叶酸代谢能力评估及亚甲基四氢叶酸还原酶基因多态性检测试剂盒性能验证

Evaluation of folate metabolism ability and performance validation of methylenetetrahydrofolate reductase gene polymorphism detection kit

目的对江西诺德医疗器械有限公司生产的叶酸代谢能力测定试剂盒进行性能验证,评估该试剂盒能否满足江苏省常熟地区患者对亚甲基四氢叶酸还原酶(MTHFR)基因A1298C(rs1801131)及C677T(rs1801133)两个多态性位点的检测需求。方法收集10例临床样本进行测序,将结果作为“金标准”,同时采用实时荧光聚合酶链反应(PCR)对标本的MTHFR基因多态性位点进行检测,与“金标准”结果比较,验证试剂盒的准确性。选择测序确定的C677T多态性位点CC及TT基因型标本各1份,A1298C多态性位点AA及CC基因型标本各1例,重复测定5次以验证试剂盒的精密度。同时对以上4份不同基因型的标本进行DNA浓度测定,根据试剂盒说明书中的性能描述,稀释至最低检出浓度5 mg/L,每例标本各检测3次,要求基因分型结果一致且符合测序分型结果,验证试剂盒的检测下限。结果所有标本的MTHFR基因多态性位点检测结果与测序结果完全一致,准确率达到100%;对不同基因型的4例标本反复检测5次的结果完全一致,且循环阈值(Ct值)的变异系数(CV)≤5%;而稀释到检测下限的4例标本经过3次重复检测后,仍能准确进行基因分型。结论采用MTHFR基因A1298C及C677T两个多态性位点检测试剂盒进行基因分型的结果准确、稳定、可靠,能满足实验室对MTHFR基因多态性检测的要求。

Objective To validate the performance of folate metabolism assay kit manufactured by Jiangxi Nordisk Medical Device Co.,Ltd.and assess its suitability for detecting two polymorphisms A1298C(rs1801131)and C677T(rs1801133)sites of methylenetetrahydrofolate reductase(MTHFR)gene in patients from Changsu area,Jiangsu Province.Methods The 10 clinical samples were sequenced to determine MTHFR gene polymorphisms as"gold standard".Real-time fluorescence polymerase chain reaction(PCR)was also used to detect the polymorphisms.The results were compared with the sequencing data of"gold standard"to verify the accuracy of the kit.Additionally,one case each of CC and TT genotypes for C677T polymorphism site and AA and CC genotypes for A1298C polymorphism site were selected based on sequencing results.The determination was repeated for five times to confirm the precision of the kit.At the same time,DNA concentration measurements were performed on the four samples with different genotypes.According to the performance description in the reagent kit manual,the samples were diluted to the minimum detection concentration of 5 mg/L.Each sample was tested for three times,and the genotyping results were required to be consistent with the sequencing results.The detection lower limit of the reagent kit was verified.Results The results obtained from all samples regarding MTHFR gene polymorphism were completely consistent with sequencing data,demonstrating an accuracy rate of 100%.The results of repeated test for five times on four specimens of different genotypes were completely consistent,and the coefficient of variation(CV)of the cycle threshold(Ct value)was≤5%.The four samples diluted to the lower limit of detection were still able to accurately genotype after repeated tests for three times.Conclusion The results of genotyping using MTHFR gene A1298C and C677T polymorphism sites detection kits are accurate,stable and reliable,which could meet the laboratory's requirements for MTHFR gene polymorphism detection.