Method for detecting genotype of SNP site in target gene independent of target and kit thereof

Abstract

The present invention provides a method and a kit for detecting the genotype of SNP sites in a target gene independent of a target. The method includes designing a first primer, a second primer, and a mediator probe for a target gene containing a SNP site to be detected, designing a detection probe according to the mediator sequence of the mediator probe, and performing PCR amplification and analysis of the amplification product in a reaction system containing the first primer, the second primer, the mediator probe, the detection probe, a nucleic acid sample containing the target gene, and a DNA polymerase. The method of the present invention is a detection method independent of a target. By detecting a melting peak having the melting point of a certain double-stranded body in melting curve analysis, the SNP typing of the target gene corresponding to the double-stranded body can be determined. The present invention can arbitrarily construct the Tm value of the double-stranded body, realizing the customization of the melting curve peak of the SNP site, thereby solving the problem that the Tm values of the wild type and mutant genotypes of the same SNP site cannot be distinguished, and reducing the design difficulty.

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 SNP sites in a target gene independent of a non-target and a kit thereof. Background Art

[0002] Drug resistance mutation refers to the phenomenon that microorganisms (such as bacteria, viruses, fungi, etc.) undergo genetic variation after contacting antibiotics or other antimicrobial drugs, resulting in a decrease or complete loss of their sensitivity to the drugs. This variation may be caused by the direct action of the drug on the genetic material (such as DNA) of the microorganism, or may be due to the change of the microorganism's own repair mechanism or metabolic pathway. Drug resistance mutation is a common phenomenon in nature and an important mechanism for microorganisms to resist external pressure. However, drug resistance mutation poses a huge challenge to clinical treatment. At present, drug resistance mutation has become a global public health problem and one of the important factors seriously threatening human health and life safety. Therefore, detecting the mutation situation of drug resistance mutation sites in microbial genes is of great significance for guiding clinical medication, reducing unnecessary drug use, promoting the realization of precision medicine, and improving the treatment effect.

[0003] On the other hand, DNA variation occurring at a specific single nucleotide in the genome, namely single nucleotide polymorphism (SNP), is one of the most common genetic variation forms in humans. SNPs are closely related to genetic diseases and have an important impact 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 disease susceptibility, disease pathogenesis, and individual differences in drug response. Therefore, detecting SNPs can promote the diagnosis, prevention, and treatment of early genetic diseases and has a guiding role in clinical diagnosis.

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

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

[0006] In addition, the TaqMan probe method is also a common detection method in clinical applications, which has the characteristics of simplicity, high sensitivity, high accuracy, etc. However, this method only detects and analyzes in one dimension, that is, using the fluorescence signal generated by fluorescence change. For one SNP site, two specific probes with different fluorescent labels at both ends need to be designed to identify different alleles. For multiplex detection, due to the limitation of the fluorescence channel, it can only be used for the analysis of a small number of SNP sites, it is difficult to distinguish multiple targets, and it is also difficult to design probes for SNP sites that are relatively close.

[0007] The multi-color melting curve analysis technology combines melting curve analysis on the basis of multi-color fluorescence, and detects and analyzes in two dimensions, namely fluorescence change and Tm value, to achieve multiplex detection in one channel. This method uses the Tm value generated by the hybridization of probes with a large number of single-stranded DNAs for melting curve analysis. Therefore, asymmetric PCR is used. By adjusting the concentration ratio of upstream and downstream primers, a large number of single-stranded products complementary to the fluorescent probes are obtained, and the asymmetric amplification is combined with the DNA melting temperature to form the characteristic peaks of the melting curve. This method avoids the fluorescence channel limitation of the PCR instrument during multiplex detection of other methods, and has the advantages of large throughput, simple operation, low cost, reliable precision, etc. However, the current multi-color melting curve analysis technology still faces many challenges. Since asymmetric amplification belongs to linear amplification rather than exponential amplification, it is prone to problems such as low amplification yield and low sensitivity. At the same time, it is difficult to optimize the ratio of upstream and downstream primers, and the design difficulty is relatively large. The molecular beacon used in the asymmetric melting curve undergoes the processes of hairpin structure, free single-stranded, hybrid double-stranded, and free single-stranded in the reaction. The fluorescence signal consists of two parts: from weak to strong and from strong to weak, and it is easy to appear problems such as uneven baseline and inverted peaks.

