CN111363792A

CN111363792A - Kit and method for detecting gene polymorphism based on shared primer probe and application

Info

Publication number: CN111363792A
Application number: CN202010339628.XA
Authority: CN
Inventors: 扶媛媛; 周洋; 苏正稳; 曹彦东; 王利; 郝俊; 杨颖�
Original assignee: Beijing Anzhiyin Biotechnology Co ltd
Current assignee: Beijing Anzhiyin Biotechnology Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-07-03
Anticipated expiration: 2039-10-31
Also published as: CN111363792B; CN110628883B; CN110628883A

Abstract

The application discloses a method and a kit for detecting gene polymorphism based on a shared primer probe and application thereof. The method is characterized in that a reporter group is marked at the 5' ends of a wild ARMS primer and a mutant ARMS primer which are designed in an isotropic way, an ARMS primer with probe property is prepared, and the ARMS primer is used for detecting gene polymorphism after being screened, in particular for detecting human CYP2C19 polymorphism. The method has the advantages of simple design, low cost, higher detection sensitivity, stronger specificity and more accurate and objective result.

Description

Kit and method for detecting gene polymorphism based on shared primer probe and application

The divisional application is based on Chinese patent application with application number of 201911055308.5, application date of 2019, 10 and 31, and invented as 'kit, method and application for detecting gene polymorphism based on shared primer probe'.

Technical Field

The application relates to the field of gene detection, in particular to PCR detection of gene polymorphism.

Technical Field

The methods currently used for detecting gene polymorphisms are mainly as follows: sequencing typing method, high resolution melting curve typing method, gene chip typing method, TaqMan probe typing method, MGB probe typing method and the like. The sequencing typing method is a gold standard for detection in the field, but is limited by the complexity of operation and the specificity of equipment, and is not widely popularized; the rest methods are derived based on PCR amplification technology and are relatively widely used. The detection process of the sequencing typing method comprises PCR amplification and sequencing, the operation is complicated, the detection time generally reaches 4-5 hours, and the PCR reaction tube needs to be opened in the operation process, so that the pollution of PCR products is easily caused; the high-resolution melting curve typing method is characterized in that the Tm value difference caused by the difference of bases of polymorphic sites is utilized, the detection is carried out by identifying different Tm values, the Tm values of wild type sites and mutant type sites are often different from 1 ℃, so equipment for accurately identifying the temperature is needed, the equipment is few, most of the equipment belongs to imports, and the price is high; the detection process of the gene chip typing method comprises PCR amplification and probe hybridization, the operation is complicated, the detection time generally reaches 4-5 hours, and the PCR reaction tube needs to be opened in the operation process, so that the pollution of PCR products is easily caused; the TaqMan probe typing method is to utilize ARMS primers to identify polymorphic sites, and the methodology needs 2 reaction holes for detecting one polymorphic site, and the flux is relatively low; MGB probe typing utilizes MGB marked probes to identify polymorphic sites, MGB probes are difficult to design and high in price, and a plurality of suitable MGB probes need to be screened, so that the cost is high.

The coding gene of CYP2C19 enzyme is CYP2C19 gene, is located on human chromosome 10, is an important member in the second subfamily of cytochrome enzyme (CYP450 enzyme), is an important drug metabolizing enzyme of human body, and has a lot of expression in liver. The gene polymorphism of CYP2C19 is closely related to individual difference of medicines, the metabolic rate type of a patient can be judged by detecting the CYP2C19 genotype of the patient, the dosage of the medicine can be reasonably adjusted, and the medicine is an effective way for improving the cure rate of related diseases and reducing toxic and side effects, particularly clopidogrel, voriconazole, antidepressant medicines, antiepileptic medicines and the like. In the prior art, the detection means for human CYP2C19 gene polymorphism are mainly the above 5 methods.

Therefore, the above methods still have many defects, and it is necessary to find a new detection method, which makes the detection effect more specific, the sensitivity higher, the flux higher, the result more objective, and the like, and also ensures simple design, low cost, simple operation, short detection time, and the like. Based on this, the present application is specifically proposed.

Disclosure of Invention

The first purpose of the application is to provide a method for preparing a shared ARMS primer probe for detecting gene polymorphism;

a second object of the present application is to provide a method for detecting gene polymorphism based on a shared primer probe;

the third purpose of the application is to provide a sharing primer probe-based gene polymorphism detection primer system, a kit and application thereof.

In order to achieve the purpose, the technical scheme adopted by the application is as follows:

the application provides a method for preparing a probe ARMS primer for detecting gene polymorphism, which comprises the steps of designing a wild type ARMS primer and a mutant type ARMS primer aiming at a wild type locus and a mutant type locus in the same direction, marking the last base at the 5' ends of the wild type ARMS primer and the mutant type ARMS primer by different report groups, sharing the same nucleotide sequence with the primers and the probes, and preparing the probe ARMS primer with the probe property (shown in figures 1-3);

preferably, the reporter gene is selected from FAM, ROX, TET, VIC, JOE, HEX, Cy3, cy3.5, Cy5, cy5.5, TAMRA, Texas Red, LC Red640 or LC Red 705;

in some embodiments, further comprising screening high amplification efficiency primers as primers for preparing a probe ARMS;

in some embodiments, further comprising quenching probe and downstream primer design;

in some embodiments, the quenching probe is a probe designed complementary nucleotide sequence for wild-type and mutant-type ARMS primers and labeled with a non-fluorescent quenching group at the 3' end of the complementary nucleotide sequence;

in some embodiments, the quenching probe has 3-9 fewer bases than the wild-type and mutant probe ARMS primers; preferably, 4 to 7 fewer; more preferably 5 to 6 less;

in some embodiments, further comprising the introduction of wild-type probe ARMS primer and mutant probe ARMS primer mismatch genes;

in some embodiments, the mismatch gene is near the 3' end of the wild-type and mutant probe ARMS primers;

