基于TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法  

Detection of three polymorphic sites of CYP2C19 gene*2,*3,and*17 based on TaqMan-MGB probe technology

作　　者：卓召振 靳倩 张亮亮 匡颖[1] 令狐克燕 ZHUO Zhaozhen;JIN Qian;ZHANG Liangliang;KUANG Ying;LINGHU Keyan(Department of Medical Genetics,Guizhou Provincial People’s Hospital,Guizhou Province,Guiyang 550002,China)

机构地区：[1]贵州省人民医院医学遗传科,贵州贵阳550002

出　　处：《中国医药导报》2023年第7期32-36,共5页China Medical Herald

基　　金：贵州省科技计划项目(黔科合成果-LC[2021]032)。

摘　　要：目的建立TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法。方法针对CYP2C19基因*2、*3和*17位点设计合成相应引物及双标记探针。选取2021年3月至2022年6月贵州省人民医院心内科门诊已知基因型的剩余全血样本42例,覆盖每个位点的每个基因型各10例,提取DNA进行聚合酶链反应(PCR)检测,产物进行电泳和Sanger法测序分析。加样1、10、100、800 ng DNA,进行PCR检测以评价灵敏度。每个位点的每个基因型5例,每周1次对其检测,连续3周,以评价重复性。结果设计的引物扩增出目的基因片段,电泳显示明亮的单一的DNA条带;90个基因型的PCR结果与测序结果完全一致(P>0.05);不同加样量PCR法均可准确判读基因型,所有基因型在1 ng水平的Ct值均<34。同一基因型3次重复检测的结果一致。结论本研究成功建立了TaqMan-MGB探针检测CYP2C19基因*2、*3和*17三个多态性位点的方法,具有快速、准确、灵敏、简便的特点。Objective To establish a method for detection of the three polymorphic sites*2,*3,and*17 of CYP2C19 based on TaqMan-MGB probe technology.Methods Corresponding primers and double-labeled probes were designed for*2,*3 and*17 sites of CYP2C19 gene.The remaining whole blood samples from the outpatient Department of Cardiology,Guizhou Provincial People’s Hospital from March 2021 to June 2022 with interested genotype were selected.The 42 samples containing 10 genotypes at each site were collected and the whole blood genomic DNA was extracted by polymerase chain reaction(PCR)detection,followed by electrophoresis and Sanger sequencing.1,10,100,800 ng DNA was added and PCR detection was conducted to evaluate the sensitivity.Five cases of each genotype of each locus were detected once a week for three consecutive weeks,to evaluate the repeatability.Results The designed primers successfully amplified the target gene fragment and the electrophoresis showed a bright single DNA band;the PCR results of 90 genotypes were consistent with the sequencing results(P>0.05);the genotypes could be accurately identified by PCR when different amount of DNA was added and the Ct values of all genotypes at 1 ng level were less than 34.The results of three repeated tests for the same genotype were consistent.Conclusion This study successfully establish a TaqMan-MGB probe to detect the three polymorphic sites,*2,*3,and*17 of CYP2C19 gene.The method is fast,accurate,sensitive and simple.

关 键 词：TAQMAN-MGB探针 CYP2C19 单核苷酸多态性 细胞色素酶P450 

分 类 号：R446.9[医药卫生—诊断学]