Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

Abstract

Background: The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR.

Methods: Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay.

Results: We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2 h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits.

Conclusion: We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.