in some embodiments, the wild-type probe ARMS primer and the mutant probe ARMS primer introduce mismatched bases at the 2 nd to 4 th bases near the 3' end, the mismatched bases introduced at the same sequence position are different, and the mismatched bases introduced at different sequence positions can be the same or different. For example: the wild type probe ARMS primer introduces a mismatched base A at the 2 nd base close to the 3 ' end, and the mutant type probe ARMS primer can introduce a mismatched base T, G or C except A at the 2 nd base close to the 3 ' end and introduce a mismatched base A, T, G or C containing A at the 3 rd or 4 th base close to the 3 ' end;

in some embodiments, the wild-type and mutant-type probing ARMS primers have a Tm of 65 to 70 ℃;

in some embodiments, the wild type site detection and the mutant site detection are PCR amplified in the same reaction system;

in some embodiments, the gene polymorphism detection is for a CYP2C19 polymorphism detection; preferably, the compounds are directed against CYP2C19 x 2 and CYP2C19 x 3; more preferably, also for CYP2C19 x 17;

in some embodiments, the wild-type probed ARMS primer 5 ' of CYP2C19 x 2 is labeled with FAM fluorophore, the mutant probed ARMS primer 5 ' of CYP2C19 x 2 is labeled with TET fluorophore, and the quenching probe 3 ' of CYP2C19 x 2 is labeled with BHQ2 quencher; the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 3 is marked with ROX fluorescent group, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 3 is marked with CY5 fluorescent group, and the 3 ' end of the quenching probe of CYP2C 19X 3 is marked with BHQ2 quenching group;

in some embodiments, the wild-type probe ARMS primer, mutant probe ARMS primer, quenching probe and downstream primer sequences of CYP2C19 x 2 are set forth in SEQ ID nos. 1-4, respectively; the sequences of the wild probe ARMS primer, the mutant probe ARMS primer, the quenching probe and the downstream primer of the CYP2C19 x 3 are respectively shown as SEQ ID NO. 5-8;

in some embodiments, the CYP2C19 x 17 wild-type probed ARMS primer is labeled with FAM fluorophore at the 5 ' end, the TET fluorophore at the 5 ' end of the mutant probed ARMS primer of CYP2C19 x 17, and the BHQ2 quencher at the 3 ' end of the quencher probe of CYP2C19 x 17;

in some embodiments, the wild-type probe ARMS primer, mutant probe ARMS primer, quenching probe and downstream primer sequences of CYP2C19 x 17 are set forth in SEQ ID nos. 9 to 12, respectively;

the application provides a detection method for detecting gene polymorphism based on a shared primer probe, which comprises the steps of preparing a probe ARMS primer for detecting gene polymorphism;

the method comprises the steps of designing a wild type ARMS primer and a mutant type ARMS primer aiming at a wild type locus and a mutant type locus in the same direction, wherein the last base at the 5' ends of the wild type ARMS primer and the mutant type ARMS primer is marked by different report groups, and the primers and a probe share the same nucleotide sequence to prepare a probe ARMS primer with probe property;

in some embodiments, the quenching probe has 3-9 fewer bases than the wild-type and mutant probe ARMS primers; preferably, 4 to 7 fewer; more preferably 5-6 less.

In some embodiments, further comprising the introduction of mismatched bases from the wild-type probe ARMS primer and the mutant probe ARMS primer;

in some embodiments, the mismatched base is near the 3' end of the wild-type and mutant probe ARMS primers;

in some embodiments, the wild-type probe ARMS primer and the mutant probe ARMS primer introduce different mismatched bases at the same sequence position, and the mismatched bases introduced at different sequence positions may be the same or different;

in some embodiments, the wild-type and mutant probe ARMS primers have a Tm of 65-70 ℃.

in some embodiments, the detection method for detecting gene polymorphism based on shared primer probe further comprises a sample processing step, a detection system preparation step, a reaction parameter setting step and a result interpretation and analysis step;

in some embodiments, in the step of preparing the detection system, the concentration of the probe ARMS primer and the downstream primer in the PCR mixed solution for amplifying the polymorphic sites during detection is 400-600 nM, preferably 500 nM; the concentration of the quenching probe is 1.2-1.8 mu M, preferably 1.5 mu M;

in some embodiments, in the detection system preparation step, the negative quality control substance during detection consists of Tris-HCl buffer solution;

in some embodiments, in the step of formulating the assay system, the positive quality control substance in the assay consists of CYP2C19 × 2 wild-type plasmid, CYP2C19 × 2 mutant-type plasmid, CYP2C19 × 3 wild-type plasmid, CYP2C19 × 3 mutant-type plasmid, CYP2C19 × 17 wild-type plasmid, CYP2C19 × 17 mutant-type plasmid; preferably, the plasmid is an artificial cloning circular plasmid, and is diluted to 2000 copies by Tris-Hcl (pH8.0); the Taq enzyme in the kit is hot-start enzyme;

in some embodiments, in the reaction parameter setting step, the reaction parameters are set to 95 ℃ for 3 minutes for pre-denaturation, 1 cycle; denaturation at 95 ℃ for 15 seconds, denaturation at 60 ℃ for 1 minute, collection of fluorescence signals, and 40 cycles; the fluorescence signal acquisition channel is provided with FAM, TET, ROX and CY 5;

in some embodiments, the results are judged and analyzed by ① positive quality control products FAM, TET, ROX and CY5 fluorescence amplification curves, the negative quality control products have no amplification curve, the detection data is valid, ② the fluorescence signals of FAM and TET in the PCR mixture of amplified CYP2C19 and CYP2C19 are judged to be the polymorphism of CYP2C19, FAM the amplification curve and TET the amplification curve are judged to be the wild type CYP2C19 homozygous, FAM the amplification curve and TET the amplification curve are judged to be the mutant type CYP2C19 homozygous, FAM and TET the amplification curve are judged to be the amplification curve, ③ the fluorescence signals of FAX and CY5 in the PCR mixture of amplified CYP2C19, CYP2C19, CYP 3 the fluorescence signals of TEX and TET 36863 are judged to be the mutant type CYP2C 8672, the fluorescence signals of TEX and TET the amplification curve and TET 368672 the fluorescence signals of the amplification curve and TEX 2C 368672, 3617 the fluorescence signals of the amplification curve and TEX 2C 36863 are judged to be the mutant type CYP2C 8672, and the fluorescence amplification curve and the amplification curve are judged to be the amplification curve respectively.

