Detection of Vibrio and ten Vibrio species in cage-cultured fish by multiplex polymerase chain reaction using house-keeping genes

Vibriosis is one of the major bacterial diseases observed in cultured marine fish worldwide. An epidemic trait of the Vibrio derived from cage-cultured marine fish is still lacking. To identify the Vibrio and discriminate ten Vibrio species from cage-cultured marine fish, we designed specific primers targeting the RNA polymerase sigma factor (rpoD), recombination protein F (recF), transcriptional regulator (toxR) genes (known as housekeeping genes), and two multiplex PCR sets (PCR set 1 for V. anguillarum, V. gigantis, V. atlanticus, V. harveyi, and V. scophthalmi; and PCR set 2 for V. lentus, V. splendidus, Photoacterium piscicola, V. alginolyticus and P. damselae). Moreover, each multiplex PCR assay comprised internal control primers that enabled the detection of the Vibrio. Designed primers yielded specific diagnostic results to target the Vibrio and ten Vibrio species in the multiplex PCR assays. The minimal detection limits of the multiplex PCR were each 5 ng DNA mu L-1, which showed ten-fold sensitivity compared to a single PCR reaction. The novel multiplex PCR assays could contribute to detecting the Vibrio and discriminate the major Vibrio species in cage-cultured marine fish, and could be used as a reliable and informative diagnostic tool in the aquaculture industry.