阅读说明：本技术 用于确定氯吡格雷用药疗效相关的基因snp检测试剂盒 (Gene SNP detection kit for determining related therapeutic effect of clopidogrel medication ) 是由 秦胜营 张素丽 朱金行 张娜 贺林 于 2019-09-11 设计创作，主要内容包括：本发明公开了一种用于确定氯吡格雷用药疗效相关的基因SNP检测试剂盒,所述试剂盒包括MassARRAY芯片、检测基因P2Y12基因的rs16863356,rs7634096和rs12497330 3个SNP位点各三条引物分别称为正向引物,反向引物以及延伸引物,其序列依次分别如SEQ ID No.1～9,其中引物是用于鉴别氯吡格雷药效和不良反应相关基因的多态性位点。本发明的试剂盒可对上述三个位点进行高通量检测,具有灵敏度高、特异性高、检测结果稳定、可靠性高等优点；同时,适用于临床疾病突变检测,药物基因组学分析、法医学鉴定等基因分析领域。(The invention discloses a gene SNP detection kit for determining clopidogrel medication curative effect correlation, which comprises a MassARRAY chip, three primers of rs16863356, rs7634096 and rs 124973303 SNP loci for detecting gene P2Y12, wherein the three primers are respectively called as a forward primer, a reverse primer and an extension primer, the sequences of the primers are respectively shown as SEQ ID No. 1-9, and the primers are polymorphic loci for identifying clopidogrel drug effect and adverse reaction correlation genes. The kit can carry out high-throughput detection on the three sites, and has the advantages of high sensitivity, high specificity, stable detection result, high reliability and the like; meanwhile, the method is suitable for the gene analysis fields of clinical disease mutation detection, pharmacogenomics analysis, forensic medicine identification and the like.)

1. A primer group for detecting gene polymorphism sites related to clopidogrel medication curative effect comprises:

detecting a PCR primer with the sequence of rs16863356 shown as SEQ ID No. 2 and SEQ ID No. 5 and an extension primer with the sequence shown as SEQ ID No. 8 in the peripheral blood DNA;

detecting a PCR primer with the sequence of rs7634096 shown as SEQ ID No.1 and SEQ ID No. 4 and an extension primer with the sequence shown as SEQ ID No. 7 in peripheral blood DNA;

detecting the PCR primer with the sequence of rs12497330 in the peripheral blood DNA as shown in SEQ ID No. 3 and SEQ ID No. 6 and the extension primer with the sequence as shown in SEQ ID No. 9.

2. A kit for SNP detection of related genes of clopidogrel personalized medicine, comprising the primer set of claim 1.

3. The kit of claim 2, further comprising a detection reagent, a detection chip, and a detection carrier.

4. The kit of claim 3, wherein the detection reagents comprise a PCR mix reaction, an SAP enzyme mix reaction, and a UEP extension mix reaction.

5. The kit of claim 3, further comprising a resin for purification, a target for spotting and mass spectrometric detection, and a human genomic DNA extraction reagent.

6. A method for using the kit for SNP detection of clopidogrel personalized medicine related genes according to claim 2 for non-diagnostic purposes, characterized in that it comprises the following steps:

s1, performing PCR primer dilution and UEP extension primer dilution by using the primer group;

s2, PCR reaction;

s3, SAP digestion reaction;

s4, UEP extension reaction;

s5, purifying resin.

7. The use of the method according to claim 6, wherein in step S2, the PCR mixture solution used in the PCR reaction comprises deionized water, PCR Buffer, MgCl2, dNTPs, heat-resistant Taq DNA polymerase, and amplification primers mix.

8. The use of claim 6, wherein in step S3, the SAP digestion reaction uses an SAP enzyme mix reaction solution comprising deionized water, SAP Buffer, and SAP enzyme.

9. The use method according to claim 6, wherein in step S4, the UEP extension reaction uses UEP extension mix reaction solution containing deionized water, Gold Buffer, Termination mix, UEP primer mix and high temperature resistant extension enzyme.

10. The use of claim 6, wherein in step S5, the purification resin is dropped into the single base extension product obtained in step S4.

Technical Field

The invention belongs to the technical field of genes, and relates to a detection kit for determining gene polymorphism (SNP) related to the therapeutic effect of clopidogrel administration.

Background

Clopidogrel (Clopidogrel) is chemically (S) -alpha- (2-chlorophenyl) -6, 7-dihydrothieno [3, 2-C)]Pyridine-5 (4H) -acetic acid methyl esterMolecular formula C₁₆H₁₆ClNO₂And S. Clopidogrel is a novel and widely used first-choice medicament for treating cardiovascular and cerebrovascular diseases, particularly acute coronary syndrome and percutaneous coronary intervention. Meanwhile, clopidogrel has certain characteristics on the structural characteristics and functions as a thienopyridine antiplatelet medicament, and clinical adverse events have certain advantages compared with other anticoagulants, so the clopidogrel is considered as the antiplatelet medicament which is considered to be important in the treatment process of cardiovascular and cerebrovascular diseases. In recent years, intensive research and clinical observation show that adverse clinical events still exist in the anti-platelet treatment of patients with cardiovascular and cerebrovascular diseases after receiving regular clopidogrel medication, wherein the most common clinical events comprise myocardial infarction, cerebral apoplexy, thrombus in a stent, even death and the like.