The application provides a primer group (or a primer system) for detecting CYP2C19 gene polymorphism, wherein the primer group (or the primer system) is prepared by a preparation method of a shared ARMS primer probe for detecting gene polymorphism;

in some embodiments, the mismatched bases are near the 3' end of the wild-type and mutant probe ARMS primers;

in some embodiments, the wild-type probe ARMS primer, mutant probe ARMS primer, quenching probe and downstream primer sequences of CYP2C19 x 2 are set forth in SEQ ID nos. 1-4, respectively; the nucleotide sequences of the wild type probe ARMS primer, the mutant probe ARMS primer, the quenching probe and the downstream primer of the CYP2C19 × 3 are respectively shown as SEQ ID NO. 5-8;

the method comprises the following specific steps:

CYP2C19 x 2 wild type probe ARMS primers:

5′-FAM-TTTTAAGTAATTTGTTATGGGTTGCC-3′SEQ ID NO.1

CYP2C19 × 2 mutant probe ARMS primers:

5′-TET-TTTTAAGTAATTTGTTATGGGTGCCT-3′SEQ ID NO.2

CYP2C19 × 2 quench probe:

5′-CCCATAACAAATTACTTAAAA-BHQ2-3′SEQ ID NO.3

CYP2C19 × 2 downstream primer:

5′-AATTTTCCCACTATCATTGATTATTTCC-3′SEQ ID NO.4

CYP2C19 × 3 wild-type probe ARMS primers:

5′-ROX-AAAAAACTTGGCCTTACCTGAATC-3′SEQ ID NO.5

CYP2C19 × 3 mutant probe ARMS primers:

5′-CY5-AAAAAACTTGGCCTTACCTGTATT-3′SEQ ID NO.6

CYP2C19 × 3 quench probe:

5′-AGGTAAGGCCAAGTTTTTT-BHQ2-3′SEQ ID NO.7

CYP2C19 × 3 downstream primer:

5′-GAAAACATCAGGATTGTAAGCACC-3′SEQ ID NO.8

in some embodiments, the wild-type probe ARMS primer, mutant probe ARMS primer, quenching probe and downstream primer of CYP2C19 x 17 have the nucleotide sequences shown in SEQ ID nos. 9 to 12, respectively;

the method comprises the following specific steps:

CYP2C19 × 17 wild-type probe ARMS primers:

5′-FAM-AATTTGTGTCTTCTGTTCTCAGAGC-3′SEQ ID NO.9

CYP2C19 × 17 mutant probe ARMS primers:

5′-TET-AATTTGTGTCTTCTGTTCTCAAGGT-3′SEQ ID NO.10

CYP2C19 × 17 quench probe:

5′-GAGAACAGAAGACACAAATT-BHQ2-3′SEQ ID NO.11

CYP2C19 × 17 downstream primer:

5′-GCGCATTATCTCTTACATCAGAG-3′SEQ ID NO.12。

a primer group (or a primer system) for detecting CYP2C19 gene polymorphism, wherein the primer sequence is shown as 1-12;

in some embodiments, the fluorescent labels of the primers are: the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 2 is marked with FAM fluorophore, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 2 is marked with TET fluorophore, and the 3 ' end of the quenching probe of CYP2C 19X 2 is marked with BHQ2 quencher; the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 3 is marked with ROX fluorescent group, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 3 is marked with CY5 fluorescent group, and the 3 ' end of the quenching probe of CYP2C 19X 3 is marked with BHQ2 quenching group; the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 17 is marked with FAM fluorescent group, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 17 is marked with TET fluorescent group, and the 3 ' end of the quenching probe of CYP2C 19X 17 is marked with BHQ2 quenching group.

The primer group (or the primer system) is applied to the preparation of a kit for detecting the polymorphism of the CYP2C19 gene.

A kit for detecting CYP2C19 gene polymorphism comprises the primer group (or primer system).

In some embodiments, the kit further comprises negative and positive quality controls, as well as conventional components such as PCR buffer, dNTP, Mg2+, Taq enzyme, and the like;

in some embodiments, the negative control consists of Tris-HCl buffer; the positive quality control product consists of CYP2C19 x 2 wild-type plasmid, CYP2C19 x 2 mutant-type plasmid, CYP2C19 x 3 wild-type plasmid, CYP2C19 x 3 mutant-type plasmid, CYP2C19 x 17 wild-type plasmid and CYP2C19 x 17 mutant-type plasmid;

in some embodiments, the plasmid is an artificial cloning circular plasmid;

in some embodiments, the Taq enzyme in the kit is a hot start enzyme;

the method for detecting CYP2C19 gene polymorphism by using the kit comprises the following steps:

⑴ sample processing, namely extracting human peripheral blood genome DNA by using a blood genome DNA extraction kit to serve as a sample to be detected;

⑵ preparing detection system, adding 18 μ l PCR mixed solution and 2 μ l sample to be detected into reaction tube, setting negative quality control system and positive quality control system, placing the reaction tube into fluorescent quantitative PCR instrument;