[0008] For the detection of gene polymorphisms, the currently widely used asymmetric melting curve method designs the mutation site on the probe, and uses the Tm value difference of a fluorescent probe to distinguish the wild type and the mutant type. It is easy to distinguish the Tm value difference of the mutation of ATGC>GC, but for the mutation of A>T, the Tm difference of probe binding is relatively small, and the genotype is difficult to distinguish. Moreover, due to the limitations of probe temperature and two peaks in a single channel, at most 2-3 SNP sites can be designed in one fluorescence 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 SNP sites in a target gene independent of non-targets, and the method includes the following steps:

[0010] A method for detecting the genotype of SNP sites in a target gene independent of non-targets, characterized in that the method includes the following steps:

[0011] Step 1: Design a first primer, a second primer and a mediator probe for the target gene containing the SNP site to be detected,

[0012] 1.1. The first primer and the second primer specifically bind to the target gene and are used for amplifying the target gene containing the SNP site to be detected;

[0013] 1.2. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, 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; a group that blocks extension is labeled at the 3'-end of the mediator probe;

[0014] Step 2: Design a detection probe according to the mediator sequence of the mediator probe. The detection probe sequentially includes a complementary sequence to the mediator sequence of the mediator probe, a base complementary to the first base of the target gene-specific binding sequence of the mediator probe along the 5'-end to the 3'-end direction, and an extension sequence. For the extension sequence, along the 3'-end to the 5'-end direction, the first base cannot be complementary to the second base of the target gene-specific binding sequence of the mediator probe along the 5'-end to the 3'-end direction;

[0015] Step 3: In a reaction system containing the first primer, the second primer, the mediator probe, the detection probe, a nucleic acid sample containing the target gene, and a DNA polymerase, perform PCR amplification and analysis of the amplification product:

[0016] 3.1. When the first base of the target gene-specific binding sequence is complementary to the wild-type base of the SNP site of the target gene:

[0017] 3.1.1. 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 complementarily binds to the wild-type target gene to form an SNP site base pair. Both the first primer and the second primer are extended for amplification. When extending along the 5'-end to the 3'-end to the last base of the mediator sequence in the mediator probe, the DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and the first adjacent base pair. The cleaved sequence fragment includes the mediator sequence and the first base, and this fragment is called the first mediator primer. The first mediator primer completely complementarily pairs with the detection probe and extends along the extension sequence part 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;

[0018] 3.1.2. When the SNP site of the target gene is mutant, at this time, the first base of the target gene specific binding sequence in the medium probe does not complementarily bind to the mutant target gene. At this time, neither the first base of the target gene specific binding sequence nor the medium sequence complementarily binds to the target gene. Both the first primer and the second primer perform extension amplification. When extending from the 5' end to the 3' end to the first base of the target gene specific binding sequence in the medium probe, DNA polymerase will cleave the phosphodiester bond between the first base and the second base that pair with the target gene in the target gene specific binding sequence. The cleaved sequence fragment contains the medium sequence, the first base of the target gene specific binding sequence, and the first base that pairs with the target gene. This fragment is called the second medium primer. The second medium primer cannot extend along the detection probe, so a second double-stranded product is obtained. 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 mutant;

[0019] 3.1.3. When the SNP site of the target gene is heterozygous, at this time, both the first double-stranded product and the second double-stranded product will be generated. Therefore, when the formation of both the first double-stranded product and 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 heterozygous;

[0020] 3.2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the SNP site of the target gene:

[0021] 3.2.1. When the SNP site of the target gene is wild-type or synonymous mutant, at this time, the first base of the target gene specific binding sequence in the medium probe does not complementarily bind to the target gene. At this time, neither the first base of the target gene specific binding sequence nor the medium sequence complementarily binds to the target gene. Just like in 3.1.2 above, 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 wild-type or synonymous mutant;

[0022] 3.2.2. When the SNP site of the target gene is missense mutant, at this time, the first base of the target gene specific binding sequence in the medium probe complementarily binds to the missense mutant target gene to form an SNP site base pair. Just like in 3.1.1 above, 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 missense mutant;

[0023] 3.2.3. Similar to that in 3.1.3 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.

