Research on the Application of TaqMan-MGB Probe Method in Detecting MTHFR Gene Polymorphisms
10.13241/j.cnki.pmb.2025.16.004
- VernacularTitle:TaqMan-MGB探针法在MTHFR基因多态性检测中的应用研究
- Author:
Hong-xuan LIANG
1
;
Liang-hui CHEN
;
Xuan-yi ZHENG
;
Qiong-lu HUANG
;
Kang ZHANG
;
Qiu-ping YE
;
Ya-qun LIU
Author Information
1. 韩山师范学院广东省粤东药食资源功能物质与治未病研究重点实验室 广东潮州 521041;湖南师范大学生命科学学院 湖南长沙 410000
- Publication Type:Journal Article
- Keywords:
TaqMan-MGB probe;
MTHFR C677T;
MTHFR A1298C;
Gene polymorphism
- From:
Progress in Modern Biomedicine
2025;25(16):2598-2607
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To establish a TaqMan-MGB probe-based method for detecting the polymorphic loci C677T and A1298C of the MTHFR gene.Methods:Specific primers and TaqMan-MGB probes targeting the C677T and A1298C polymorphic loci of the MTHFR gene were designed and optimized based on the gene sequence information.A real-time quantitative PCR detection system was established.Gradient dilution experiments were conducted to determine the limit of detection,and reproducibility experiments were performed to evaluate detection consistency.Specificity was validated using wild-type and mutant plasmid templates.The method was applied to detect 56 clinical samples,and its accuracy and practicality were assessed through comparison with traditional Sanger sequencing.Results:The TaqMan-MGB probe method demonstrated high specificity for detecting the C677T and A1298C loci,with no cross-reactivity between wild-type and mutant probes,enabling accurate genotype differentiation.Sensitivity experiments revealed detection limits of 1.13 × 103 copies/μL for C677T and 8.39 × 101 copies/μL for A1298C.Reproducibility experiments showed coefficients of variation below 1％,indicating stable and reliable results.Among the 56 clinical samples,the overall detection rate for the C677T locus was 86.99％,and for the A1298C locus,it was 97.92％.The TaqMan-MGB method exhibited good concordance with Sanger sequencing results.Conclusion:The TaqMan-MGB method exhibits high specificity,sensitivity,and excellent reproducibility in detecting the polymorphic loci C677T and A1298C of the MTHFR gene,making it suitable for rapid detection in large-scale clinical samples.This method provides an effective molecular diagnostic tool for the early diagnosis and prevention of folate-related diseases.