⑶ setting reaction parameters including pre-denaturation at 95 deg.c for 3 min for 1 cycle, denaturation at 95 deg.c for 15 sec for 1 min at 60 deg.c, collecting fluorescent signal for 40 cycles, FAM, TET, ROX and CY5 in the fluorescent signal collecting passage;

the ⑷ result is judged and analyzed, wherein ① positive quality control products FAM, TET, ROX and CY5 fluorescence amplification curve and negative quality control products do not have amplification curve, the detection data is effective, ② the FAM and TET fluorescence signals in PCR mixed liquor of amplified CYP2C 19X 2 and CYP2C 19C 3 are used for judging the polymorphism of CYP2C 19X 2, FAM the amplification curve and TET the amplification curve are used for judging the result is CYP2C19 homozygous wild type, FAM the amplification curve is not used for judging the mutation type of CYP2C 42C 2, TET the amplification curve is used for judging the result is CYP2C19 heterozygous, FAM and TET the amplification curve are used for judging the result is CYP2C 695 2C 6862, 9 the result is CYP2C 3653 fluorescent signal homozygous mutant type, TEX and TET 36863, and TEX 368672 the result is used for judging the result is pure mutant type of FAM, TEX and TET 36863, and TEX 36863 the result is used for judging the result is 19 the result is pure mutant type FAC 2C19, and TEX fluorescent amplification curve and TET 36863, and the result is used for judging the result is 368672.

Compared with the prior art, the beneficial effects of the application lie in that:

1) the method improves the design of an ARMS primer and a TaqMan or MGB probe, a report group on the TaqMan or MGB probe is directly marked on the last base at the 5' end of the AMRS primer, the primer and the probe share the same nucleotide sequence, the probe ARMS primer with the probe property is prepared, compared with the common ARMS primer, the Tm value is high, the specificity and the sensitivity are greatly improved, and the detection result is more accurate (shown in a figure 1-3);

2) the probe ARMS primers share the same nucleotide sequence, have dual functions of the primers and the probes, do not need to additionally design a TaqMan probe, an MGB probe or other probes, save the design and the cost of the TaqMan or MGB probe, and have simple design and low cost;

3) the probe AMRS primer and the quenching probe are complementarily matched, the reporter group and the quenching group are contacted with each other, and compared with a TaqMan probe and an MGB probe, the background signal is lower, and the detection result is more objective;

4) the method is designed aiming at CYP2C19 polymorphic sites, a wild type probe ARMS primer and a mutant probe ARMS primer are designed in the same direction, and has the advantages that the wild type probe AMRS primer with mismatched bases amplifies a wild type template, and the PCR product generates base mutation at the same position, so that the number of mismatched bases of the wild type PCR product and the mutant probe ARMS primer is increased, the non-specific amplification probability of the mutant probe is further reduced, and the amplification specificity of the mutant probe ARMS primer is improved; similarly, wild-type probe ARMS primers can also improve specificity;

6) the probe ARMS primer group suitable for CYP2C19 polymorphism detection is screened out through optimization, the detection sensitivity is high, the specificity is strong, and the detection of multiple types of polymorphisms can be realized in the same reaction system.

7) The method adopts FAM marked CYP2C19 x 2 wild type ARMS primer, TET marked CYP2C19 x 2 mutant ARMS primer, ROX marked CYP2C19 x 3 wild type ARMS primer and CY5 marked CYP2C19 x 3 mutant ARMS primer, can realize the detection of 2 gene polymorphism sites in the same reaction hole, and obviously improve the flux;

8) the wild type and mutant detection system of CYP2C19 x 2, the wild type and mutant detection system of CYP2C19 x 3 can be arranged in the same PCR mixed solution, the wild type and mutant detection system of CYP2C19 x 17 are arranged in the same PCR mixed solution, and an internal standard system is not required to be arranged according to the principle that only a sample is homozygous for the wild type, the homozygous mutant and the heterozygous type;

9) the method can adopt a form of premix, and mixes ARMS primers, quenching probes, downstream primers, PCR buffer solution, dNTP, Mg2+ and Taq enzyme required for detection in advance, does not need to be prepared when in use, only dispenses and adds template DNA for on-machine detection, and has simple and convenient operation and short time;

10) according to the method, the result is interpreted according to the fluorescence signals collected by the fluorescence quantitative PCR instrument, the ct value does not need to be counted, and the result analysis is simple;

11) the detection method and the kit fill up the market blank in the field, have good market feedback, and are suitable for industrial application.

Drawings

FIG. 1 is a schematic diagram a of the method for detecting gene polymorphism described in the present application;

FIG. 2 is a schematic diagram b of the method for detecting gene polymorphism according to the present application;

FIG. 3 is a schematic diagram c of the method for detecting gene polymorphism according to the present application;

FIG. 4 marker optimization of CYP2C19 x 2 mutant ARMS primers of the present application;

FIG. 5 marker optimization of CYP2C19 x 3 mutant ARMS primers of the present application;

figure 6 marker optimisation of CYP2C19 x 17 mutant ARMS primers of the present application;

FIG. 7 optimized results of the sequence of the present CYP2C19 x 2 quenching probe;

FIG. 8 optimized results of the sequence of the present CYP2C19 × 3 quenching probe;

FIG. 9 optimized results of the sequence of the present CYP2C19 × 17 quenching probe;

figure 10 assay results for CYP2C19 x 2 homozygous wild type according to the invention;

FIG. 11 shows the results of the CYP2C19 x 2 homozygous mutant assay of the present invention;

figure 12 CYP2C19 x 2 heterozygote assay results according to the invention;

figure 13 assay of CYP2C19 x 3 homozygous wild type according to the invention;

FIG. 14 is a result of testing CYP2C19 x 3 homozygous mutant according to the present invention;

FIG. 15 is a result of detection of CYP2C19 x 3 heterozygous according to the invention;

figure 16 CYP2C19 x 17 homozygous wild type assay of the invention;

FIG. 17 is a result of detection of CYP2C19 x 17 homozygous mutant according to the present invention;

fig. 18 shows the results of the CYP2C19 x 17 heterozygote assay of the present invention.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and should not be construed as limiting the scope of the present application. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by manufacturers, and are all conventional products available on the market.

