Tetra-primers ARMS-PCR with GoldMag Lateral Flow Assay for Genotyping: A Simultaneous Visual Detection of Both Alleles

Rapid and simple detection of single nucleotide polymorphism (SNP) is vital for individualized diagnosis and eventual treatment in current clinical setting. In this study, we developed a tetra-primers ARMS-PCR with lateral flow assay (T-ARMS-LFA) for simultaneous visual detection of two alleles. By using four primers labeled with digoxin, biotin and Cy5 separately in one PCR reaction, the amplified allele-specific products could be captured by streptavidin and anti-Cy5 antibody on two separated test lines of LFA strip, which allows presentation of both alleles within single LFA strip. Both DNA and whole blood can be applied as templates to this genotyping method with whole detection process within 75 minutes. The performance assay of T-ARMS-LFA demonstrates accuracy, specificity and sensitivity of this method. One hundred of human whole blood samples were used for MTHFR C677T genotyping with T-ARMS-LFA. The concordance rate of the results detected was up to 100% when compared with the sequencing results. Collectively, this newly developed method is highly applicable for SNP screening in clinical practices.