[0024] In one embodiment, when there are multiple SNP sites in the target gene, corresponding mediator probes and detection probes are designed respectively for each SNP site, so that the first double-stranded product and the second double-stranded product corresponding to each SNP site are formed respectively.

[0025] In one embodiment, the method of melting curve is used to detect whether the first double-stranded product and / or the second double-stranded product is formed.

[0026] In one embodiment, a fluorescent group is labeled on the mediator sequence, and a quenching group corresponding to the fluorescent group labeled on the mediator sequence is labeled on the target gene specific binding sequence; a quenching group corresponding to the fluorescent group labeled on the mediator sequence of the mediator probe is modified on the detection probe, and the method of melting curve is used to detect whether the first double-stranded product and / or the second double-stranded product is formed.

[0027] In one embodiment, a fluorescent group and a corresponding quenching group are labeled on the detection probe, and the method of melting curve is used to detect whether the first double-stranded product and / or the second double-stranded product is formed.

[0028] In one embodiment, the mediator probe sequence and / or the detection probe sequence comprises or consists of naturally occurring nucleotides, modified nucleotides, non-natural nucleotides, or any combination thereof.

[0029] 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.

[0030] In one embodiment, a kit for detecting the genotype of SNP sites in a target gene independent of non-targets is provided, and the kit includes: DNA polymerase, a first primer, a second primer, a mediator probe and a detection probe designed for a target gene containing a SNP site to be detected;

[0031] 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 detected;

[0032] b. The media probe sequentially includes a media sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the media sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, 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 media probe is labeled with a group that blocks extension;

[0033] d. Design a detection probe according to the media sequence of the media probe. The detection probe sequentially includes a complementary sequence to the media sequence of the media probe, a base complementary to the first base of the target gene-specific binding sequence of the media probe along the 5'-end to the 3'-end direction, and an extension sequence. For the extension sequence, along the 3'-end to 5'-end direction, the first base cannot be complementary to the second base of the target gene-specific binding sequence of the media probe along the 5'-end to 3'-end direction.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] 1. The method of the present invention is a non-target-dependent detection method, that is, the sequences of each media primer and the detection probe are artificially designed, known or predetermined. Therefore, 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 double-stranded body can be calculated in advance. Thus, by detecting the melting peak with the melting point (Tm value) of a certain double-stranded body in the melting curve analysis, the SNP typing of the target gene corresponding to the double-stranded body can be judged, which solves the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target gene sequence to be detected, and well solves the problem that the melting curve analysis method is not applicable to RNA samples.

[0036] 2. In the currently widely used asymmetric melting curve method, the mutation site is designed on the probe, and the wild type and mutant type are distinguished by the difference in the Tm value of a single fluorescent probe. For mutations such as ATGC>GC, the difference in Tm values is easy to distinguish. However, for mutations such as A>T, the difference in Tm values of probe binding is small, and it is difficult to distinguish genotypes. Currently, the asymmetric melting curve is widely used in SNP genotyping. In this method, the mutation site is designed on the probe. Since the probe has no selectivity, only one probe is designed for one SNP site to distinguish two genotypes, and the genotype is distinguished by the single-base Tm value of the probe binding to the single strand in the same fluorescence channel. For mutations such as ATGC>GC, the difference in Tm values is easy to distinguish. However, for mutations such as A>T, the difference in Tm values of probe binding is small, and it is difficult to distinguish genotypes. Moreover, due to the limitations of the probe temperature and the two peaks in a single channel, at most 2-3 SNP sites can be designed in one fluorescence channel. In the method of the present invention, the first base of the specific binding sequence between the mediator probe and the target gene is designed as the SNP site. By using whether the SNP site of the target gene to be detected is complementary to the mediator probe, two different mediator primers are generated by cleavage. Due to the difference in the 3'-terminal sequence of different mediator primers, after binding to the detection probe, only one of the mediator primers can extend along the detection probe, while the other mediator primer can only bind to the detection probe but cannot extend. Therefore, the melting point (Tm value) of the double-stranded body can be arbitrarily constructed, realizing the self-definition of the melting curve peak of the SNP site, thus solving the problem that the Tm values of the wild type and mutant genotypes of the same SNP site cannot be distinguished, reducing the design difficulty, and enabling the specific distinction of the target site with A>T base mutation. For different SNP sites, the Tm values are distinguished by controlling the amplicon length and GC content between the mediator primer and the detection probe, and different SNP sites are distinguished by different melting curve Tm values, so the number of detectable SNP sites is increased.

