Rapid SNP genotyping detection method based on PCR-lateral flow dipstick detection technique

This study established a polymerase chain reaction-lateral flow dipstick (PCR-LFD) method for the visual detection of SNP genotypes. Targeting the MC4R gene SNP g.732 C > G, highly specific primers were designed for the mutation site, incorporating a Locked Nucleic Acid (LNA) modification at the 3' terminal nucleotide of the SNP, a BIOTIN modification at the 5' end of the upstream primer, and a fluorescein isothiocyanate (FITC) modification at the 5' end of the downstream primer. The detection primers were used for PCR amplification with the sample, and the reaction system was optimized. The amplification products were subsequently detected using LFD. The results demonstrated that the optimized reaction system and modified primers effectively distinguished among CC, CG, and GG genotypes at the g.732 C > G. Blood samples from 24 Hu sheep were analyzed using the PCR-LFD assay specific to this SNP. The genotyping results from PCR-LFD were completely consistent with those obtained from the mutation analysis of the same blood samples. The PCR-LFD method established in this study did not require genomic DNA extraction; whole blood could be directly used as a template for PCR amplification combined with LFD, enabling on-site visual detection. This positions PCR-LFD as a rapid, simple, and visually interpretable tool for on-site SNP genotyping.