高分辨率熔解曲线法检测CBS基因SNP的技术优化

目的：探索高分辨率熔解曲线法（highresolutionmelting，HRM）检测胱硫醚β-合酶（CBS）基因SNP位点所需的最佳反应体系和条件，建立快速高效的对CBS基因分型的方法。方法通过普通PCR初步确立引物的退火温度范围，在实时荧光定量PCR（qPCR）-HRM反应中调整确定最佳退火温度。对影响qPCR-HRM的因素包括反应程序、模板DNA、MgCl2再分别进行优化，确立最佳反应体系和反应条件，对在此基础上得出的基因分型结果通过测序验证其准确性，从而建立系统的HRM检测CBS基因SNP的方法。结果HRM检测CBS基因该片段最佳退火温度为62℃，qPCR-HRM最佳反应体系为20μl，引物浓度为0．2μmol/L，Mg2＋为2．5μmol/L，模板DNA为60ng。在优化的体系条件下筛选出的突变型样本经测序验证与实验结果一致。结论HRM可以作为检测SNP准确、经济、高效的方法。

The Optimization of High Resolution Melting Analysis for SNP of CBS Gene Detection

Objective To explore the optimal reaction condition of high resolution melting （HRM）for detecting SNPs of Cystathionine Beta Synthetase（CBS）,and establish the fast and effective way of genotyping.Methods The annealing temperature range was firstly determined by common polymerase chain reaction,then optimal annealing tem-perature was adjusted by qPCR-HRM.The qPCR-HRM reaction system,including reaction process,DNA template,and MgCl2 concentration,were optimized to obtain the best result.Finally,the genotyping results were varified by Sanger sequencing.In this way,the qPCR-HRM of detecting CBS SNP was established.Results The optimal annealing tem-perature was 62℃;the best reaction system of qPCR-HRM includs primers 0.2μmol/L,Mg2＋ 2.5μmol/L,and DNA template 60 ng in 20μl.The results of HRM and direct sequencing were consistent.Conclusion HRM could serve as an accurate,fast,and effective method for detecting SNPs of genes.