Multiplex detection of single nucleotide polymorphism loci of folate metabolism enzyme in extract-free samples with fluorescence labeled probe

Abstract

Objective To establish a method of detecting folic acid metabolism-related enzyme gene polymorphism using triple fluorescently labeled probe. Methods Specific amplification primers and fluorescently labeled probes were designed according to the sequence of MTHFR C677T, A1298C and MTRR A66G. Polymorphisms were detected by qPCR amplification process combined with probe melting curve analysis. Results A detection method with triple fluorescently labeled probe was established in this study. Wild, mutant and heterozygous types can be recognized based on melting curve Tm valuein a single tube.The method was optimized and validated in DNA samples, oral swabs and filter paper blood stain samples with extract-free method. Conclusion The SNP typing method for the key enzyme genes of folic acid metabolism has been established with high specificity, sensitivity, repeatability; the method is also high throughput without high quality template requirements. It could be a clinical guidance to folate supplementation for women of child bearing age, which is of great importance to the prevention of neonatal and cardiovascular diseases caused by folate metabolism abnormalities. Key words: Fluorescently labeled probe; Folic acid metabolism; Single nucleotide polymorphism; Melting curve analysis; Extracted-free sample; MTHFR; MTRR