, and the double-stranded chain formed after the media sequence binds to the media sequence-specific binding sequence is flush with the double-stranded chain of the auxiliary sequence, that is, there is no intervening base between the two double-stranded chains; the end of the single-stranded media sequence-specific binding sequence is labeled with a group that prevents extension; d. Designing a detection probe according to the single-stranded reporter sequence of the amplification probe, the detection probe sequentially comprises a complementary sequence to the single-stranded reporter sequence of the amplification probe, a base complementary to the first base at the 5' end of the auxiliary sequence, and an extension sequence, wherein the first base of the extension sequence along the direction from the 3' end to the 5' end cannot be complementary to the second base of the auxiliary sequence along the direction from the 5' end to the 3' end; the detection probe is modified with a quenching group.

9. The kit according to claim 8, characterized in that The intermediary probe sequence, the amplification probe and / or the detection probe sequence comprise or consist of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

10. The kit according to claim 8, characterized in that The DNA polymerase includes at least one of Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Tfi DNA polymerase, pfu DNA polymerase, KOD DNA polymerase or Tgo DNA polymerase.

11. The kit according to claim 8, characterized in that The single-stranded reporter sequence of the amplification probe is labeled with a fluorescent group; the double-stranded auxiliary sequence of the amplification probe is labeled with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the single-stranded reporter sequence of the amplification probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

12. The kit according to claim 8, characterized in that The detection probe is labeled with a fluorescent group and a corresponding quenching group, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by a melting curve method.

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