[0037] 3. In the method of the present invention, when using the melting curve method to detect the first double-stranded product and the second-stranded product, during the melting curve analysis process, the fluorescence signal fluctuations of the mediator primer and the detection probe are more prominently reflected, obtaining a flatter baseline, reducing the appearance of non-specific peaks, and improving the correctness and accuracy of the detection method. At the same time, this method is not limited by the number of fluorescence channel detections. Different target genes are distinguished according to the melting point and fluorescence color, increasing the number of target detections per well and improving the detection throughput. With the method of the present invention, multiple target detections can be performed. The baselines of the melting peaks can all remain flat, the peaks are higher, there are no non-specific peaks, and the sensitivity is higher; at the same time, the detection range is also wider, and the melting curve detection T m value range is: 40~80°C;

[0038] 4. When using the melting curve method for detection, the method for detecting the target nucleic acid in the present invention is a symmetric amplification melting curve method. Compared with the common asymmetric amplification melting curve method (a linear amplification method), this method utilizes the advantage of PCR exponential amplification to enrich a large amount of target nucleic acid and improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0040] Figure 1 Schematic diagram of the basic principle of the method of the present invention;

[0041] Figure 2 Melting curve analysis diagram for the wild-type CC genotype of MTHFR (677C>T);

[0042] Figure 3 Melting curve analysis diagram for the heterozygous mutant CT genotype of MTHFR (677C>T);

[0043] Figure 4 Melting curve analysis diagram for the wild-type AA genotype of MTHFR (1298A>C);

[0044] Figure 5 Melting curve analysis diagram for the heterozygous mutant AC genotype of MTHFR (1298A>C);

[0045] Figure 6 Melting curve analysis diagram for the wild-type of both SNP loci of MTHFR (677C>T) and MTHFR (1298A>C);

[0046] Figure 7 Melting curve analysis diagram for the wild-type of MTHFR (677C>T) and the heterozygous type of MTHFR (1298A>C);

[0047] Figure 8 Melting curve analysis diagram for the heterozygous type of MTHFR (677C>T) and the wild-type of MTHFR (1298A>C);

[0048] Figure 9 Melting curve analysis diagram for the heterozygous type of both SNP loci of MTHFR (677C>T) and MTHFR (1298A>C). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0049] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of this application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0050] Embodiment 1 The basic principle of the present invention

[0051] As Figure 1 shown, the present invention provides a method for detecting the genotype of SNP sites in a target gene independent of a non-target, and the method includes the following steps:

[0052] Step 1: Design a first primer, a second primer and a mediator probe for a target gene containing a SNP site to be detected,

[0053] 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;

[0054] 1.2. The mediator probe sequentially includes a mediator sequence and a target gene-specific binding sequence from the 5'-end to the 3'-end direction, wherein the mediator sequence cannot bind to the target gene; the target gene-specific binding sequence can specifically and complementarily bind to the target gene, and along the 5'-end to the 3'-end direction, 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 the mutant base of the SNP site of the target gene; a group that prevents extension is labeled at the 3'-end of the mediator probe;

[0055] Step 2: Design a detection probe according to the mediator sequence of the mediator probe, and the detection probe sequentially includes a complementary sequence to the mediator sequence of the mediator probe, a base complementary to the first base of the target gene-specific binding sequence of the mediator probe along the 5'-end to the 3'-end direction, and an extension sequence. Along the 3'-end to the 5'-end direction, the first base of the extension sequence cannot be complementary to the second base of the target gene-specific binding sequence of the mediator probe along the 5'-end to the 3'-end direction;

[0056] Step 3: In a reaction system containing the upstream first primer, second primer, mediator probe, detection probe, a nucleic acid sample containing the target gene and a DNA polymerase, perform PCR amplification and analysis of the amplification product:

