1887

Abstract

In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore for the detection of was developed. Probe specificity was confirmed by amplification of 16 strain templates and by the lack of a PCR product with 26 non- strains. With a pure culture of , 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 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 in fish tissue.

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2007-03-01
2019-11-15
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References

  1. Adams, A. & Thompson, K. ( 1990; ). Development of an enzyme-linked immunosorbent assay (ELISA) for the detection of Aeromonas salmonicida in fish tissue. J Aquat Anim Health 2, 281–288.[CrossRef]
    [Google Scholar]
  2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  3. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  4. Austin, B. & Austin, D. A. (editors) ( 1999; ). Bacterial Fish Pathogens: Diseases of Farmed and Wild Fish, 3rd edn. Chichester: Springer – Praxis.
  5. Best, E. L., Fox, A. J., Frost, J. A. & Bolton, F. J. ( 2005; ). Real-time single-nucleotide polymorphism profiling using TaqMan technology for rapid recognition of Campylobacter jejuni clonal complexes. J Med Microbiol 54, 919–925.[CrossRef]
    [Google Scholar]
  6. Byers, H. K., Gudkovs, N. & Crane, M. S. J. ( 2002; ). PCR-based assays for the fish pathogen Aeromonas salmonicida. I. Evaluation of three PCR primer sets for detection and identification. Dis Aquat Org 49, 129–138.[CrossRef]
    [Google Scholar]
  7. Del Cerro, A., Marquez, I. & Guijarro, J. A. ( 2002; ). Simultaneous detection of Aeromonas salmonicida, Flavobacterium psychrophilum, and Yersinia ruckeri, three major fish pathogens, by multiplex PCR. Appl Environ Microbiol 68, 5177–5180.[CrossRef]
    [Google Scholar]
  8. De Medici, D., Croci, L., Delibato, E., Di Pasquale, S., Filetici, E. & Toti, L. ( 2003; ). Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype enteritidis in poultry. Appl Environ Microbiol 69, 3456–3461.[CrossRef]
    [Google Scholar]
  9. Gilroy, D. & Smith, P. ( 2003; ). Application-dependent, laboratory-based validation of an enzyme-linked immunosorbent assay for Aeromonas salmonicida. Aquaculture 217, 23–38.[CrossRef]
    [Google Scholar]
  10. Gustafson, C. E., Thomas, C. J. & Trust, T. J. ( 1992; ). Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene. Appl Environ Microbiol 58, 3816–3825.
    [Google Scholar]
  11. Hiney, M. & Olivier, G. ( 1999; ). Furunculosis (Aeromonas salmonicida). In Fish Diseases and Disorders, vol. 3: Viral, Bacterial and Fungal Infections, pp. 341–425. Edited by P. T. K. Woo & D. W. Bruno. Wallingford: CABI Publishing.
  12. Hiney, M., Dawson, M. T., Heery, D. M., Smith, P. R., Gannon, F. & Powell, R. ( 1992; ). DNA probe for Aeromonas salmonicida. Appl Environ Microbiol 58, 1039–1042.
    [Google Scholar]
  13. Lowe, B., Avila, H. A., Bloom, F. R., Gleeson, M. & Kusser, W. ( 2003; ). Quantitation of gene expression in neural precursors by reverse-transcription polymerase chain reaction using self-quenched, fluorogenic primers. Anal Biochem 315, 95–105.[CrossRef]
    [Google Scholar]
  14. Martínez-Murcia, A. J., Saavedra, M. J., Chacón, M. R., Guarro, J., Stackebrandt, E. & Figueras, M. J. ( 2005; ). Phenotypic, genotypic, and phylogenetic discrepancies to differentiate Aeromonas salmonicida from Aeromonas bestiarum. Int Microbiol 8, 259–269.
    [Google Scholar]
  15. McCarthy, D. H. & Roberts, R. J. ( 1980; ). Furunculosis of fish – the present state of our knowledge. In Advances in Aquatic Microbiology, pp. 293–341. Edited by M. R. Droop & H. W. Jannasch. London: Academic Press.
  16. Mills, S. D., Boland, A., Sory, M. P., van der Smissen, P., Kerbourch, C., Finlay, B. B. & Cornelis, G. R. ( 1997; ). Yersinia enterocolitica induces apoptosis in macrophages by a process requiring functional type III secretion and translocation mechanisms and involving YopP, presumably acting as an effector protein. Proc Natl Acad Sci U S A 94, 12638–12643.[CrossRef]
    [Google Scholar]
  17. Miyata, M., Inglis, V. & Aoki, T. ( 1996; ). Rapid identification of Aeromonas salmonicida subspecies salmonicida by the polymerase chain reaction. Aquaculture 141, 13–24.[CrossRef]
    [Google Scholar]
  18. Mooney, J., Powell, E., Clabby, C. & Powell, R. ( 1995; ). Detection of Aeromonas salmonicida in wild Atlantic salmon using a specific DNA probe test. Dis Aquat Org 21, 131–135.[CrossRef]
    [Google Scholar]
  19. Nazarenko, I., Lowe, B., Darfler, M., Ikonomi, P., Schuster, D. & Rashtchian, A. ( 2002a; ). Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res 30, e37.[CrossRef]
    [Google Scholar]
  20. Nazarenko, I., Pires, R., Lowe, B., Obaidy, M. & Rashtchian, A. ( 2002b; ). Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res 30, 2089–2195.[CrossRef]
    [Google Scholar]
  21. O'Brien, D., Mooney, J., Ryan, D., Powell, E., Hiney, M., Smith, P. R. & Powell, R. ( 1994; ). Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared atlantic salmon smolts. Appl Environ Microbiol 60, 3874–3877.
    [Google Scholar]
  22. Sharkey, F. H., Banat, I. M. & Marchant, R. ( 2004; ). Detection and quantification of gene expression in environmental bacteriology. Appl Environ Microbiol 70, 3795–3806.[CrossRef]
    [Google Scholar]
  23. Sørum, H., Kvello, J. H. & Hastein, T. ( 1993; ). Occurrence and stability of plasmids in Aeromonas salmonicida ss salmonicida isolated from salmonids with furunculosis. Dis Aquat Org 16, 199–206.[CrossRef]
    [Google Scholar]
  24. Suzuki, K., Sasaki, J., Uramoto, M., Nakase, T. & Komagata, K. ( 1996; ). Agromyces mediolanus sp. nov., nom. rev., comb. nov., a species for “Corynebacterium mediolanum” Mamoli 1939 and for some aniline-assimilating bacteria which contain 2,4-diaminobutyric acid in the cell wall peptidoglycan. Int J Syst Bacteriol 46, 88–93.[CrossRef]
    [Google Scholar]
  25. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ( 1994; ). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.[CrossRef]
    [Google Scholar]
  26. Xi, C., Balberg, M., Boppart, S. A. & Raskin, L. ( 2003; ). Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells. Appl Environ Microbiol 69, 5673–5678.[CrossRef]
    [Google Scholar]
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