A probe directed recombinase amplification assay for detection of MTHER A1298C polymorphis associated with congenital heart disease

Single nucleotide polymorphisms (SNPs) play an important role in susceptibility to complex dis eases, treat.ment efficacy and adverse drug responses. Conventional methods to detect SNPs are usually based onPCR or DNA sequencing, which are typically time-consuming and require sophisticated equipment. in thisproof-of-concept study, a probe-directed recombinase amplification (PDRA) assay was developed to detectthe A1 298C polymorphism of 5, 10-methylenetetrahydrofolate reductase (MTHFR). The PDRA assay includ.ed two real-time reactions to detect the A and C nucleotides of A1298C polymorphism. Each reaction con-tained only one primer and one probe and was finished at 39'C within 35 min. The results of genotyping of1 50 clinical samples using PDRA were completely consistent with those by direct sequencing. Additionally,when the 1000 Genomes Project HCB freauencies were used as the control group, MTHFR A1298C wasfound to be associated with congenital heart disease. in condusion, the proposed novel PDRA assay isa valuable tool for the detection of SNPs and demonstrates significant potential to be widely applicable inboth research and clinical settings.

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