Objective: To develop a rapid PCR method to detect pathogenic Aeromonas hydrophila in fish.
Methods: For multiple PCR, three pairs of primers were designed based on the conservative sequences of 16SrRNA genes, aerolysin (aer) gens and serine-protease (ahp) genes of Aeromonas hydrophila. By optimization of PCR conditions and estimation of specificity, sensitivity, detection rate, a triplex PCR assay was established.
Results: The assay had a high specificity detecting only pathogenic strains of Aeromonas hydrophila but not other irrelative bacteria. The assay had a high sensitivity with the detection limit as low as 100 fg, the detection rate of suspicious clinical samples by this assay was 81.8%, which was noticeably higher than that by bacterial isolation method (40.9%). The detection rate of mimic challenge samples by this assay was 87.5%, which was also noticeably higher than that by bacterial isolation method (67.5%).
Conclusion: The simultaneous detection of 16SrRNA gene and two virulent genes in one PCR assay could avoid missed detection possibly caused by PCR with single virulent gene, and provided a useful tool for rapid diagnosis, large-scale quarantine, and epidemiological investigation of the pathogenic Aeromonas hydrophila.