[Detecting pathogenic Aeromonas hydrophila in fish by triplex PCR]

To develop a rapid PCR method to detect pathogenic Aeromonas hydrophila in fish.

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.

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%).

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.