[0057] 3.1. When the first base of the target gene-specific binding sequence is complementary to the wild-type base of the SNP site of the target gene:

[0058] 3.1.1. When 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 medium probe forms a SNP site base pair by complementary binding with the wild-type target gene. Both 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 medium sequence in the medium probe, DNA polymerase will cleave the phosphodiester bond between the SNP site base pair and the first adjacent base pair. The cleaved sequence fragment contains the medium sequence and the first base, and this fragment is called the first medium primer. The first medium primer is completely complementary paired with the detection probe and extends along the extended sequence part 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;

[0059] 3.1.2. When the SNP site of the target gene is mutant, at this time, the first base of the target gene specific binding sequence in the medium probe does not bind complementarily with the mutant target gene. At this time, neither the first base of the target gene specific binding sequence nor the medium sequence binds complementarily with the target gene. Both the first primer and the second primer are 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 medium probe, DNA polymerase will cleave the phosphodiester bond between the first base and the second base that pair with the target gene in the target gene specific binding sequence. The cleaved sequence fragment contains the medium sequence, the first 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 medium primer. The last base at the 3'-end of the second medium primer cannot bind complementarily with the detection probe, and the second medium primer cannot extend along the detection probe. Therefore, a second double-stranded product is obtained. 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 mutant;

[0060] 3.1.3. When the SNP site of the target gene is heterozygous, at this time, both the first double-stranded product and the second double-stranded product will be generated. Therefore, when the formation of both the first double-stranded product and 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 heterozygous;

[0061] 3.2. When the first base sequence of the target gene specific binding sequence is complementary to the missense mutant base of the SNP site of the target gene:

[0062] 3.2.1. When the SNP site of the target gene is wild-type or synonymous mutant, at this time, the first base of the target gene-specific binding sequence in the medium probe does not complementarily bind to the target gene. At this time, the first base of the target gene-specific binding sequence and the medium sequence do not complementarily bind to the target gene. Just like in 3.1.2 above, only when the formation of the second double-stranded product is detected can it be determined that the SNP site in the nucleic acid sample containing the target gene is wild-type or synonymous mutant;

[0063] 3.2.2. When the SNP site of the target gene is missense mutant, at this time, the first base of the target gene-specific binding sequence in the medium probe complementarily binds to the missense mutant target gene to form a SNP site base pair. Just like in 3.1.1 above, only when the formation of the first double-stranded product is detected can it be determined that the SNP site in the nucleic acid sample containing the target gene is missense mutant;

[0064] 3.2.3. Just like in 3.1.3 above, when the formation of both the first double-stranded product and 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 heterozygous.

[0065] In the method of the present invention, the medium sequence is labeled with a fluorescent group, and the target gene-specific binding sequence is labeled with a quenching group corresponding to the fluorescent group labeled in the medium sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the medium sequence of the medium probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by means of a melting curve.

[0066] 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 means of a melting curve.

[0067] In the method of the present invention, in addition to containing a DNA polymerase, the amplification reaction system may also contain a nuclease and / or an exonuclease.

[0068] In the present invention, since the base sequence and length of the detection probe can be designed in advance, the first double-stranded product obtained by the extension of the first medium primer and the detection probe has a predictable length and structure. Correspondingly, the first double-stranded product has a predictable melting point (T m1 value). Similarly, the second double-stranded product obtained by the binding of the second medium primer and the detection probe also has a predictable length and structure. Correspondingly, the second double-stranded product has a predictable melting point (T m2 value), and since the first double-stranded product has a longer base sequence and structure than the second double-stranded product, the Tm1 >T m2 , the melting curve analysis and detection are performed on the double-stranded product obtained in the fourth step. By detecting the peak position of the melting curve of the double-stranded product, the corresponding genotype can be determined.

[0069] In the present invention, the fluorescent group includes various commonly used fluorescent markers at present, but is 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 at present, but is not limited to these quenchers, such as Dabcyl, Eclipse, BHQ-1, BHQ-2, QYS-7, etc.

[0070] Example 2 Genotyping of the MTHFR C677T Locus

[0071] Taking the detection of the MTHFR C677T locus as an example, the method of the present invention is used to detect the MTHFR gene. The specific method includes the following steps.

