Colorimetric loop-mediated isothermal amplification (cLAMP) assay for the genotyping of a thrombophilia genetic risk factor, MTHFR (C677T)

Abstract

Methylenetetrahydrofolate reductase (MTHFR) is an enzyme involved in regulating serum homocysteine and folate levels. A single nucleotide polymorphism (SNP) of MTHFR (rs1801133; C677T) is strongly associated with an increased risk of thrombophilia, heart attack, stroke, and dementia. Genotyping this SNP is vital; however, techniques need to be simplified. This study developed a colorimetric loop-mediated isothermal amplification (cLAMP) technique to genotype the C677T in thrombophilia-susceptible individuals. The results were compared with those of kompetitive allele-specific PCR (KASP) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays. The cLAMP assay successfully detected the C677T SNPs in the MTHFR gene. The results agreed with the KASP and PCR-RFLP results. The MTHFR-cLAMP assay is a simple, rapid, and affordable technique for colorimetric genotyping. This is the first study describing visual detection of a SNP associated with thrombophilia. The assay can also be used in screening other SNPs at low-resource centers and hospitals for point-of-care testing.