实时荧光定量PCR检测CYP2C9* 3及VKORC1_C1173T基因多态性方法的建立  

Establishment of Quantitative Real-time PCR for Detecting CYP2C9* 3 and VKORC1_C1173T Genes Polymorphism

作　　者：顾梦月 张希根 张楚麟 康楠楠[1] 陈楚圆 刘煜[1] 

机构地区：[1]中国药科大学生命科学与技术学院,江苏南京211198 [2]南京仁天生物科技有限公司,江苏南京211500

出　　处：《药物生物技术》2016年第3期195-200,共6页Pharmaceutical Biotechnology

基　　金：江苏高校品牌专业建设工程资助项目(No.PPZY2015A057);江苏省重点高校学术程序开发资助项目(PAPD)

摘　　要：建立实时荧光定量PCR法检测人药物代谢基因CYP2C9*3及VKORC1_C1173T多态性,并确定最适反应条件。该方法采用Taq Man-MGB(Minor groove binder,小沟结合物)探针技术,针对每个待检测位点设计特定的引物和探针,并优化引物、探针浓度;建立和优化反应体系,设计合理的PCR Enhancer组分;并对Taq ManMGB探针特异性、敏感性和重复性进行评价,同时对部分实时荧光定量PCR产物样品进行测序验证。结果表明,Taq Man-MGB探针法对2个等位基因检测结果准确,检测方法特异性高;所构建方法线性关系良好(R2≥0.991),扩增效率为95.2%~109.9%,标准曲线斜率为-3^-3.5;检测灵敏度可达10拷贝/反应;随机样品Taq ManMGB探针检测结果与测序结果一致。所建立用于基因多态性检测的Taq Man-MGB探针法具有简便、快速、准确、高效等特点,检测原理可用于各种SNP分型检测,具有广泛的应用前景。This study focused on establishing a method of TaqMan-MGB quantitative real-time PCR assay for the detection of CYP2C9 ＊ 3 and VKORC1_C1173T polymorphism. Specific primers and TaqMan Minor groove binder （TaqMan-MGB） probes were designed, and the rapid real-time PCR assay was established and optimized. The specificity, sensitivity and repetitiveness of TaqMan- MGB quantitative real-time PCR assay were evaluated, and the PCR products were also subjected to gene sequence analysis to validate the results of TaqMan-MGB real-time PCR. Detection of CYP2C9 ＊ 3 and VKORCl_C1173T polymorphism was specifically and accurately finished by TaqMan-MGB quantitative real-time PCR. The relative coefficients of the quantitative standard curve were above 0.991 and the amplification efficiencies of the quantitative standard curve were 95.2% - 109.9%. The slopes of the quantita- tive standard curve were - 3 - - 3.5 and the sensitivity of detection reached 10 copies per reaction. The results of random sample in TaqMan-MGB probe genotyping were consistent with the results of gene sequence analysis. For the purpose of SNPs detection, the established TaqMan-MGB assay was an accurate, simple and rapid method. And it is also an available method to assess the effect of SNPs on drug pharmacokinetics.

关 键 词：细胞色素P450 2C9 维生素K环氧化物还原酶复合体1 基因多态性 华法林 TAQMAN-MGB探针 实时荧光定量PCR 

分 类 号：Q503[生物学—生物化学]