Simultaneous Detection of Streptococcus spp. and Aeromonas spp. from Diseased Tilapia (Oreochromis niloticus) using Multiplex-Polymerase Chain Reaction

Abstract

A multiplex Polymerase Chain Reaction (m-PCR) assay was developed for simultaneous detection of two major pathogens, Streptococcal and Aeromonad bacteria, in farmed tilapia. DNA fragments of Streptococcus spp. and Aeromonas spp. were amplified using genus specific primers, C1/C2 and AERF/AERR, which produced PCR of 207 bp and 953 bp, respectively. The lowest concentration of each Streptococcus and Aeromonas spp. extracted genomic DNA from a colony detected by m-PCR was 2 ng. The m-PCR assay was proven applicable for detection of bacterial genomic DNA in tissues (brain and posterior kidney) of infected fish. Specificity of the assay tested with other Gram-positive (Staphylococcus aureus) and Gram-negative water borne bacteria (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Vibrio harveyi and Vibrio parahemolyticus) showed no amplification. As Streptococcal and Aeromonad infections are common concurrent bacterial diseases, the m-PCR assay established in this study enabled effective simultaneous detection of these two major bacterial infections responsible for current economic losses in tilapia farming in Thailand.