Development of Multiple Real-Time Fluorescent Quantitative PCR for Vibrio Pathogen Detection in Aquaculture

The <i>Vibrio</i> genus represents a critical group of bacterial pathogens in the marine environment globally, leading to massive mortality in the aquaculture industry. Diagnosing vibriosis, an infection caused by <i>Vibrio</i> species, in clinical samples poses challenges due to its non-specific clinical manifestations. In this study, we developed a TaqMan probe-based multiplex real-time PCR method for the simultaneous detection and quantification of four <i>Vibrio</i> pathogens: <i>Vibrio anguillarum</i> (Va), <i>Vibrio alginolyticus</i> (Val), <i>Vibrio harveyi</i> (Vh), and <i>Vibrio scophthalmi</i> (Vsc). The assay targets conserved intra-species regions and specific inter-species regions using specific primers and TaqMan probes to ensure specificity. Sensitivity analysis demonstrated that the multiplex real-time PCR assay could simultaneously detect the four different bacteria, with detection limits of 26-60 copies per reaction, making it 100 times more sensitive than conventional PCR assays. Additionally, the assay exhibited high reproducibility, with intra- and inter-group coefficients of variation below 1.4%. A total of 63 clinical samples was analyzed using this established assay, which successfully detected both single and mixed infections. These results demonstrate that the multiplex quantitative PCR assay is a rapid, specific, and sensitive diagnostic tool for the detection of Va, Val, Vh, and Vsc, making it suitable for monitoring these bacteria in both single- and co-infected clinical samples.