PfAgo-Enhanced LAMP Assay for Rapid and Specific Detection of Vibrio alginolyticus and Vibrio vulnificus.

Rapid and accurate detection of Vibrio alginolyticus and Vibrio vulnificus is important for seafood safety and foodborne infection control. In this study, we developed a detection platform that combines loop-mediated isothermal amplification (LAMP) with Pyrococcus furiosus Argonaute (PfAgo)-mediated sequence-specific cleavage. The LAMP rapidly amplified pathogen-specific target sequences under isothermal conditions, while PfAgo provides an additional layer of sequence verification through a programmable guide DNA-directed cleavage step to reduce false-positive signals caused by nonspecific amplification. Under optimized conditions, the assay achieved limits of detection of 3 × 101 CFU·mL-1 for V. alginolyticus and 102 CFU·mL-1 for V. vulnificus, with no positive signals observed among the tested non-target bacteria. In artificially contaminated salmon samples, both V. alginolyticus and V. vulnificus were detected without enrichment after simple boiling-based DNA extraction, with a practical detection limit of 2.9 × 102 CFU·mL-1 in the final homogenate. Compared with culture-based methods and instrument-intensive molecular assays, the proposed LAMP-PfAgo platform exhibits a rapid two-step workflow with an added sequence-verification layer. These results support LAMP coupled with Argonaute-mediated cleavage as a practical strategy for foodborne pathogen monitoring in seafood.