Duplex PCR for imultaneous and unambiguous detection of Streptococcus iniae and Streptococcus agalactiae associated with streptococcosis of cultured tilapia in Thailand

Abstract

Warm-water streptococcosis outbreaks in Thai cultured tilapia are caused by Streptococcus iniae and S. agalactiae. However, distinguishing between these two species of streptococcus are very difficult due to their similar microbiological appearance and clinical signs of infected fish. In this study, we proposed a new duplex-PCR based method for simultaneous detection of these pathogens. The duplex-PCR amplified partial lctO and 16s rRNA gene of S. iniae and S. agalactiae at 870 bp and 220 bp, respectively. This technique gave 100% specificity while sensitivity of reaction was 100 fg of each bacterial genomic DNA. Detection limit of duplex-PCR applicable to clinical specimens is also evaluated as 106 bacterial cells per gram of fish tissue. This study suggested that this duplex-PCR based method might be a good candidate for easy, sensitive, specific and rapid detection of S. iniae and S. agalactiae associated with warm-water streptococcosis of cultured tilapia in Thailand.