焦磷酸测序法检测ALDH2*2和MTHFR 677基因多态性的性能验证

Performance verification of pyrosequencing for detecting ALDH2*2 and MTHFR 677 gene polymorphism

目的对实验室自建焦磷酸测序法检测乙醛脱氢酶2(ALDH2)*2和亚甲基四氢叶酸还原酶(MTHFR)677基因多态性的方法进行性能评价。方法对不同浓度(10.0、2.0、0.4、0.0ng/μL)的杂合型DNA标本进行检测,评价方法的检出限;采用经焦磷酸测序法验证的DNA标本的PCR产物各20例,与Sanger测序法进行比较,评价方法的准确度;对3种(野生、杂合和突变型)基因型标本重复检测4次,评价方法的重复性。结果焦磷酸测序法检测ALDH2*2和MTHFR 677多态性的检出限为2ng/μL基因组DNA;焦磷酸测序法的分型结果与Sanger测序法一致,准确度为100%;批内重复4次检测结果完全一致。结论焦磷酸测序法检测ALDH2*2和MTHFR 677基因多态性稳定可靠,满足江苏省临床检验中心的要求,可用于临床检测。

Objective To evaluate performance verification of laboratory developed pyrosequencing for detecting ALDH22 and MTHFR 677 gene polymorphism.Methods Different concentration of heterozygous DNA samples(10.0,2.0,0.4 and 0.0 ng/μL)were detected to evaluate the detection limit.Separated 20 PCR products genotyped by pyrosequencing were used to compare with Sanger sequencing to evaluate the accuracy.Samples with three genotypes(wild type,heterozygous type and mutant type)were detected for 4 times for evaluating precision.Results The detection limit of pyrosequencing detecting ALDH22 and MTHFR 677 polymorphism was 2 ng/μL genomic DNA.The results of pyrosequencing were in consistent with Sanger sequencing,so the accuracy was 100%.The detection results in one run for 4 times were completely same.Conclusion Pyrosequencing is a stable and reliable method for detecting ALDH22 and MTHFR 677 polymorphism,which satisfies with the needs of Jiangsu Center for Clinical Laboratory and is suitable for clinical detection.