Primer probe composition and kit for detecting polymorphism of human CYP2C19 gene and application

A technology of CYP2C19 and gene polymorphism, applied in the field of molecular biology, can solve the problems of certain sample quantity requirements, easy pollution of amplification products, and difficult optimization of primer design, achieving high sensitivity and accuracy, effective It is beneficial to clinical operation and avoids the effect of mutual interference

Active Publication Date: 2018-09-04
SHANDONG VIGENE BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the most commonly used sequencing method can directly detect the location and type of the mutation site, but the method has cumbersome operation steps, a long detection cycle, and the amplification product is prone to contamination; the chip method involves gene-specific amplification, hybridization, detection, etc. High-throughput analysis can be carried out in one step, but the cost is high, the detection steps are complicated, and there are certain requirements fo

Method used

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  • Primer probe composition and kit for detecting polymorphism of human CYP2C19 gene and application
  • Primer probe composition and kit for detecting polymorphism of human CYP2C19 gene and application
  • Primer probe composition and kit for detecting polymorphism of human CYP2C19 gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0088] Embodiment 1 The clinical application of detection kit of the present invention

[0089] 1. Design of specific primers and fluorescent probes for polymorphic sites of CYP2C19 gene

[0090] According to the dbSNP database (https: / / www.ncbi.nlm.nih.gov / snp / term=) published by NCBI (National Center for Biotechnology Information) ), CYP2C19*3 (rs4986893, c.636G>A) and CYP2C19*17 (rs12248560, c.-806C>T) SNP site information, design multiple specific primers for CYP2C19 gene polymorphism sites And specific fluorescent probes, specific primers are designed at both ends of the mutation site of the detected gene, and the specific fluorescent probe covers the mutation site of the detected gene. After a large number of experimental screening, the final product with high sensitivity and good specificity Three pairs of specific primers for amplifying CYP2C19*2, CYP2C19*3, and CYP2C19*17 and specific fluorescence for detecting CYP2C19*2, CYP2C19*3, and CYP2C19*17 Probes CYP2C19*2-P...

Embodiment 2

[0145] Example 2 Verification of CYP2C19 gene polymorphism detection results

[0146] The 250 clinical samples to be tested in Example 1 were sequenced using the gold standard sequencing method while using the above primers and probes for qPCR testing. The results showed that the genotype obtained by the gold standard sequencing method for each sample to be tested was consistent with The genotypes obtained by the method in Example 1 were consistent, with 0 missed detection and an accuracy of 100%.

[0147] The minimum detection limit of the primer and probe composition of the present invention is detected, and the steps are as follows:

[0148] 1. Randomly select 10 cases of DNA samples extracted in Example 1, and dilute their concentrations to 1ng / ul, 0.5ng / ul, and 0.25ng / ul respectively. The DNA templates of 3 concentrations of each sample were loaded into 3 replicate PCR wells respectively, and the loading volume was 4ul, and the minimum detection limit test was carried ou...

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Abstract

The invention discloses a primer probe composition and kit for detecting the polymorphism of a human CYP2C19 gene and application. The primer probe composition comprises three pairs of specific primers for amplifying CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites and three specific fluorescent probes. The primers and the probes have high sensitivity, strong specificity and strong anti-interference capability; the polymorphism of the gene is detected by adopting a manner combining asymmetric PCR (Polymerase Chain Reaction) amplification and a fluorescent probe melting curve analysis technology; different gene types can be effectively distinguished according to the quantity of melting peaks and Tm value and result interpretation is convenient, clear and objective. Single-tube sampling can be usedfor detecting 6 mutation types of 3 gene sites at the same time; the operation is simple and convenient and the detection efficiency is improved; a large batch of samples can be detected and clinicaloperation is facilitated.

Description

technical field [0001] The present invention relates to a primer, probe, kit and application for detecting human CYP2C19 gene polymorphism, in particular to a primer for detecting human CYP2C19 gene *2, *3, *17 polymorphism The probe composition, kit and application belong to the technical field of molecular biology. Background technique [0002] Clopidogrel (trade name: Plavix) is a novel thienopyridine derivative that irreversibly inhibits platelet aggregation by selectively binding to the ADP receptor coupled to adenylate cyclase on the surface of platelets. Clopidogrel is currently the most commonly used antiplatelet drug in the world because it can effectively reduce the risk and mortality of diseases such as myocardial infarction, ischemic stroke and acute coronary syndrome, and has good safety. drug. However, 4% to 40% of patients will develop resistance to clopidogrel, leading to adverse cardiovascular events, and the incidence of adverse clinical events in Asian p...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2537/143C12Q2563/107C12Q2547/101C12Q2545/113
Inventor 孙秀莲苏桂锋李轶群
Owner SHANDONG VIGENE BIOSCI
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