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

Xu Yeqiong; Zhao Yilin; Gao Manqiu

doi:10.3969/j.issn.1674-7151.2024.01.019

Abstract：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.