Example 1 optimization of primer design, optimization screening of markers

Designing and screening wild type and mutant probe ARMS primers, which comprises the following steps:

1) a series of ARMS primers are preliminarily designed aiming at CYP2C19 x 2, CYP2C19 x 3, CYP2C19 x 17 wild type and mutant sites, the Tm value is 58-62 ℃, and the sequences are as shown in the following table 1:

table 1 preliminary design of CYP2C19 x 2, CYP2C19 x 3, CYP2C19 x 17 wild type and mutant ARMS primers

2) Preparing PCR reaction liquid by CYP2C19 x 2 wild type ARMS primer, downstream primer and SYBR-premix buffer solution, and respectively detecting CYP2C19 x 2 wild type plasmid and mutant type plasmid with 5000 copy/mu l; preparing PCR reaction liquid by CYP2C19 × 2 mutant ARMS primer, downstream primer and SYBR-premix buffer solution, and respectively detecting 5000copy/μ l CYP2C19 × 2 wild type plasmid and mutant plasmid; preparing PCR reaction liquid by CYP2C19 x 3 wild type ARMS primer, downstream primer and SYBR-premix buffer solution, and respectively detecting 5000 copy/mu l CYP2C19 x 3 wild type plasmid and mutant type plasmid; preparing PCR reaction liquid by CYP2C19 × 3 mutant ARMS primer, downstream primer and SYBR-premix buffer solution, and respectively detecting 5000copy/μ l CYP2C19 × 3 wild type plasmid and mutant plasmid; preparing PCR reaction liquid by CYP2C19 x 17 wild type ARMS primer, downstream primer and SYBR-premix buffer solution, and respectively detecting 5000 copy/mu l CYP2C19 x 17 wild type plasmid and mutant type plasmid; preparing PCR reaction liquid by CYP2C19 × 17 mutant ARMS primers, downstream primers and SYBR-premix buffer solution, and respectively detecting 5000copy/μ l of CYP2C19 × 17 wild-type plasmids and mutant plasmids; the reaction parameters were set as follows:

TABLE 2 PCR amplification parameter design

Counting CYP2C19 x 2 wild type PCR reaction liquid to detect the amplification Ct values and △ Ct values of 5000 copy/mu l wild type plasmids and mutant type plasmids, counting CYP2C19 x 2 mutant type PCR reaction liquid to detect the amplification Ct values and △ Ct values of 5000 copy/mu l wild type plasmids and mutant type plasmids, counting CYP2C19 x 3 wild type PCR reaction liquid to detect the amplification Ct values and △ Ct values of 5000 copy/mu l wild type plasmids and mutant type plasmids, counting CYP2C19 x 3 PCR reaction liquid to detect the amplification Ct values and △ Ct values of 5000 copy/mu l wild type plasmids and mutant type plasmids, counting CYP2C19 x 17 wild type PCR reaction liquid to detect the amplification Ct values and △ Ct values of 5000 copy/mu l wild type plasmids and mutant type plasmids, counting CYP2C 3617 copy 5000 copy/mu 2 x 17 mutant type plasmids, and mutation △ Ct values:

preferably amplifying wild type ARMS primers with relatively small Ct value and relatively large △ Ct value in a wild type PCR reaction solution of CYP2C19 x 2, preferably amplifying mutant type ARMS primers with relatively small Ct value and relatively large △ Ct value in a mutant type PCR reaction solution of CYP2C19 x 2, preferably amplifying wild type ARMS primers with relatively small Ct value and relatively large △ Ct value in a wild type PCR reaction solution of CYP2C19 x 3, preferably amplifying mutant type ARMS primers with relatively small Ct value and relatively large △ Ct value in a mutant type PCR reaction solution of CYP2C19 x 3;

the wild-type ARMS primer with relatively small Ct value of the amplified wild-type plasmid template and relatively large Ct value △ Ct value is preferably selected from CYP2C19 x 17 wild-type PCR reaction solution, and the mutant ARMS primer with relatively small Ct value of the amplified mutant-type plasmid template and relatively large Ct value △ Ct value is preferably selected from CYP2C19 x 17 mutant-type PCR reaction solution.

TABLE 3 ARMS primers for optimized screening

Secondly, optimizing and screening the alternative ARMS primer reporter group mark, which comprises the following specific steps:

1) different types of markers such as FAM, VIC, HEX, TET, NED, CY3, ROX, CY5 and the like are tried on the 5' end of the alternative AMRS primer, FAM and ROX are common probe markers and have stable performance, so that the FAM and ROX are used as wild type site group markers and are mainly used for screening different reporter group markers of the mutant ARMS primer, the marker of the reporter group is completed by Shanghai workers, and the specific marking conditions are as follows:

TABLE 4 labeling and screening of alternative ARMS primers

2) Preparing PCR reaction liquid by ARMS primers marked by CYP2C19 x 2 mutant different reporter groups TET, VIC and HEX respectively with a quenching probe, a downstream primer and a PCR-premix buffer solution, and detecting the CYP2C19 x 2 mutant plasmid with 5000 copy/mu l; CYP2C19 x 3 mutant different reporter groups CY3, CY5 and NED marked ARMS primers are respectively mixed with a quenching probe, a downstream primer and a PCR-premix buffer solution to prepare a PCR reaction solution, and 5000 copy/mu l of CYP2C19 x 3 mutant plasmid is detected; CYP2C19 x 17 mutant ARMS primers marked by different reporter groups TET, VIC and HEX are respectively mixed with a quenching probe, a downstream primer and a PCR-premix buffer solution to prepare a PCR reaction solution, and 5000 copy/mu l of CYP2C19 x 17 mutant plasmid is detected.

TABLE 5 PCR reaction parameter settings

3) Analysis of results

CYP2C19 x 2 mutant ARMS primer mark is preferably TET mark, and the test result is shown in figure 4;

CYP2C19 x 3 mutant ARMS primer markers preferably CY5 markers, results of which are shown in figure 5;

CYP2C19 × 17 mutant ARMS primer marker is preferably TET marker, and the test result is shown in figure 6.