Cardiovascular and cerebrovascular diseases are relatively complex chronic diseases, which are the result of interaction of non-genetic factors (age, height, weight, interaction of drugs, dietary habits and the like) such as environmental factors and a plurality of factors such as genetic factors, wherein the genetic factors play a core leading role in the process to cause individual differences, and the individual differences are gene mutations of proteins or enzymes related to pharmacokinetics and pharmacodynamics of clopidogrel, effective protein expression and enzyme activity, so that the curative effect of clopidogrel is enhanced or reduced. Therefore, in order to achieve the optimal treatment effect of the drug, namely, to improve the drug effectiveness or reduce the occurrence of adverse drug reactions, the deep excavation of the drug effect of the clopidogrel and the biomarkers related to the adverse reactions is more and more important, and the social health problem to be solved is also formed.

The clopidogrel is firstly absorbed by oral administration into intestinal tracts, the whole process is accompanied with P-glycoprotein (ABCB1 gene coding) transportation, the finally activated metabolite is directionally selected to a certain extent to be directly and irreversibly combined with a platelet surface ADP receptor P2Y12 by utilizing the activation effect of cytochrome enzyme CYP450, so that the aim of blocking the activity enhancement effect of platelets is fulfilled, the subsequent activation of ADP mediated GP IIb/IIIa compound is further inhibited, and the activation and aggregation of the platelets are finally inhibited. In the search of the existing literature, the P2Y12 gene is located on the human chromosome 3 q25.1 and is used as the final binding receptor in the clopidogrel metabolic process, namely the polymorphism of the drug target P2Y12 gene, but the related reports that P2Y12rs16863356, rs7634096 and rs12497330 are clopidogrel drug effect molecular markers are not found in China.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a gene SNP detection kit for determining the related curative effect of clopidogrel medication; in particular to a method for detecting 3 gene polymorphic sites of P2Y12 gene related to therapeutic effect and adverse reaction of clopidogrel drugs by utilizing a multiplex PCR technology, a single base extension technology and a mass spectrum technology and a corresponding kit.

The purpose of the invention is realized by the following technical scheme,

in a first aspect, the present invention relates to a primer set for detecting a gene polymorphism site related to clopidogrel medication efficacy, comprising:

detecting a PCR primer with the sequence of rs16863356 shown as SEQ ID No. 2 and SEQ ID No. 5 and an extension primer with the sequence shown as SEQ ID No. 8 in the peripheral blood DNA;

detecting a PCR primer with the sequence of rs7634096 shown as SEQ ID No.1 and SEQ ID No. 4 and an extension primer with the sequence shown as SEQ ID No. 7 in peripheral blood DNA;

detecting the PCR primer with the sequence of rs12497330 in the peripheral blood DNA as shown in SEQ ID No. 3 and SEQ ID No. 6 and the extension primer with the sequence as shown in SEQ ID No. 9.

The corresponding molecular weights of the extension primers and the extension primers at each site are shown in table 1 below:

extension primer	Site of the body	Molecular weight of extended primer
			SEQ ID No:7	rs7634096	6106
SEQ ID No:8	rs16863356	7369.8
			SEQ ID No:9	rs12497330	8526.6

The primer is designed on a gene typing MassARRAY platform, and is more efficient, accurate and high-flux compared with the prior art.

In a second aspect, the invention relates to a kit for SNP detection of related genes of clopidogrel personalized medicine, which comprises the primer set.

As one embodiment of the kit, the kit further comprises a detection reagent, a detection chip and a detection carrier.

The detection reagent comprises a PCR mixed reaction solution, an SAP enzyme mix reaction solution and a UEP extension mix reaction solution.

As an embodiment of the kit, the kit also comprises a resin for purification, a target sheet for spotting and mass spectrometry and a human genome DNA extraction reagent.

In a third aspect, the invention also relates to a non-diagnostic purpose use method of the kit for carrying out SNP detection on related genes of clopidogrel personalized medication, which comprises the following steps:

s1, performing PCR primer dilution and UEP extension primer dilution by using the primer group;

s2, PCR reaction;

s3, SAP digestion reaction;

s4, UEP extension reaction;

s5, purifying resin.

In step S2, the PCR mixed reaction solution adopted in the PCR reaction includes deionized water, PCR Buffer, MgCl2, dNTPs, thermostable Taq DNA polymerase, and amplification primer mix.

In step S3, the SAP enzyme mix reaction solution used in the SAP digestion reaction includes deionized water, SAP Buffer, and SAP enzyme.

In step S4, the UEP extension mix reaction solution used in the UEP extension reaction includes deionized water, Gold Buffer, Termination mix, UEP primer mix, and high temperature resistant extension enzyme.

In step S5, the resin for purification is dropped into the single-base-charged extension product obtained in step S4.

Compared with the prior art, the invention has the following beneficial effects:

1. the invention integrates the technologies of multiplex PCR, single base extension, mass spectrum detection and the like, can detect trace samples while amplifying a detection template, and has very high detection sensitivity;

2. single base extension is also called micro sequencing, and uses a specific probe to identify DNA, so that the method has the advantages of good specificity, low false positive and the like;

3. the high-throughput sequencing technology has high speed and high efficiency, and can complete hundreds of sample detections in 3-4 hours;

4. the operation is simple, and meanwhile, the pollution is reduced under the automatic operation;

5. the invention can detect a plurality of patients and can obtain different gene locus information at the same time;

6. the invention overcomes the defect of high cost of detecting a small amount of SNP loci at one time in the prior art;

7. the invention lays a foundation for researching the relation between the polymorphism of the P2Y12 gene of the population in China and the safety of clinical medication, provides a theoretical basis for clinical individualized medication, and provides a guiding basis for the research and development of new drugs based on the pharmacogenomics concept.

Detailed Description

The present invention will be described in detail with reference to examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally following conventional conditions, such as molecular cloning by Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations.