Simultaneous detection of Pathogenic Vibrio species using multiplex real-time PCR

We developed a multiplex real-time (RTi) PCR method for the simultaneous detection of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus using zot, vmrA, and vuuA as the respective target genes. A set of primer pairs specific for those target genes was designed and employed in the SYBR Green-based multiplex RTi-PCR assay. Quantitative analyses with ten-fold serially diluted genomic DNA of each target organism resulted in a linear correlation between CT values and the amount of each target genome per reaction, with a lower detection level of less than ten genome copies per reaction. Similar sensitivities were observed for Vibrio-spiked seafood samples (oyster, crab meat, and raw fish). After 8 h of enrichment culture of the seafood homogenate in alkaline peptone water, our optimized multiplex RTi-PCR was shown to achieve theoretical maximum sensitivity (ca. 100 CFU/gram food homogenate). Our proposed method is simple, robust and readily adaptable in routine laboratories, allowing for high-throughput surveillance of pathogenic Vibrio species in seafood.