Thirdly, designing, optimizing and screening a quenching probe sequence, and specifically comprising the following steps:

1) according to the sequence of the probe ARMS primer, a series of quenching probes with different lengths are designed to be completely complementary, and BHQ2 labeling is carried out at the 3' end, and the specific conditions are shown in the following table.

TABLE 6 quenching Probe marker sequences

2) CYP2C19 x 2 wild type and mutant probe ARMS primers are respectively mixed with quenching probes, downstream primers and PCR-premix buffer solution with different lengths to prepare PCR reaction solution, and 5000 copy/mu l of CYP2C19 x 2 wild type plasmid and mutant plasmid are detected; CYP2C19 x 3 wild type and mutant probe ARMS primers are respectively mixed with quenching probes, downstream primers and PCR-premix buffer solution with different lengths to prepare PCR reaction solution, and 5000 copy/mu l CYP2C19 x 3 wild type plasmids and mutant plasmids are detected; CYP2C19 x 17 wild type and mutant probe ARMS primers are respectively mixed with quenching probes, downstream primers and PCR-premix buffer solution with different lengths to prepare PCR reaction solution, and 5000 copy/mu l CYP2C19 x 17 wild type plasmids and mutant plasmids are detected; the reaction parameters were set as follows:

TABLE 7 PCR reaction parameters

3) Analysis of results

CYP2C19 x 2 preferably amplifies quenching probes with relatively small Ct value and relatively high fluorescence signal value, and the test result is shown in FIG. 7;

CYP2C19 x 3 preferably amplifies quenching probes with relatively small Ct value and relatively high fluorescence signal value, and the test result is shown in FIG. 8:

CYP2C19 × 17 preferably amplifies a quenching probe with a relatively small Ct value and a relatively high fluorescence signal value, and the test results are shown in FIG. 9.

The final preferred quenching probe sequence is as follows:

CYP2C19 x 2 quench probe 5 '-CCCATAACAAATTACTTAAAA-BHQ 2-3';

CYP2C19 x 3 quench probe 5 '-AGGTAAGGCCAAGTTTTTT-BHQ 2-3';

CYP2C19 × 17 quenching probe 5 '-GAGAACAGAAGACACAAATT-BHQ 2-3'.

Example 2 primer preparation and kit Assembly

First, synthesis and fluorescence labeling of the probe ARMS primer, the quenching probe and the downstream primer

Primer synthesis and fluorescence labeling are carried out according to the optimized screened probe ARMS primers, quenching probes, downstream primers and labels in example 1, wherein the labels are specifically marked as CYP2C19 x 2 wild type probe ARMS primer 5 ' end labeled FAM, CYP2C19 x 2 mutant probe ARMS primer 5 ' end labeled TET, CYP2C19 x 2 quenching probe 3 ' end labeled BHQ2 and CYP2C19 x 2 downstream primer; the 5 ' end of the CYP2C19 × 3 wild-type probe ARMS primer is marked with ROX, the 5 ' end of the CYP2C19 × 3 mutant probe ARMS primer is marked with CY5, the 3 ' end of the CYP2C19 × 3 quenching probe is marked with BHQ2 and a CYP2C19 downstream primer; 5 ' end of CYP2C19 × 17 wild type probe ARMS primer is marked with FAM, 5 ' end of CYP2C19 × 17 mutant probe ARMS primer is marked with TET, 3 ' end of CYP2C19 × 17 quenching probe is marked with BHQ2, and CYP2C19 × 17 downstream primer.

The specific sequence is as follows:

CYP2C19 x 2 wild type probe ARMS primers:

5′-FAM-TTTTAAGTAATTTGTTATGGGTTGCC-3′SEQ ID NO.1；

CYP2C19 × 2 mutant probe ARMS primers:

5′-TET-TTTTAAGTAATTTGTTATGGGTGCCT-3′SEQ ID NO.2；

CYP2C19 × 2 quench probe:

5′-CCCATAACAAATTACTTAAAA-BHQ2-3′SEQ ID NO.3；

CYP2C19 × 2 downstream primer:

5′-AATTTTCCCACTATCATTGATTATTTCC-3′SEQ ID NO.4；

CYP2C19 × 3 wild-type probe ARMS primers:

5′-ROX-AAAAAACTTGGCCTTACCTGAATC-3′SEQ ID NO.5；

CYP2C19 × 3 mutant probe ARMS primers:

5′-CY5-AAAAAACTTGGCCTTACCTGTATT-3′SEQ ID NO.6；

CYP2C19 × 3 quench probe:

5′-AGGTAAGGCCAAGTTTTTT-BHQ2-3′SEQ ID NO.7；

CYP2C19 × 3 downstream primer:

5′-GAAAACATCAGGATTGTAAGCACC-3′SEQ ID NO.8；

CYP2C19 × 17 wild-type probe ARMS primers:

5′-FAM-AATTTGTGTCTTCTGTTCTCAGAGC-3′SEQ ID NO.9；

CYP2C19 × 17 mutant probe ARMS primers:

5′-TET-AATTTGTGTCTTCTGTTCTCAAGGT-3′SEQ ID NO.10；

CYP2C19 × 17 quench probe:

5′-GAGAACAGAAGACACAAATT-BHQ2-3′SEQ ID NO.11；

CYP2C19 × 17 downstream primer:

5′-GCGCATTATCTCTTACATCAGAG-3′SEQ ID NO.12。

second, PCR mixed liquor preparation for gene polymorphism detection

1. The PCR mixed solution for amplifying CYP2C19 x 2 and CYP2C19 x 3 comprises CYP2C19 x 2 wild type probe ARMS primer, CYP2C19 x 2 mutant probe ARMS primer, CYP2C19 x 2 quenching probe, CYP2C19 x 2 downstream primer, CYP2C19 x 3 wild type probe ARMS primer, CYP2C19 x 3 mutant probe ARMS primer, CYP2C19 x 3 quenching probe and CYP2C19 x 3 downstream primer, the sequences are SEQ ID NO. 1-SEQ ID NO.8, PCR buffer solution, dNTP, Mg2+ and Taq enzyme, and the specific components are as follows.

