Development of 16S rRNA Targeted PCR Method for the Detection of Escherichia coli in Rainbow Trout (Oncorhynchus mykiss)

& Objective: The presence of E. coli in fish intended for human consumption may constitute a potential danger, not only on causing disease, but also because of the possible transfer of antibiotic resistance from aquatic bacteria to those infecting humans. The objective of this study was to develop an improved PCR method based on species – specific 16 S rRNA gene primers (FES, RES) for detection of E. coli from agar plates and fish tissues.Materials and Methods: For the rapid detection of E. coli from fish, a set of primers (FES, RES), targeting 16S rRNA gene sequences of the specific microorganism was designed, and fifty two rainbow trout were obtained. Then 1mL of bacterial concentration of 106 CFU/ml was injected into intraperitoneal cavity. Samples were collected from liver and kidney after 48h injection. The PCR reaction conditions were optimized to permit detection of organism from agar plates and fish tissue in a day.Results: All tissue samples were positive for microbiological and PCR identification. DNA was successfully extracted by a boiled – extraction method or by phenol – chloroform – isoamyl alcohol. The BLAST analysis from sequencing of 4 amplicons randomly selected showed similar results, with the match being E .coli with a 100% similarity.Conclusion: This method is fast, specific and sensitive to detect E. coli in infected and asymptomatic animals, fish product, and may have a positive impact on public and environmental health.

Keywords:

16S Ribosomal RNA , E. coli , PCR , Rainbow Trout