Abstract
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 10(6) 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.