TABLE 8 PCR mixture Components and amounts of CYP2C19 x 2 and CYP2C19 x 3

2. The PCR mixed solution for amplifying CYP2C19 x 17 is prepared from CYP2C19 x 17 wild type probe ARMS primer, CYP2C19 x 17 mutant probe ARMS primer, CYP2C19 x 17 quenching probe, CYP2C19 x 17 downstream primer with the sequence of SEQ ID NO. 9-SEQ ID NO.12, PCR buffer solution, dNTP, Mg2+ and Taq enzyme. The concrete components are as follows:

TABLE 9 PCR mixture composition and content of CYP2C19 x 17

Components	Content (wt.)
		PCR buffer solution	1×
dNTP	150μM
		Mg2+	2mM
Taq enzyme	1U
		CYP2C19 x 17 wild-type probe ARMS primer	500nM
CYP2C19 x 17 mutant-type ARMS primer	500nM
		CYP2C19 x 17 quenching probe	1.5μM
CYP2C19 x 17 downstream primer	500nM
		Deionized water	To 18. mu.l

3. And preparing a negative quality control product, namely preparing a conventional buffer solution containing Tris-HCl (pH8.0).

4. The positive quality control product is prepared from CYP2C19 x 2 wild-type plasmid, CYP2C19 x 2 mutant plasmid, CYP2C19 x 3 wild-type plasmid, CYP2C19 x 3 mutant plasmid, CYP2C19 x 17 wild-type plasmid and CYP2C19 x 17 mutant plasmid, and comprises the following specific components:

TABLE 10 wild type plasmid Components

Species of	Number of copies
		CYP2C19 x 2 wild-type plasmid	2000 copies
CYP2C19 x 2 mutant plasmids	2000 copies
		CYP2C19 x 3 wild-type plasmid	2000 copies
CYP2C19 × 3 mutant plasmid	2000 copies
		CYP2C19 x 17 wild-type plasmid	2000 copies
CYP2C19 × 17 mutant plasmid	2000 copies

Thirdly, assembling the kit

The kit comprises 4 reagents: PCR mixed liquor used for amplifying CYP2C19 x 2 and CYP2C19 x 3, PCR mixed liquor used for amplifying CYP2C19 x 17, a negative quality control product and a positive quality control product comprise the following specific components:

TABLE 11 kit compositions

PCR for amplification of CYP2C19 x 2, CYP2C19 x 3Mixed solution	450μl
		PCR mixture for amplifying CYP2C19 x 17	450μl
Negative quality control product	100μl
		Positive quality control product	100μl

Example 3 detection sensitivity

The kit a for detecting the polymorphism of the human CYP2C19 gene by using the shared primer probe prepared in the example 2 is compared with the kit b for detecting the polymorphism of the CYP2C19 gene by using the ARMS primer + TaqMan probe and the kit C for detecting the polymorphism of the CYP2C19 gene by using the MGB probe, and the specific comparison test steps are as follows:

preparing a sensitivity template, wherein the sensitivity template comprises CYP2C19 x 2 homozygous wild type 2 ng/mu l, 1 ng/mu l, 0.5 ng/mu l, CYP2C19 x 2 homozygous mutant type 2 ng/mu l, 1 ng/mu l, 0.5 ng/mu l, CYP2C19 x 2 heterozygous type 2 ng/mu l, 1 ng/mu l and 0.5 ng/mu l; CYP2C19 x 3 homozygous wild type 2ng/μ l, 1ng/μ l, 0.5ng/μ l, CYP2C19 x 3 homozygous mutant type 2ng/μ l, 1ng/μ l, 0.5ng/μ l, CYP2C19 x 3 heterozygous type 2ng/μ l, 1ng/μ l, 0.5ng/μ l; CYP2C19 x 17 homozygous wild type 2ng/μ l, 1ng/μ l, 0.5ng/μ l, CYP2C19 x 17 homozygous mutant type 2ng/μ l, 1ng/μ l, 0.5ng/μ l, CYP2C19 x 17 heterozygous type 2ng/μ l, 1ng/μ l, 0.5ng/μ l;

step two, preparing a kit a, a kit b and a kit c according to the instruction, adding a template, repeating 20 reaction holes, uniformly mixing, and placing in a fluorescent quantitative PCR instrument;

step three, setting reaction parameters, which are specifically shown in the following table:

TABLE 12 PCR reaction parameters

Step four, analyzing the detection results as shown in the following table

TABLE 13 results of sensitivity detection

The kit a for detecting the human CYP2C19 gene polymorphism based on the shared primer probe, the kit b for detecting the CYP2C19 gene polymorphism based on the ARMS primer + TaqMan probe, and the kit C for detecting the CYP2C19 gene polymorphism based on the MGB probe are used for detecting sensitivity templates of 2 ng/mul and 1 ng/mul, wherein the detection rates are all 100%, and when 0.5 ng/mul with lower concentration is detected, the kit a for detecting the human CYP2C19 gene polymorphism based on the shared primer probe has higher detection rate, so that the kit prepared by the shared primer probe has higher detection sensitivity.

