title: Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers.
abstract: 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.
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Balcázar
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Daniel
authors.initials: D
authors.lastname: Vendrell
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: de Blas
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Imanol
authors.initials: I
authors.lastname: Ruiz-Zarzuela
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: Olivia
authors.initials: O
authors.lastname: Gironés
authors.affiliation: Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
authors.forename: José Luis
authors.initials: JL
authors.lastname: Múzquiz
mesh_terms: D048409:Aeromonas salmonicida / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D015169:Colony Count, Microbial / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D005399:Fishes / Q000382:microbiology*; D005453:Fluorescence; D005667:Furunculosis / Q000382:microbiology / Q000662:veterinary; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology / Q000662:veterinary*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S