Detection of Aeromonas spp. and virulence gene aerolysin in Nile tilapia (Oreochromis niloticus) using PCR technique

Infections caused by bacteria of the genus Aeromonas are among the most common diseases in fish farming systems worldwide, and this disease occurs in all countries which have Nile tilapia (Oreochromis niloticus) farmed. The present work describes the development of a new multiplex PCR (mPCR) technique that diagnosis the genus Aeromonas and detects aerolysin gene (aerA). Reference strains of several Aeromonas species and other genera were used for standardization of mPCR. Strains of A. hydrophila from "pacaman" fish (Lophiosilurus alexandri) and Aeromonas spp. from Nile tilapia from farming systems were used too. Primers were designed based on the 16S rRNA region and aerA (aerolysin toxin). To verify a better annealing temperature were used gradients between 59 degrees C and 61 degrees C with 40ng of the DNA template. The 16S rRNA gene and the aerA gene amplification products showed 786 and 550 bp, respectively. The mPCR showed better annealing temperature at 57.6 degrees C, and the detection limit for both genes (16S rRNA and aerA) was 10-(10)g/mu L of the DNA. The standardized mPCR is quick, sensitive, and specific for Aeromonas spp. diagnosis and to detect aerolysin gene. This method showed advantages when compared to the conventional diagnostic methods and can be used in Nile tilapia or other fish farming systems. The detection of aerolysin gene is an important tool to determine the potential pathogenicity of Aeromonas spp. isolates.