Development of a quadruple Taqman probe-based real-time fluorescent quantitative PCR for the detection of bacterial pathogens in a marine fish.

Bacterial diseases are the most common diseases in aquaculture worldwide, resulting in severe economic losses in the aquaculture industry. However, bacterial pathogens usually lead to non-specific clinical symptoms and are difficult to diagnose in clinical samples. Therefore, specific, sensitive, and time-saving detection methods are crucial for earlier prevention and accurate treatment of bacterial pathogens. This study developed a TaqMan probe-based multiplex real-time PCR method for simultaneous detection and quantification of four bacterial pathogens, including Edwardsiella piscicida, Photobacterium damselae subsp. piscicida, Vibrio rotiferianus, and Pseudomonas fluorescens. The conserved regions of the intra-species and the specific regions of the inter-species were targeted using specific primers and Taqman probes to ensure specificity. Sensitivity analysis showed that the four bacteria were simultaneously detected in the multiplex real-time PCR assay, with detection limits of 32-76 copies/reaction, which is 100 times higher than that of the conventional PCR assay. Furthermore, the assay had good reproducibility, with intra- and inter-group coefficients of variation below 1 %. A total of 63 clinical samples were analyzed using this established assay, of which either single or mixed infection samples could be correctly detected. These findings indicate that this multiplex qPCR assay can serve as a quick, specific, sensitive diagnostic tool for E. piscicida, P. damselae subsp. piscicida, V. rotiferianus, and P. fluorescens detection, thus can be utilized to monitor these bacteria in single or co-infected clinical samples.