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 VKORC1-1693 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.%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.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201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.
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