实时荧光PCR用于CYP2C19基因多态性检测的性能验证

摘要

目的 评价实时荧光聚合酶链反应(PCR)检测细胞色素P450家族成员2C19(CYP2C19)基因多态性的精密度、准确度和检测限.方法 采用实时荧光PCR对基因型为*1/*1、*1/*2、*1/*3和*1/*17的DNA样本分别作批内重复扩增10次,以评价方法的精密度;比较实时荧光PCR与Sanger测序法的检测结果,以评价方法的准确性;对2倍梯度稀释的DNA样本(基因型为*1/*1、*1/*2、*1/*3和*1/*17)进行实时荧光PCR扩增,以评价方法的最低检测限.结果 批内重复10次的分型检测结果完全一致,所有PCR的Ct值变异系数(CV)均<2.5％;实时荧光PCR的分型结果与Sanger测序法结果的符合率为100％(20/20),其中*1/*17型1例、*1/*1型3例、*1/*2型5例、*1/*3型5例、*2/*2型4例、*2/*3型2例;实时荧光PCR的最低检测限为1.875 ng/μL DNA.结论 实时荧光PCR的基因分型结果稳定、可靠,适用于医学实验室CYP2C19基因多态性的检测.%ObjectiveTo evaluate the precision,accuracy and limit of detection of real-time fluorescence polymerase chain reaction(PCR) for cytochrome P4502C19(CYP2C19) polymorphisms.MethodsDNA samples with differentCYP2C19 genotypes(*1/*1,*1/*2,*1/*3 and *1/*17) were determined for 10 times in 1 run for evaluating precision. The results of real-timefluorescence PCR and Sanger sequencing were used and compared for evaluating accuracy. The DNA samples with different genotypes(*1/*1,*1/*2,*1/*3 and *1/*17) were diluted in a 2-fold serial pattern and determined by real-timefluorescence PCR for evaluating limit of detection.Results Real-time fluorescence PCR showed low coefficient of variation(CV) of Ct values(<2.5％),a low limit of detection of 1.875 ng/μL and accuracy of 100％(20/20) compared to the results of Sanger sequencing,including 6 kinds ofCYP2C19 genotypes in 20 cases(1 case of *1/*17,3 cases of *1/*1,5 cases of *1/*2,5 cases of *1/*3, 4 cases of *2/*2 and 2 cases of *2/*3).ConclusionsReal-timefluorescence PCR is precise,accurate and suitable forCYP2C19 polymorphisms.