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J Med Microbiol 56 (2007), 323-328; DOI: 10.1099/jmm.0.46647-0
© 2007 Society for General Microbiology
ISSN 1473-5644

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers

José Luis Balcázar, Daniel Vendrell, Ignacio de Blas, Imanol Ruiz-Zarzuela, Olivia Gironés and José Luis Múzquiz

Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain

Correspondence
José Luis Balcázar
balcazar{at}unizar.es

Received 27 March 2006
Accepted 6 November 2006

In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore for the detection of Aeromonas salmonicida was developed. Probe specificity was confirmed by amplification of 16 A. salmonicida strain templates and by the lack of a PCR product with 26 non-A. salmonicida strains. With a pure culture of A. salmonicida, the assay was linear over a range of 0.5 pg to 50 ng and was able to detect 16 c.f.u. per reaction. A similar sensitivity was observed in DNA extracted from a mixture of A. salmonicida and fish tissue. Results using artificially inoculated tissues and diseased fish from outbreaks indicated that the assay can provide sensitive species-specific detection and quantification of A. salmonicida in fish tissue.

Abbreviations: Ct, cycle threshold; FAM, 6-carboxyfluorescein; LUX, Light Upon eXtension.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of the A. salmonicida strains determined in this study are AM296501–AM296510.




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A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish
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