Development of a new multiplex-PCR tool for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda

Disease assessment and management in cultured aquatic animals is a major concern in commercial aquaculture. Disease outbreaks have direct effects on fish production, causing serious economic losses in this industry. This can be overcome by early detection through molecular high sensitivity tools such as PCR (polymerase chain reaction). One of the most critical steps in the study of bacterial fish diseases is the precise identification of the infectious agent. Considering the damage that some bacteria can cause to fish and humans, the development of a rapid detection method for the four target species that demonstrates to be simple, accurate and low cost is an essential step for the prevention and early treatment of these diseases. Edwardsiella tarda (ACC 36.1), Vibrio harveyi (DSM 19023), Vibrio anguillarum (AQV 55.1) and Vibrio alginolyticus (CECT 521) were selected as targets of a multiplex PCR tool. The multiplex PCR reactions were performed in various reaction conditions, including different annealing temperatures (between 49 degrees C and 55 degrees C) and changes in the MgCl2 concentration from 2mMto 8 mM. Best results were obtained at 51 degrees C and MgCl2 concentration from 4mM to 6 mM. Primers were tested using purified DNA from the respective bacterial strains, yielding bands at expected sizes, which can be identified on agarose gels with clearness and without overlapping sizes. Results indicate that this multiplex PCR tool is suitable for the detection of target pathogens and may, in the future, have extended practical application in commercial aquaculture.