Example 450 sample assays

The human CYP2C19 gene polymorphism detection kit prepared in example 2 is used for detecting 50 venous whole blood samples to be detected, and the detection steps are as follows:

step one, sample processing: the outsourcing whole blood genome DNA extraction kit is used for extracting nucleic acid DNA from 50 samples, OD260/280 is between 1.8 and 2.0, OD260/230 is between 1.8 and 2.2, and the concentration is more than 5 ng/mu l;

step two, preparation of a detection reagent: the PCR mixed solution for amplifying CYP2C19 x 2 and CYP2C19 x 3 and the PCR mixed solution for amplifying CYP2C19 x 17 prepared in example 2 are balanced to room temperature, inverted and mixed uniformly, 18 mul of PCR mixed solution is respectively taken to a PCR reaction tube, then 2 mul of nucleic acid DNA sample purified in the step one, 2 mul of negative quality control product and 2 mul of positive quality control product are added, mixed uniformly and placed in a fluorescent quantitative PCR instrument;

setting reaction parameters as follows:

TABLE 14 PCR reaction parameters

Step four, analyzing the detection result:

1. the results interpretation criteria are given in the following table:

TABLE 15 results interpretation criteria

Note: positive indicates that there is an S-type amplification curve; negative means no sigmoidal amplification curve

2. The results of 50 sample analyses were analyzed according to the results interpretation criteria, as shown in the following table:

test results of 50 samples in tables 16

3. The results of third party sequencing of 50 samples are as follows and are used as a result reference.

Third party sequencing results for 50 samples in tables 17

	Homozygous wild type	Heterozygote type	Homozygous mutant
				CYP2C19*2	28	15	7
CYP2C19*3	40	7	3
				CYP2C19*17	47	2	1

Therefore, compared with the third-party sequencing result, the analysis results of the 50 samples to be tested have the coincidence rate of 100%. The above results show that: the kit designed by the invention can be used for detecting the polymorphism of the human CYP2C19 gene, the result is accurate and reliable, the specificity is strong, and the sequencing detection effect is achieved; meanwhile, the method is simple and convenient to operate, short in detection time, simple in result analysis and low in cost.

The above description of the specific embodiments of the present application is not intended to limit the present application, and those skilled in the art may make various changes and modifications according to the present application without departing from the spirit of the present application, which is intended to fall within the scope of the appended claims.

Sequence listing

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Claims

1. A method for preparing a probe ARMS primer for detecting gene polymorphism is characterized by comprising the step of designing a wild type ARMS primer and a mutant type ARMS primer in the same direction aiming at a wild type locus and a mutant type locus, wherein the last base at the 5' ends of the wild type ARMS primer and the mutant type ARMS primer is marked by different reporter groups.

2. The method for preparing a probe ARMS primer for detecting gene polymorphism according to claim 1, characterized in that: the method further comprises the design of a quenching probe and a downstream primer, wherein the quenching probe is used for designing a complementary nucleotide sequence aiming at the wild probe ARMS primer and the mutant probe ARMS primer and marking a non-fluorescence quenching group at the 3' end of the complementary nucleotide sequence; preferably, the number of bases of the quenching probe is 3 to 9 less than the number of bases of the wild-type probe ARMS primer and the mutant probe ARMS primer.

3. The method for preparing a probe ARMS primer for detecting gene polymorphism according to any one of claims 1-2, characterized in that: further comprising the introduction of mismatched bases of the wild type probed ARMS primer and the mutant probed ARMS primer near the 3' ends of the wild type probed ARMS primer and the mutant probed ARMS primer; preferably, the wild type probe ARMS primer and the mutant probe ARMS primer have different mismatched bases introduced at the same sequence position, and the mismatched bases introduced at different sequence positions can be the same or different; more preferably, the mismatched bases are introduced into the 2 nd to 4 th bases at the 3' ends of the wild type probe ARMS primer and the mutant type probe ARMS primer, and the Tm values of the wild type probe ARMS primer and the mutant type probe ARMS primer are 65 to 70 ℃.

4. The method for preparing a probe ARMS primer for detecting gene polymorphism according to any one of claims 1 to 3, characterized in that: the gene polymorphism detection is directed at CYP2C 19; preferably, the compounds are directed against CYP2C19 x 2, CYP2C19 x 3; more preferably, CYP2C19 x 17 is also targeted.

5. The method for preparing a probe ARMS primer for detecting gene polymorphism according to claim 4, characterized in that: the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 2 is marked with FAM fluorophore, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 2 is marked with TET fluorophore, and the 3 ' end of the quenching probe of CYP2C 19X 2 is marked with BHQ2 quencher; the 5 ' end of the CYP2C 19X 3 wild-type probe ARMS primer is marked with a ROX fluorescent group, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 3 is marked with a CY5 fluorescent group, the 3 ' end of the quenching probe of CYP2C 19X 3 is marked with a BHQ2 quenching group, and the nucleotide sequences of the wild-type probe ARMS primer, the mutant probe ARMS primer, the quenching probe and the downstream primer of CYP2C 19X 2 are respectively shown in SEQ ID NO. 1-4; the nucleotide sequences of the wild probe ARMS primer, the mutant probe ARMS primer, the quenching probe and the downstream primer of the CYP2C19 x 3 are respectively shown as SEQ ID NO. 5-8; preferably, the 5 ' end of the wild-type probe ARMS primer of CYP2C 19X 17 is marked with FAM fluorophore, the 5 ' end of the mutant probe ARMS primer of CYP2C 19X 17 is marked with TET fluorophore, and the 3 ' end of the quenching probe of CYP2C 19X 17 is marked with BHQ2 quencher; the sequences of the wild-type probe ARMS primer, the mutant probe ARMS primer, the quenching probe and the downstream primer of CYP2C19 x 17 are respectively shown as SEQ ID NO. 9-12.

6. A method for detecting gene polymorphism based on shared primer probe comprises the following steps: the method comprises the primer preparation method of any one of claims 1 to 5, and further comprises a sample processing step, a detection system preparation step, a reaction parameter setting step, a result interpretation and analysis step; preferably, in the detection system preparation step, the concentrations of the probe ARMS primer and the downstream primer are between 400 and 600nM, and the concentration of the quenching probe is between 1.2 and 1.8 mu M; more preferably, the amplification parameters of the reaction parameter setting step are pre-denaturation at 95 ℃ for 3 minutes for 1 cycle; denaturation at 95 ℃ for 15 seconds, denaturation at 60 ℃ for 1 minute, collection of fluorescence signals, and 40 cycles; the fluorescence signal acquisition channel is provided with FAM, TET, ROX and CY5.

7. A primer set for detecting gene polymorphism, characterized in that: the primer set is prepared by the method of any one of claims 1 to 5.