Development of a novel multiplex-PCR technology for simultaneous detection of five major aquaculture pathogens

Abstract

Early and precise pathogen identification and corresponding disease management are primary concerns in aquaculture. Here, we attempted at diagnostic methods that can simultaneously identify multiple pathogens, where many samples, several pathogens, and concurrent infections are to be handled. Hence, a multiplex PCR assay targeting five major aquaculture pathogens, viz. Vibrio parahaemolyticus, Vibrio anguillarum, Vibrio alginolyticus, Vibrio vulnificus and Vibrio harveyi was developed for the first time. The primers targeting toxR of V. parahaemolyticus, amiB of V. anguillarum, col of
V. alginolyticus, vvhA of V. vulnificus, and topA of V. harveyi were applied. Furthermore, the reaction included an internal amplification control against prokaryotic 16S rRNA to perceive false-negative results. The assay showed 100% specificity against 56 unique bacteria. The sensitivity was 0.25 ng for V. harveyi, 0.5 ng for V. vulnificus, 1 ng for V. parahaemolyticus and V. anguillarum, and 2 ng for V. alginolyticus DNA per µL assay. Sensitivity regarding CFU was 1.2, 5.2, 10, 5.6×10¹ and 3.8×10² per µL, for V. harveyi, V. vulnificus, V. anguillarum, V. parahaemolyticus and V. alginolyticus, respectively. The results suggest that the optimized method can be applied for sensitive and specific identification of five aquaculture pathogens through a single PCR