Development of a Multiplex Quantitative PCR Assay for Simultaneous Detection of Multiple Vibrio Species in Aquaculture Samples

ABSTRACT Aquaculture associated Vibrio species are important bacterial pathogens causing significant economic losses in aquaculture and zoonotic infections in humans. Rapid and accurate detection of Vibrio species is therefore essential for diagnosis, surveillance and control. In this study, a multiplex quantitative PCR (qPCR) assay was developed for the simultaneous detection of Vibrio spp. together with V. alginolyticus , V. anguillarum , V. harveyi , V. ordalii , V. parahaemolyticus , V. vulnificus and V. cholerae . Species‐specific primers and probes targeting ompA, glutamate synthase, mreB, vohB, dnaJ, gyrB and lolB genes were designed, while the rpoA gene was selected for genus level Vibrio detection. The assay demonstrated 100% inclusivity for all target strains without cross reactivity against non‐target bacterial species. PCR efficiency/inhibition analyses showed stable amplification dynamics across serial dilutions, with R 2 values above 0.99, amplification efficiencies between 96.8% and 98.2%, and ΔCq values ≤ 0.13 for all targets. Limit of detection (LOD) studies performed using artificially contaminated matrices demonstrated a detection limit of 10 1 –10 0 CFU/mL for all targets, with detection performance comparable to the culture‐based recovery method. Overall, the developed multiplex qPCR assay provides a rapid, sensitive and analytically reliable approach for simultaneous detection of multiple Vibrio species associated with aquaculture and zoonotic infections.