AIMS: Development of a PCR assay for detection of aeromonads carrying the hlyA and/or aerA genes in fish. METHODS AND RESULTS: The protocol involves an overnight selective enrichment step in tryptic soy broth yeast extract containing 10 microg ml(-1) of ampicillin followed by extraction of DNA and PCR amplification of two haemolysin genes that contribute to the virulence of Aer. hydrophila. This procedure can detect initial populations of 1-10 cfu g(-1) within 24 h in artificially contaminated samples. In naturally contaminated fish, both genes were detected in 13 out of 14 fresh fish lots (aeromonads levels between < 1 and 5.42 log cfu g(-1)) and in 4 out of 16 lots of vacuum-packed cold-smoked fish (aeromonads levels between < 1 and 3.37 log cfu g(-1)). Before enrichment, dominant species were Aer. hydrophila HG1 (aerA+hlyA+), Aer. bestiarum HG2 (aerA+hlyA+) and Aer. caviae HG4 (aerA-hlyA-). After enrichment, Aer. hydrophila HG1 (aerA+hlyA+) was dominant. CONCLUSIONS: Fresh fish and even smoked fish carry hlyA+ and/or aerA+ aeromonads that can be detected by PCR within 24 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assay described offers considerable potential as a rapid method with specificity, sensitivity and simplicity.
AIMS: Development of a PCR assay for detection of aeromonads carrying the hlyA and/or aerA genes in fish. METHODS AND RESULTS: The protocol involves an overnight selective enrichment step in tryptic soy broth yeast extract containing 10 microg ml(-1) of ampicillin followed by extraction of DNA and PCR amplification of two haemolysin genes that contribute to the virulence of Aer. hydrophila. This procedure can detect initial populations of 1-10 cfu g(-1) within 24 h in artificially contaminated samples. In naturally contaminated fish, both genes were detected in 13 out of 14 fresh fish lots (aeromonads levels between < 1 and 5.42 log cfu g(-1)) and in 4 out of 16 lots of vacuum-packed cold-smoked fish (aeromonads levels between < 1 and 3.37 log cfu g(-1)). Before enrichment, dominant species were Aer. hydrophila HG1 (aerA+hlyA+), Aer. bestiarum HG2 (aerA+hlyA+) and Aer. caviae HG4 (aerA-hlyA-). After enrichment, Aer. hydrophila HG1 (aerA+hlyA+) was dominant. CONCLUSIONS: Fresh fish and even smoked fish carry hlyA+ and/or aerA+ aeromonads that can be detected by PCR within 24 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR assay described offers considerable potential as a rapid method with specificity, sensitivity and simplicity.