华法林相关维生素K环氧化物还原酶复合体亚单位1及细胞色素P450 2C9基因型快速检测的方法研究  

The efficient method for detecting vitamin K epoxide reductase complex subunit 1 and cytochrome P450 2C9 after the treatment of warfarin.

作　　者：李杰[1] 刘双[1] 郭伟[1] 陈勇[1] 高元明[1] 

机构地区：[1]首都医科大学附属北京安贞医院呼吸科北京市心肺血管疾病研究所,100029

出　　处：《中国医药》2013年第4期472-474,共3页China Medicine

基　　金：北京市科技计划课题（Z101107050210044）

摘　　要：目的建立一种快速、准确、简便、经济的检测维生素K环氧化物还原酶复合体亚单位1（VKORC1）和细胞色素P450 2C9（CYP2C9）基因型的方法。方法提取220例肺栓塞患者外周血的DNA，针对2个基因的SNP位点设计特异性引物，并进行荧光聚合酶链反应（PCR），根据荧光PCR的熔解曲线分析样本的基因型。同时对样本进行传统的限制性酶切片段长度多态性（PCR—RFLP）基因分型，从时间、通量、费用、准确性等方面对2种方法进行比较。结果VKORC1 1693G〉A和CYP2C91 075A〉C两个SNP位点的不同基因型具有不同的熔解曲线峰型。220例样本中，VKORC1基因AA型184例，AG型34例，GG型2例，各占83．6％、15．5％、0．9％；CYP2C9基因＊1／＊1型加1例，＊1／＊3型19例，各占91．4，％、8．6％，无＊3／＊3型纯合子。PCR-RFLP检测结果与上述结果基本相同。2种方法相比，前者较后者操作步骤少，用时少，费用相当，结果更准确。结论利用荧光PCR检测VKORC1及CYP2C9的SNP分型操作简便、快速、经济、准确，适用于临床实验室开展基因分型。Objective To establish a rapid, accurate, simple and economical method for detecting gene polymorphisms of vitamin K epoxide reductase complex subunit 1 （VKORC1） and cytochrome P450 2C9 （CYP2C9）. Methods The DNA extracted from the peripheral blood from 220 patients with pulmonary embolism were examined. The specific primers were designed regarding of the two gene SNP sites, and the real tiem PCR was performed. According to the fluorescence PCR melting curve, sample genotypes were analyzed. At the same time, the genotypes of these samples were detected by the traditional restriction enzyme fragment length polymorphism （ PCR - RFLP）. The expense of the time, the cost and the accuracy of two methods were compared. Results Different genotypes of VKORCl-1693 G 〉 A and CYP2C9 1075 A 〉 C has different melt curve peak patterns. Among 220 samples, there were 177 cases VKORC1 AA of 34 cases, AG 2cases GG, which represente 83.6%, 15.5%, 0. 9% respectively; CYP2C9 ＊ 1/＊ 1 201 cases, 19 cases ＊ 1/＊ 3 type, each accounted for 91.4 % , 8.6%, no ＊ 3/＊ 3 homozygote. The testing results of PCR - RFLP were basically the same with the former. Comparing the two kinds of methods, the PCR method need less operation steps, spent less time, cost equal, and the results are more accurate than the latter. Conclusion Fluorescence PCR is the fast, simple, economic and accurate method to detect the genotypes of VKORC1 and CYP2C9 which is suitable for the genotyping in clinical laboratory.

关 键 词：荧光聚合酶链反应 维生素K环氧化物还原酶 细胞色素P450 2C9 

分 类 号：R332[医药卫生—人体生理学]