[0072] I. Primer and Probe Sequence Information

[0073] According to the target nucleic acid site to be detected, the first primer, the second primer, the mediator probe and the detection probe are designed. Among them, the first base sequence of the target gene-specific binding sequence is used to design the mediator probe complementary to the missense mutant base of the SNP site of the target gene. The sequence information is shown in Table 1 below.

[0074] Table 1

[0075]

[0076] Note: The bold part of the mediator probe is the mediator sequence, the italic part is the detection site, and the lowercase letters are the target gene-specific sequences.

[0077] II. PCR Amplification Reaction System and Program

[0078] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. 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 mediator probe, 100 nM detection probe 1, 5 μL nucleic acid template.

[0079] The fluorescence PCR reaction program of the present invention is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0080] III. Melting curve analysis

[0081] The results of the melting curve analysis are shown in Table 2 below.

[0082] Table 2

[0083]

[0084] For the MTHFR C677T mutation site, a peak appears at the T m value of 49.4 ± 1°C in the FAM channel, which is judged as wild type, as Figure 2 ; a peak appears simultaneously at the T m values of 49.4 ± 1°C and 65.0 ± 1°C in the FAM channel, which is judged as heterozygous, as Figure 3 .

[0085] Example III Genotyping of the MTHFR A1298C Site

[0086] Taking the detection of the MTHFR A1298C mutation site as an example, the method of the present invention is used to detect the mutation of the MTHFR gene. The specific method includes the following steps.

[0087] I. Primer and probe sequence information

[0088] The first primer, second primer, mediator probe and detection probe are designed according to the target nucleic acid site to be detected. Among them, the first base sequence of the specific binding sequence of the target gene is designed as the mediator probe complementary to the missense mutant base of the SNP site of the target gene. The sequence information is shown in Table 3 below.

[0089] Table 3

[0090]

[0091] Note: For the media probe, the bold part is the media sequence, the italic part is the detection site, and the lowercase letters are the target gene-specific sequences.

[0092] II. PCR Amplification Reaction System and Procedure

[0093] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. 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 media probe, 100 nM detection probe 2, and 5 μL nucleic acid template.

[0094] The fluorescence PCR reaction procedure of the present invention is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve procedure is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 real-time fluorescence PCR instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0095] III. Melting Curve Analysis

[0096] The results of the melting curve analysis are shown in Table 4 below.

[0097] Table 4

[0098]

[0099] For the MTHFR A1298C mutation site, a peak appears at the T m value of 53.2 ± 1°C in the Cy5 channel, which is judged as the wild type, as Figure 4 ; A peak appears simultaneously at the T m values of 53.4 ± 1°C and 69.0 ± 1°C in the Cy5 channel, which is judged as the heterozygous type, as Figure 5 .

[0100] Example 3 Genotyping of MTHFR C677T and A1298C Double Loci

[0101] Taking the detection of the MTHFR C677T and MTHFR A1298C double mutation sites as an example, the method of the present invention is used to detect the mutations of the MTHFR gene. The specific method includes the following steps:

[0102] I. Primer and Probe Sequence Information

[0103] Design the first primer, the second primer, the mediator probe, and the detection probe according to the target nucleic acid site to be detected. Among them, design the mediator probe according to the first base sequence of the target gene-specific binding sequence to be complementary to the missense mutant base of the target gene SNP site, and the sequence information is shown in Table 5 below.

[0104] Table 5

[0105]

[0106] Note: The bold part of the mediator probe is the mediator sequence, the italic part is the detection site, and the lowercase letters are the target gene-specific sequences.

[0107] II. PCR Amplification Reaction System and Procedure

[0108] The present invention uses a 25 μL PCR reaction system for PCR amplification and melting curve analysis. 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 mediator probe, 100 nM detection probe 1 and 100 nM detection probe 2, 5 μL nucleic acid template.

[0109] The fluorescence PCR reaction program of the present invention is pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing and extension at 60°C for 45 s while collecting fluorescence, repeating 45 cycles; the melting curve program is 95°C for 2 min, 40°C for 5 min, and 40 - 80°C for melting curve analysis, detecting the fluorescence signal every 0.04°C. The instrument used in this example is the SLAN 96 Real-Time Fluorescence PCR Instrument (Shanghai Hongshi Medical Technology Co., Ltd.).

