Single probe PCR melting curve analysis MTHFR C677T SNP sites

Abstract

BackgroundThe detection and analysis of methylene tetrahydrofolate reductase (MTHFR) C677T single nucleotide polymorphism (SNP) from blood samples is time-consuming and costly. We aimed to establish a method to detect these SNPs by direct whole blood PCR and without DNA extraction. MethodsProbes modified by different fluorescent groups on the same sequence were designed. Various MTHFR genotypes from direct blood PCR experiments were used to verify the similarity of the obtained and sequencing results. The SNP sites adjacent to the MTHFR C677T SNP were used to verify whether the method can accurately distinguish these sites. ResultsThe ROX probe was found to be the most suitable for this study. We tested 291 samples with 1 μL whole blood as a template, and obtained 126, 43, and 122 cases of C677C, C677T, and C677 C/T genotypes, respectively. The melting curve was consistent with the sequencing results. The detection limit was approximately 1000 white blood cells/μL. Through PCR and the melting curve method, the adjacent sites were accurately distinguished. ConclusionWe established a reliable, simple, rapid, and low-cost direct blood PCR method for the detection of MTHFR C677T SNPs. This could also be used as a potential diagnostic tool for a variety of diseases.