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Clopidogrel drug-resistant gene detection method based on multiple HRM analysis

A technology of clopidogrel and drug-resistant genes, applied in the field of molecular biology, can solve the problems of low detection accuracy and achieve the effects of reduced reagent consumption, high accuracy, and shortened detection time

Active Publication Date: 2017-05-10
SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the accuracy of clinical medical detection is very important, and the high-throughput detection based on HRM technology often has low detection accuracy due to the large number of primers in the same reaction system and other reaction components. It is necessary to detect primers and methods for clopidogrel resistance gene with high accuracy

Method used

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  • Clopidogrel drug-resistant gene detection method based on multiple HRM analysis
  • Clopidogrel drug-resistant gene detection method based on multiple HRM analysis
  • Clopidogrel drug-resistant gene detection method based on multiple HRM analysis

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 Clopidogrel resistance gene detection method of the present invention

[0024] Material

[0025] Rotor-Gene Q real-time fluorescent quantitative PCR analyzer (Qiagen), rTaq enzyme (Takara), Syto9 fluorescent dye (Invitrogen), primers (Shanghai Sangon Biology), human genomic DNA samples (extracted from percutaneous coronary intervention surgery) patient).

[0026] method

[0027] 1. Primer design: For five SNP loci related to clopidogrel resistance: rs4244285, rs4986893, rs12248560, rs1045642 and rs662, design five pairs of PCR amplification primers, so that the amplicon generated by each pair of primers contains Corresponding SNP loci. Among them, in order to increase the Tm value of the rs4986893 amplicon and separate it from the melting peak of the rs12248560 amplicon on the HRM curve, a GC sequence (underlined part) was added to the 5' end of the rs4986893 primer. The primer sequences are listed in Table 1.

[0028]2. PCR amplification system: the a...

Embodiment 2

[0037] Example 2 Verification of the accuracy of the clopidogrel-resistant gene detection method of the present invention

[0038] The clopidogrel resistance gene detection method of the present invention is used to detect 316 samples of unknown genotypes. All the samples were from consecutive patients who underwent percutaneous coronary intervention for chest tightness and chest pain in Renji Hospital from April to December 2015 and needed to be given dual antiplatelet therapy of aspirin and clopidogrel, 77 of which were female, and 239 The sample was male with an age range of 35 to 94 years. The detection results were compared with the Sanger sequencing method, and the detection accuracy rates of rs4244285, rs4986893, rs12248560, rs1045642, and rs662 were 100%, 99.37%, 99.37%, 100%, and 100%, respectively, and the accuracy rates were significantly higher than conventional Level.

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Abstract

The invention relates to a clopidogrel drug-resistant gene detection method based on multiple HRM analysis. A primer in the method is shown as SEQ ID NO: 1-10. The invention further provides a primer combination related to HRM analysis and a kit. The clopidogrel drug-resistant gene detection method has the advantages that a method of HRM typing aiming at multiple SNP sites is established, three sites of rs4244285, rs4986893 and rs12248560 and / or two sites of rs1045642 and rs662 can be detected in a same reaction system, so that reagent consumption is reduced, and detection time is shortened. The method is used to detect samples of patients receiving percutaneous coronary interventional angiography, and a Sanger sequencing method is used for verification, so that accuracy is high.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for detecting clopidogrel drug-resistant genes based on multiplex HRM analysis. Background technique [0002] Clopidogrel is used to prevent thrombotic events in patients with coronary heart disease after percutaneous coronary intervention (PCI). In clinical application, 4% to 30% of patients cannot achieve the expected antiplatelet effect in conventional dose therapy, and severe cases may lead to cardiovascular adverse events such as myocardial infarction, thrombosis, and death. This phenomenon is called clopidogrel resistance ( Clopidogrel resistance, CR). Clopidogrel is a prodrug that is inactive in vitro and is derivatized into thiol metabolites through enzymatic reactions to exert clinical efficacy. The genes related to drug transformation mainly include ABCB1, which affects drug absorption, and liver drug-metabolizing enzyme genes, which affect metaboli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/106C12Q2600/156C12Q2563/107C12Q2545/114C12Q2531/113C12Q2527/107
Inventor 傅启华张立辰张晓青游国岭杨海鸥
Owner SHANGHAI CHILDRENS MEDICAL CENT AFFILIATED TO SHANGHAI JIAOTONG UNIV SCHOOL OF MEDICINE
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