Multiplex one-step direct asymmetric PCR of blood and dual-labelled probe-mediated melting curve for genotyping of MTHFR and MTRR polymorphisms.

Accurate, rapid, and multiplex SNP analysis holds significant clinical value. However, the inevitable nucleic acid extraction, involving centrifugation, heating, and magnetic separation, is often time-consuming. In this study, direct blood PCR was combined with dual-labelled probe-mediated melting curves to identify SNPs corresponding to MTHFR (C677T, rs#1801133 and A1298C, rs#1801131) and MTRR (A66G, rs#1801394) in a single tube. Our results indicated that nucleic acid extraction does not enhance gDNA concentrations. The results of the gDNA and whole blood samples were perfectly aligned with each other. The maximum volume tolerance for whole blood is 10%. Our method demonstrated robustness to hemolysis and temperature variations. The turnaround time of the test is <100 min with great potential value in clinical settings. Furthermore, this approach eliminates the need for complex preprocessing steps, simplifies the workflow and reduces potential sources of error. The efficiency and accuracy of this method make it a promising tool for routine clinical diagnostics and personalized medical applications. Additionally, the method's reliability and speed are crucial for effective patient management. The ability to obtain results quickly and accurately supports timely treatment decisions, which can be critical in many clinical scenarios. As a result, this technique has the potential to be widely adopted in clinical laboratories due to its practicality and effectiveness.