Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers.
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.
Balcázar
José Luis
JL
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
Vendrell
Daniel
D
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
de Blas
Ignacio
I
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
Ruiz-Zarzuela
Imanol
I
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
Gironés
Olivia
O
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
Múzquiz
José Luis
JL
Laboratory of Fish Pathology, Faculty of Veterinary Sciences, University of Zaragoza, 50013 Zaragoza, Spain.
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
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S