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。
Objective To evaluate the precision,accuracy and limit of detection of real-time fluorescencepolymerase chain reaction(PCR)for cytochrome P4502C19(CYP2C19)polymorphisms.Methods DNAsamples with different CYP2C19genotypes(*1/*1,*1/*2,*1/*3and*1/*17)were determined for10times in1run for evaluating precision.The results of real-time fluorescence PCR and Sanger sequencing were used and comparedfor evaluating accuracy.The DNA samples with different genotypes(*1/*1,*1/*2,*1/*3and*1/*17)were dilutedin a2-fold serial pattern and determined by real-time fluorescence 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 ofdetection of1.875ng/μL and accuracy of100%(20/20)compared to the results of Sanger sequencing,including6kinds of CYP2C19genotypes in20cases(1case of*1/*17,3cases of*1/*1,5cases of*1/*2,5cases of*1/*3,4cases of*2/*2and2cases of*2/*3).Conclusions Real-time fluorescence PCR is precise,accurate and suitablefor CYP2C19polymorphisms.
作者
陈丹
徐婷
张洁心
赵鸿
戎国栋
潘世扬
王芳
黄珮珺
CHEN Dan;XU Ting;ZHANG Jiexin;ZHAO Hong;RONG Guodong;PAN Shiyang;WANG Fang;HUANG Peijun(Department of Clinical Laboratory,the First Affiliated Hospital of Nanjing Medical University/the National Key Clinical Department of Laboratory Medicine,Nanjing 210029,Jiangsu,China)
出处
《检验医学》
CAS
2017年第9期801-805,共5页
Laboratory Medicine
基金
江苏省实验诊断学重点实验室基金项目(XK201114)
