PCR detection, characterization, and distribution of virulence genes in Aeromonas spp.

We found 73.1 to 96.9% similarity by aligning the cytolytic enterotoxin gen e of Aeromonas hydrophila SSU (AHCYTOEN; GenBank accession no. M84709) agai nst aerolysin genes of Aeromonas spp,, suggesting the possibility of select ing common primers. Identities of 90 to 100% were found among the eight sel ected primers from those genes, Amplicons obtained from Aeromonas sp, refer ence strains by using specific primers for each gene or a cocktail of prime rs were 232 bp long. Of hybridization group 4/5A/5B (HG4/5A/5B), HG9, and H G12 or non-Aeromonas reference strains, none were positive. PCR-restriction fragment Length polymorphism (PCR-RFLP) with HpaII yielded three types of patterns. PCR-RFLP 1 contained two fragments (66 and 166 bp) found in HG6, HG7, HCS, HG10, and HG11. PCR-RFLP 2 contained three fragments (18, 66, and 148 bp) found in HG1, HG2, HG3, and HG11. PCR-RFLP 3, with four fragments (7, 20, 66, and 139 bp), was observed only in HG13, PCR-amplicon sequence a nalysis (PCR-ASA) revealed three main types. PCR-ASA 1 had 76 to 78% homolo gy with AHCYTOEN and included strains in HG6, HG7, HG8, HG10, and HG11. PCR -ASA 2, with 82% homology, was found only in HG13. PCR-ASA 3, with 91 to 99 % homology, contained the strains in HG1, HG2, HG3, and HG11. This method i ndicated that 37 (61%) of the 61 reference strains were positive with the p rimer cocktail master mixture, and 34 (58%) of 59 environmental isolates, 9 3 (66%) of 141 food isolates, and 100 (67%) of 150 clinical isolates from a round the world carried a virulence factor when primers AHCF1 and AHCR1 wer e used. In conclusion, this PCR-based method is rapid, sensitive, and speci fic for the detection of virulence factors of Aeromonas spp. It overcomes t he handicap of time-consuming biochemical and other DNA-based methods.