Simultaneous detection of Aeromonas hydrophila, and Escherichia coli in Rainbow trout (Oncorhynchus mykiss) by Duplex PCR

Abstract

Rapid and accurate identification of microorganisms have a significant impact on strategies and fish health management programs. Hence, in this study a duplex PCR assay based on the 16s rRNA gene for simultaneous detection of Aeromonas hydrophila RTICC 1032 and Escherichia coli RTICC 2325 from pure cultures, and challenged fish tissues was performed and their results were compared with the results of single PCR assays for each bacterium. For this purpose, an experiment with three treatments including artificially infected with A. hydrophila, E. coli and a mixture of them with a control group was designed. Fish were injected intraperitoneally with 1 ml of sterile physiological saline containing 106CFU/ml of the corresponding bacteria. Samples were collected from liver, kidney and spleen 48 hrs post-injection. A duplex PCR based 16S rRNA genes was developed for the simultaneous detection of A. hydrophila and E. coli. The PCR reaction conditions were optimized to permit detection of organisms from agar plates and fish tissues in less than 8 hrs. Each of the two pairs of oligonucleotide primers exclusively targeted 16S rRNA gene of the specific microorganism. When duplex PCR assay was used to simultaneous detection of the pathogens in asymptomatic fish, spleen and liver were negative for A. hydrophila, whereas kidney was positive for two bacteria. Samples that were duplex PCR negative were also negative by the culture method. On the whole, the duplex PCR has advantages in terms of its accuracy, sensitivity, ease of use, time of length analysis and cost-effectiveness compared to the single PCR and traditional method.

Keywords

• Duplex PCR, Rainbow trout, simultaneous detection, 16SrRNA gene.