MTHFR基因多态性检测试剂盒的性能验证

The Performance Verification of SKY Kit for MTHFR Polymorphisms

目的对人亚甲基四氢叶酸还原酶MTHFR基因多态性检测试剂盒通过实时荧光聚合酶链式反应(PCR)检测MTHFR基因多态性的准确性、精密度和检测限进行验证。方法利用实时荧光PCR对20例标本的MTHFR基因多态性进行检测,将结果与测序(金标准)结果进行比较,以验证准确性;采用杂合型DNA标本重复扩增10次,以验证精密度;将杂合型DNA标本稀释至检测限(10ng/μL)下的浓度进行检测,重复3次,以验证检测限。结果20例标本的MTHFR基因多态性检测结果,与其测序结果完全符合,符合率为100%(20/20);重复扩增10次的分型检测结果与实际基因型完全相符,且所有通道的CT值变异系数CV≤5%;稀释到检测限下的三次重复实验均能准确进行分型。结论该人MTHFR基因多态性检测试剂盒通过PCR方法进行的基因分型结果稳定、可靠,适用于医学实验室对MTHFR基因多态性的检测。

Objective To verify the accuracy,precision and detection limit of real-time fluorescence polymerase chain reaction (PCR) for Methylene tetrahydrofolate reductase polymorphism.Methods 20 specimens were detected for the study,and the results of real time fluorescence PCR and sequencing were compared for evaluating the accuracy.Heterozygous DNA specimens were determined 10 times for evaluating the precision.Heterozygous DNA specimens were diluted to the detect limit and determined for evaluating the limit of detection.Results The PCR results of 20 specimens were in complete agreement with the sequencing results and the coincidence rate was 100%(20/20).The results of the amplification from 10 times of measurements were fully consistent with the actual genotype,and the CV of all CT values were less than 5%.The diluted specimens were all accurately typed.Conclusion This MTHFR gene polymorphism detection kit is stable and reliable by PCR method and suitable for the detection of MTHFR gene polymorphism in medical laboratory.