MTHFR C677T基因多态性方法学比较
A Comparison of Detection Methods of MTHFR C677T Gene Polymorphism
刘紫薇 1王玉飞 1刘淑静 1姜文灿 1李斯文 1王利娟 1陈柯霖1
作者信息
- 1. 首都医科大学附属北京天坛医院实验诊断中心,国家药监局体外诊断试剂质量控制重点实验室,北京市免疫试剂临床工程技术研究中心,北京 100070
- 折叠
摘要
目的 评价比较实时荧光定量PCR法与PCR-金磁微粒层析法两种方法检测5,10-亚甲基四氢叶酸还原酶(MTHFR)C677T基因多态性的一致性、检测限、特异性.方法 随机选取首都医科大学附属北京天坛医院采集的50例静脉抗凝全血,均通过磁珠法提取基因组DNA,依照最新版分子诊断检验程序性能验证指南(CNAS-GL039)要求对两种方法学的一致性、检测限、特异性进行性能评价.结果 实时荧光定量PCR与PCR-金磁微粒层析具有高度的一致性,采用两种方法检测50例样本的一致性为100％;两种方法检测交叉反应中检测结果未受影响,基因型之间无交叉污染情况;检测限验证中前者的灵敏度更高,最低检测限可达0.078125ng/μL,后者的最低检测限为0.625ng/μL.结论 实时荧光定量PCR法与PCR-金磁微粒层析法均具有操作简便、灵敏度高,特异性强的特点,可用于人类MTHFR基因多态性检测,同时能够对叶酸代谢作出有效的诊断,需根据临床实验室的需求,选择合理有效的检测方法.
Abstract
Objective To evaluate and compare the consistency,detection limit and specificity of 5,10-methylenetetrahydrofolate reductase(MTHFR)C677T gene polymorphism by using real-time fluorescence quantitative PCR method and PCR-gold magnetic chromatography.Methods A total of 50 cases of whole blood samples were collected from Beijing Tiantan Hospital affiliated to Capital Medical University,which were then randomly selected to extract genomic DNA by paramagnetic particle method.The consistency,detection limit and specificity of these two methods were evaluated according to the requirements of the latest version of Guidance on the Performance Verification for Molecular Diagnostic Procedures(CNAS-GL039).Results The comparison of Real-time fluorescence quantitative PCR method and PCR-gold magnetic chromatography showed a high consistency in results,and the consistency rate of 50 samples detected by two methods was 100％.The cross-reactivity of two methods had no effect on the results,and there was no cross contamination detected between genotypes.During the detection limit verification,the former method had a higher sensitivity and the minimum detection limit was up to 0.078125 ng/μL,while the minimum detection limit of the latter method was 0.625ng/μL.Conclusion Real-time fluorescence quantitative PCR method and PCR-gold magnetic chromatography have promising advantages,including simple operation,high sensitivity,strong specificity,which can be better applied to the detection of human MTHFR gene polymorphism and effectively diagnose folic acid metabolism.In summary,our study suggests that reasonable and effective detection methods should be selected according to specific needs of clinical laboratories.
关键词
5,10-亚甲基四氢叶酸还原酶/实时荧光定量PCR法/PCR-金磁微粒层析/方法学比较Key words
5,10-methylenetetrahydrofolate reductase/Real-time fluorescence quantitative PCR method/PCR-gold magnetic chromatography/Methodological comparison引用本文复制引用
基金项目
北京市科学技术协会青年人才托举工程(BYESS2022170)
出版年
2024
NETL
NSTL
万方数据