PCR-金磁微粒层析法在叶酸代谢相关基因检测中的应用研究

Application of PCR-gold magnetic particle chromatography in detection of genes related to folic acid metabolism

目的:对PCR-金磁微粒层析法检测人叶酸代谢相关基因进行方法学评价,并初步应用于临床检测。方法:同时应用PCR-金磁微粒层析法和Sanger测序法两种方法分别检测221例临床样本的5,10-亚甲基四氢叶酸还原酶(MTHFR)C677T、A1298C位点及甲硫氨酸合成酶还原酶(MTRR)A66G位点的基因型,并对两种方法检测的结果进行比较,以评估PCR-金磁微粒层析法的准确性、灵敏度及特异性。结果:两种方法检测同一批样本的结果完全一致,准确性达100%。PCR-金磁微粒层析法的灵敏度高,对MTHFR C677T、A1298C位点的最低检测限分别为2.5、1.25 ng·μl^-1,MTRR A66G位点的最低检测限为1.25 ng·μl^-1;特异性强,扩增试剂的扩增反应呈高度专一性。结论:PCR-金磁微粒层析法检测人叶酸代谢相关基因快速、简便,结果准确,适合应用于各级具有PCR实验室资质的医疗机构。

Objective: To evaluate the method of PCR-gold magnetic particle chromatography for detecting the folic acid metabolism-related genes, and initially apply it to clinical testing. Methods: The genotypes of 5,10-methylenetetrahydrofolate reductase(MTHFR C677 T,A1298 C) and methionine synthetase reductase(MTRR A66 G) from 221 clinical samples were detemined by the method of PCR-gold magnetic particle chromatography and Sanger sequencing, and then the results of two different detection methods were compared to evaluate the accuracy, sensitivity and specificity of PCR-gold magnetic particle chromatography. Results: The results of testing from the same batch detected by two ways were completely consistent with 100% accuracy. The sensitivity of PCR-gold magnetic particle chromatography was high, the minimum detection limits for MTHFR C677 T and A1298 C sites were 2.5 ng·μl^-1 and 1.25 ng·μl^-1, respectively, the minimum detection limit of MTRR A66 G site was 1.25 ng·μl-1;Besides, its specificity was strong and the amplification reaction was highly specific. Conclusion: PCR-gold magnetic particle chromatography for detecting the folic acid metabolism-related genes has many advantages, such as rapid analysis, easy operation, accurate and stable, suitable for large-scale clinical samples, which can be highly applicable to all levels of medical institutions with PCR lab.