V. parahaemolyticus is one of the most important foodborne pathogens and poses a serious threat to public health because of its high incidence and transferable antimicrobial resistance genes (ARGs). In this study, a novel approach for simultaneous detection of V. parahaemolyticus and the tetracycline resistance gene tetA was developed by reasonably integrating rapid loop-mediated isothermal amplification (LAMP) with the powerful cleavage ability of Pyrococcus furiosus Argonaute (PfAgo). The guide DNAs (gDNAs) were carefully designed to complete continuous signal transduction. With this strategy, the dual-target LAMP amplicons that were originally difficult to distinguish could be ingeniously converted into fluorescent signals. The duplex LAMP-PfAgo method was used to complete the entire process within 45 min at constant temperature and showed no cross-reactivity with non-target pathogens. The results demonstrated superior analytical performance, with a limit of detection (LOD) of as low as 6.68 CFU/mL for the V. parahaemolyticus carrying the tetA gene and a dynamic range from 10° to 10⁶ CFU/mL. Furthermore, the proposed method exhibited satisfactory sensitivity and feasibility for use in identifying contaminated samples (water, salmon and oysters). In summary, this study provides effective technical support for the on-site detection of pathogenic bacteria and tetracycline resistance genes, presenting promising prospects for food safety testing, clinical diagnosis, and environmental monitoring in the One Health context.

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.

Vibrio spp., known as typical marine pathogens, pose serious threats to human health worldwide due to their high incidence and prevalence. In this study, a quantitative detection method was developed for Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio cholerae and Vibrio alginolyticus in aquatic products. A set of multiplex chamber-based digital PCR (cdPCR) primers and probes was designed based on new molecular targets of Vibrio species. The conditions of multiplex cdPCR were optimized and compared with those of multiplex qPCR. Under optimal conditions, the performance indicators, including the specificity, linear range, and detection limit, were systematically evaluated. The results indicate that the established method had 100% specificity among all tested strains and did not cross-react with other foodborne pathogens tested in parallel. The linear dynamic range was 10²–10⁶ CFU/mL, and the limits of detection (LODs) for V. parahaemolyticus, V. vulnificus, V. cholerae and V. alginolyticus were 5.40 × 10² CFU/mL, 3.05 × 10² CFU/mL, 4.55 × 10² CFU/mL and 1.32 × 10² CFU/mL, respectively. Furthermore, the feasibility of the proposed method was assessed in contaminated food samples, with a 10-fold improvement in detection sensitivity over that of the qPCR method under the conditions of this study. The entire detection process could be completed within 2 h. Taken together, the results of this study offer a readily adaptable diagnostic method, allowing for the simultaneous and quantitative detection of four pathogenic Vibrio species, which is promising for food safety and environmental monitoring.