SYBR GreenⅠ实时荧光PCR检测VKORC1和CYP2C9基因型

Detection of VKORC1 and CYP2C9 genotypes by SYBR Green Ⅰ-based real-time PCR

目的建立一种检测维生素K环氧化物还原酶复合体亚单位1(VKORC1)和细胞色素P450 2C9(CYP2C9)基因型的SYBR GreenⅠ实时荧光PCR法。方法提取134例心脏瓣膜置换术患者的外周血基因组DNA。针对VKORC1 1173C>T和CYP2C9 1075A>C两个单核苷酸多态性(SNP)位点,分别设计一套等位基因特异性引物。PCR反应体系中加入荧光染料SYBR GreenⅠ,样本经扩增后结合熔解曲线分析,确定每个样本的基因型。结果 VKORC1 1173C>T和CYP2C9 1075A>C两个SNP位点的不同基因型具有不同的熔解曲线峰型。134例患者中,VKORC1 1173C>T基因型TT、TC和CC分别有111例、21例和2例,各占82.8%、15.7%和1.5%;CYP2C9 1075A>C基因型*1/*1和*1/*3分别有121例和13例,各占90.3%和9.7%,未检出纯合子*3/*3基因型。结论基于荧光染料SYBR GreenⅠ的实时荧光PCR分型方法操作简便、价格低廉、结果准确可靠,适于临床实验室对VKORC1 1173C>T和CYP2C9 1075A>C的基因分型。

Objective To establish a SYBR Green Ⅰ-based real-time PCR assay for detecting gene polymorphisms of vitamin K epoxide reductase complex subunit 1（VKORC1） and cytochrome P450 2C9（CYP2C9）.Methods Genomic DNA of peripheral blood samples from 134 patients with heart valve replacement surgery was extracted.A set of allele-specific PCR primers was designed for each single nucleotide polymorphism（SNP） of VKORC1 C1173C〉T and CYP2C9 1075A〉C.The fluorescent dye of SYBR Green Ⅰ was added into the PCR reaction system,and the genotype of each sample was determined by melting curve analysis after amplification.Results Each genotype of VKORC1 1173C〉T and CYP2C9 1075A〉C had its respective melting curve patterns.Among the 134 patients,the cases of TT,TC and CC genotype of VKORC1 1173C〉T were 111（82.8%）,21（15.7%） and 2（1.5%）,and the cases of ＊1/＊1 and ＊1/＊3 genotype of CYP2C9 1075A〉C were 121（90.3%） and 13（9.7%）,respectively.But homozygote ＊3/＊3 genotype was not detected.ConclusionThe SYBR Green Ⅰ-based real-time PCR assay is convenient,inexpensive,accurate and will be useful for VKORC1 1173CT and CYP2C9 1075AC genotyping in a clinic laboratory.