Rapid and Sensitive Visual Detection of Gene Mutations via LIVE-SNP

As the most prevalent form of genetic variation, single nucleotide polymorphisms (SNPs) are strongly associated with disease pathogenesis, microbial resistance, and pathogenicity. The rapid mutation rates of viruses such as influenza A and SARS-CoV-2 highlight the urgent need for multifunctional diagnostic tools capable of effectively distinguishing emerging variants. However, current clinical SNPs detection methods often require complex primer/probe designs and sophisticated instruments, which limits their wide application in resource-constrained settings. Here, we developed the LIVE-SNP method, a rapid ligation-mediated isothermal assay for the visual evaluation of SNPs with high sensitivity and specificity. This method employs multicolor fluorescent probes to establish multiplex assays for the simultaneous identification of different SNPs, allowing for direct discrimination of target types through visual observation of fluorescent signals. The established method exhibits high specificity in identifying SNPs in both RNA and DNA. It can accurately detect drug-resistant single-nucleotide mutations in influenza A virus and efficiently identify antimicrobial resistance mutations in bacteria. Furthermore, we have developed a compact hand-held device integrating heating and fluorescence detection modules, which facilitates rapid on-site detection of pathogenic microorganisms and their variants, providing a powerful technical platform for applications in food safety, genotyping, and infectious disease control.