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.

Bacillus cereus is a common Gram-positive bacterium that poses a significant threat to food safety due to its environmental adaptability, spore-forming ability, and production of harmful toxins. Traditional detection methods for B. cereus are time-consuming and inaccurate. This study aimed to develop a rapid enrichment medium for B. cereus to improve detection efficiency. Five B. cereus strains and five non-B. cereus strains were used. The TSB medium was selected as the basic medium as it supported the best growth and spore germination of B. cereus among the tested media. Magnesium sulfate and inosine were identified as the most effective promoters for the growth of vegetative cells and spore germination respectively, while glycine and sodium nitrite were chosen as suitable inhibitors against non-B. cereus bacteria. Through orthogonal experiments, the optimal formulation of the rapid enrichment medium (BC-TSB) was determined. BC-TSB effectively inhibited the growth of non-target bacteria and significantly promoted the growth and spore germination of B. cereus compared to the TSB basic medium. It also efficiently facilitated the recovery of thermally injured B. cereus cells, with a 6 h recovery rate of 87%—shortening the incubation time required by traditional method from 48 h to 6 h. In the detection of artificially contaminated dairy samples, BC-TSB could effectively pre-enrich B. cereus, achieving a 100% detection rate in UHT milk, modified milk, and pasteurized milk using both traditional and PMAxx-qPCR methods. Overall, the developed BC-TSB medium has great potential for the rapid and accurate detection of B. cereus in food, which can help enhance food safety monitoring.