Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae. The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila, 62 CFU for E. tarda, 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.