Genotyping MTHFR C677T By Direct ARMS-PCR Combine With Gold-magnetic Nanoparticle Based Lateral Flow Assay And Its Clinical Application

Studies in molecular genomics require a high-throughput, low cost, and reliable approach of biological sample collection and processing. PCR is usually performed on purified DNA. However, the extraction of DNA from whole blood or other samples is time consuming and involves the risk of contamination at every step. We described a novel approach to directly amplify genomic DNA from whole blood or buccal swabs in combination with GoldMag Nanoparticles-based nucleic acid lateral flow assay for Detection of MTHFRC677T. Methylenetetrahydrofolate reductase (MTHFR), MTHFR is folic acid and homocysteine (homocysteine, Hey) is one of the key metabolic enzymes. MTHFR binding capacity, activity and thermal stability of MTHFR, and genetic predisposition may be associated with neural tube defects, cardiovascular disease, cancers and pregnancy complications.Our laboratory has established a polymerase chain reaction-Gold-Magnetic nanoparticle-based lateral flow assay platform that enables rapid visual detection of genetic polymorphisms without instrumentation for genotyping. In this study, we developed a rapid method for genotyping the methylenetetrahydrofolate reductase C677T gene by using buccal swabs or whole blood without DNA extraction. The genotyping results were further validated by direct sequencing. This method is rapid, noninvasive, economical and reliable. It was successfully used for analysis of the MTHFR C677T gene polymorphism. Our quick and easy test method, and optimize health for whole blood spread before has not reached 5 ul, time for 1h 20min total augmentation, amplification for the visual results after 5 minutes, without professionals can interpretation result, convenient for operation and clinical application. Now for 200 cases of clinical blood samples, coincidence rate was 100%.50 cases of buccal swab samples, coincidence rate was 100%. This new method is highly applicable for C677T SNP screening in the laboratories and clinical practice; more promisingly, it could also be extended for the detection of SNPs of other genes associated with medical conditions or diseases. In conclusion, as compared to the existing protocols for gene detection. Our direct PCR technique is relatively novel, simple, requires minimal steps and eliminates the need for additional standardizations. Furthermore, it can also be extended for research on other gene polymorphisms.