Rapid and simple lateral flow detection for ADH1B and ALDH2 genotyping: PCR-restriction digestion-adapter...

Single nucleotide polymorphisms (SNPs) in alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase 2 (ALDH2) significantly impact ethanol metabolism and alcohol-related health risks. Rapid and simple genotyping methods for these genes are crucial for clinical diagnostics and point-of-care testing (POCT), but existing techniques often require complex procedures or specialized equipment. We developed and validated a novel genotyping method, termed a PCR-restriction digestion-adapter ligation-lateral flow (PRALL) assay. The PRALL assay integrates: (i) template preparation from buccal swab, (ii) duplex PCR amplification using biotinylated primers incorporating a BslI recognition site, (iii) simultaneous BslI restriction digestion and allele-specific ligation of hapten-labeled adapters in a single tube, and (iv) visual detection by lateral flow assay (LFA). The method was successfully validated using model templates and human genomic DNA. The complete PRALL assay, from crude lysate preparation to visual result, was performed in approximately 1 h. The assay demonstrated high sensitivity (limit of detection: ∼100 pg genomic DNA, corresponding to ∼15 allele copies) and achieved 100 % concordance with Sanger genotyping for both SNPs across 25 human samples. The unique integration of streamlined enzymatic steps with an instrument-free visual LFA readout overcomes key limitations of previous methods. This makes PRALL a promising and practical tool for facilitating personalized alcohol risk assessment and enhancing clinical diagnostics, demonstrating strong potential for deployment in various settings, including those closer to the point of care.