[0110] For the one-tube detection of the MTHFR C677T and MTHFR A1298C double sites, the melting curve results of the random template are as shown in the appendix Figures 6 - 9 ;

[0111] If peaks only appear at 49.4 ± 1°C in the FAM channel and only at 53.4 ± 1°C in the Cy5 channel, then both sites are wild-type ( Figure 6 );

[0112] If peaks only appear at 49.4 ± 1°C in the FAM channel and peaks appear simultaneously at 53.4 ± 1°C and 69.0 ± 1°C in the Cy5 channel, then C677T is wild-type and the A1298C site is heterozygous mutant ( Figure 7 );

[0113] If the FAM channel peaks only at 49.4 ± 1 °C and 65.0 ± 1 °C simultaneously, and the Cy5 channel peaks only at 53.4 ± 1 °C, then C677T is a heterozygous mutant type, and the A1298C locus is wild type ( Figure 8 );

[0114] If the FAM channel peaks at 49.4 ± 1 °C and 65.0 ± 1 °C simultaneously, and the Cy5 channel peaks at 53.4 ± 1 °C and 69.0 ± 1 °C simultaneously, then both the C677T and A1298C loci are heterozygous mutant types ( Figure 9 ).

[0115] III. Melting Curve Analysis

[0116] The results of the melting curve analysis are shown in Table 6 below.

[0117] Table 6

[0118]

[0119] It should be understood that the disclosed invention is not limited to the specific methods, protocols, and materials described, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit the scope of the invention, which is limited only by the appended claims.

[0120] 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. These equivalents are also encompassed by the appended claims.

Claims

1. A method for detecting the genotype of a SNP site in a target gene for non-diagnostic purposes and without target dependence, 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 a detection probe according to the mediator sequence of the mediator probe, wherein the detection probe sequentially comprises a complementary sequence to the mediator sequence of the mediator probe, a base complementary to the first base of the target gene specific binding sequence of the mediator probe along the direction from 5' to 3', and an extension sequence, wherein the first base of the extension sequence along the direction from 3' to 5' cannot be complementary to the second base of the target gene specific binding sequence of the mediator probe along the direction from 5' to 3'; Step 3: Perform PCR amplification and analysis of amplification products in a reaction system containing the first primer, the second primer, the mediator probe, the detection probe, the nucleic acid sample containing the target gene, and a DNA polymerase: 3.

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: 3.1.

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 first mediator 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; 3.1.

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 cannot extend along the detection probe, thus 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. 3.1.

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; 3.

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: 3.2.

1. If the SNP site of the target gene is a wild type or a synonymous mutation, then 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 3.1.2 above, only the formation of the second double-stranded product is detected, and it can be determined that the SNP site in the nucleic acid sample containing the target gene is a wild type or a synonymous mutation; 3.2.

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 3.1.1 above, 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; 3.2.

3. As in 3.1.3 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, a corresponding mediator probe and a detection probe 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 media sequence is labeled with a fluorescent group, and the target gene-specific binding sequence is labeled with a quenching group corresponding to the fluorescent group labeled in the media sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the media sequence of the media probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the 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 and / or the detection probe sequence comprises or consists 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 without target dependence, 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, a mediator 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 a detection probe according to the mediator sequence of the mediator probe, wherein the detection probe sequentially comprises a complementary sequence to the mediator sequence of the mediator probe, a base complementary to the first base of the target gene specific binding sequence of the mediator probe along the 5' end to the 3' end, and an extension sequence, wherein the first base of the extension sequence along the 3' end to the 5' end cannot be complementary to the second base of the target gene specific binding sequence of the mediator probe along the 5' end to the 3' end.

9. The kit according to claim 8, characterized in that The intermediary probe sequence and 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 media sequence is labeled with a fluorescent group, and the target gene-specific binding sequence is labeled with a quenching group corresponding to the fluorescent group labeled in the media sequence; the detection probe is modified with a quenching group corresponding to the fluorescent group labeled in the media sequence of the media probe, and whether the first double-stranded product and / or the second double-stranded product is formed is detected by the 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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