应用焦磷酸测序技术检测VKORC1和CYP2C9基因型  

Application of the pyrosequencing technique for detection of VKORC1 and CYP2C9 genotypes

作　　者：徐琴 黄盛文[2] 杨楠楠 王仕敏 罗振元[2] 安邦权[2] XU Qin;HUANG Shengwen;YANG Nannan;WANG Shimin;LUO Zhenyuan;AN Bangquan(School of Clinical Laboratory science,Guizhou Medical University,Guiyang,Guizhou 550004,China;Guizhou Provincial People′s Hospital,Guiyang,Guizhou 550002,China)

机构地区：[1]贵州医科大学医学检验学院,贵州贵阳550004 [2]贵州省人民医院检验科,贵州贵阳550002

出　　处：《国际检验医学杂志》2018年第7期769-772,共4页International Journal of Laboratory Medicine

基　　金：贵州省高层次创新型人才培养计划--"百"层次人才(黔科合平台人才[2016]5670号);贵阳市科技创新平台计划([20161001]35号)

摘　　要：目的建立基于焦磷酸测序技术的维生素K环氧化物还原酶复合物亚单位1(VKORC1)和细胞色素P450 2C9(CYP2C9)基因分型的方法,用于检测华法林药物代谢酶相关基因多态性。方法收集50份健康人外周血样本,提取全血基因组DNA。针对VKORC1-1639G>A、CYP2C9 430C>T和CYP2C91075A>C 3个单核苷酸多态性(SNP)位点,分别设计一套带生物素标记的扩增引物和测序引物。样本经PCR扩增后进行焦磷酸测序,结合信号峰型分析每个样本基因型,以Sanger测序法为对照,比较两种方法检测基因型结果的一致性。结果根据待测位点出峰的碱基和峰高,可以清晰判断3个SNP位点的基因型,50份样本中,VKORC1-1639G>A基因型AA和AG分别有41例和9例,各占82%和12%;CYP2C9基因型*1/*1和*1/*3分别有45例和5例,各占90%和10%,未检出等位基因CYP2C9*2。焦磷酸测序技术与Sanger测序法所检测的基因型结果一致。结论焦磷酸测序技术在SNP基因分型中具有操作简便、耗时短、成本低和结果准确可靠等优点,该检测方法可快速对CYP2C9和VKORC1的SNP位点进行分型。Objective To establish genotyping methods for vitamin K epoxide reductase complex subunit 1(VKORC1)and cytochrome P450 2C9(CYP2C9)based on pyrosequencing technique to detection of warfarin metabolizing enzyme related gene polymorphisms.Methods A total of 50 peripheral blood samples from healthy adults were collected and the whole blood genomic DNA was extracted.A set of biotin-labeled amplification primers and sequencing primers were designed respectively for three SNP sites:VKORC1-1639 G>A,CYP2C9 430C>T and CYP2C9 1075A>C.After PCR amplification of the samples,pyrophosphoric acid sequencing was conducted.And then the signal peaks form were combined to analyze and determine each sample genotype.Genotyping results were verified by Sanger sequencing,and the consistency of the two sequencing methods was compared.Results Genotypes of the three SNPs can be clearly determined according to the bases and height of the signal peaks.Among the 50 samples,there were 41 AA and nine AG for VKORC1-1639G>A,accounting for 82%and 12%respectively,and there were 45*1/*1,five*1/*3 for CYP2C9,accounting for 90%and 10%respectively,no CYP2C9*2 allele detected.Genotype results detected by pyrosequencing and Sanger sequencing were consistent with each other.Conclusion In SNP genotyping,Pyrosequencing has the advantages of convenience,time-saving,cheap with accurate and reliable results,which can quickly determine the genotypes of CYP2C9 and VKORC1.

关 键 词：焦磷酸测序 华法林 CYP2C9 VKORC1 单核苷酸多态性 

分 类 号：R596.2[医药卫生—内科学]