亚甲基四氢叶酸还原酶基因多态性荧光PCR检测方法的建立与应用

Establishment and application of fluorescent PCR for detection of methylenetetrahydrofolate reductase gene polymorphism

目的建立检测人亚甲基四氢叶酸还原酶(MTHFR)基因多态性荧光PCR的检测方法。方法用Primer Premier 5.0和Primer Express 3.0软件针对人MTHFR基因677多态性位点分别设计1对引物和2条TaqMan-MGB探针,通过GenBank在线比对分析评估引物的特异性,优化建立荧光PCR反应体系。以2个检测通道的循环阈值(Ct值)差值判断检测位点的基因型。评价该方法的最低检测限、准确度和精密度。用该方法检测300例临床样本,用核酸序列测定法对准确度进行评价。结果建立的荧光PCR法对MTHFR 677位点检测具有较高的准确度,最低检测限到达2 ng/反应。300例临床样本的检测结果与测序法的检测结果的一致性为100%。结论建立的MTHFR基因多态荧光PCR检测方法具有很好的准确度和精密度,适用于临床精准预测脑卒中发生风险和预后及指导孕妇个体化补充叶酸。

Objective To establish a fluorescent PCR method for detecting the polymorphism of human methylenetetrahydrofolate reductase(MTHFR)gene.Methods Primer Premier 5.0 and Primer Express 3.0 software were used to design a pair of primers and two TaqMan-MGB probes for the 677 polymorphic locus of human MTHFR gene,respectively.The specificity of the primers was evaluated by GenBank on-line comparison analysis,and the establishment of fluorescent PCR reaction system was opptimized.The genotype of the locus was determined by the difference of cycle threshold(CT)values of two detection channels.The minimum detection limit,accuracy and precision of the method were evaluated.A total of 300 clinical samples were detected by this method,and the accuracy was evaluated by nucleic acid sequencing.Results The established fluorescent PCR method had high accuracy in detecting MTHFR 677 locus,and the minimum detection limit was 2 ng/response.The consistency between the results of 300 clinical samples and those of sequencing was 100%.Conclusion The established fluorescent PCR assay for MTHFR gene polymorphism has good accuracy and precision.It can be used to accurately predict the risk and prognosis of stroke and to guide pregnant women to individualize folic acid supplementation.