Simultaneous Detection of Three Genotypes of Gene Methylene Tetrahydrofolate Reductase and Methionine Synthase Reductase Based on Multiplex Asymmetric Real-Time PCR-HRM Biosensing

Abstract

Genotyping of folate metabolism genes is of great importance in disease diagnosis and prevention. However, most current detection methods used for folate metabolism gene genotyping are based on sequencing and chips, which suffer from a high cost and laborious and time-consuming procedures. Herein, we reported a multiplex asymmetric PCR-HRM strategy for identifying genotypes of folate metabolism genes in a single tube. The proposed multiplex PCR-HRM assay has been successfully applied to identify the genotypes of folate metabolism genes, methylene tetrahydrofolate reductase (C677T, A1298C) and methionine synthase reductase A66G, on 1 μL of genomic DNA (gDNA) samples directly released from blood specimens, and the genotyping results were 100% consistent with the results of sequencing. The assay allows us to accurately detect the genotypes of gDNA with the detection limit down to 1 ng, which meets the clinical requirement. What is more, the capacity of resistance to aerosol pollution of the multiplex asymmetric PCR-HRM biosensing was first addressed and has been evaluated as it can withstand contamination of roughly 12.5-25% interfering nucleic acids. Because of the advantages of multiplex detection, high accuracy, and resistance to aerosol pollution and having no open tube procedure, this approach would pave the way for establishing a fast and cost-effective platform for folate metabolism gene genotyping and other SNP genotyping in clinical diagnostics.