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.
AdamsA.,
ThompsonK.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]
AltschulS. F.,
MaddenT. L.,
SchäfferA. A.,
ZhangJ.,
ZhangZ.,
MillerW.,
LipmanD. J.1997; Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402[CrossRef]
BestE. L.,
FoxA. J.,
FrostJ. A.,
BoltonF. 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]
ByersH. K.,
GudkovsN.,
CraneM. 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]
Del CerroA.,
MarquezI.,
GuijarroJ. 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]
De MediciD.,
CrociL.,
DelibatoE.,
Di PasqualeS.,
FileticiE.,
TotiL.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]
GustafsonC. E.,
ThomasC. J.,
TrustT. 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
HineyM.,
OlivierG.1999; Furunculosis ( Aeromonas salmonicida ). In Fish Diseases and Disorders , vol. 3: Viral, Bacterial and Fungal Infections pp 341–425 Edited by
WooP. T. K.,
BrunoD. W.
Wallingford: CABI Publishing;
LoweB.,
AvilaH. A.,
BloomF. R.,
GleesonM.,
KusserW.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]
McCarthyD. H.,
RobertsR. J.1980; Furunculosis of fish – the present state of our knowledge. In Advances in Aquatic Microbiology pp 293–341 Edited by
DroopM. R.,
JannaschH. W.
London: Academic Press;
MillsS. D.,
BolandA.,
SoryM. P.,
van der SmissenP.,
KerbourchC.,
FinlayB. B.,
CornelisG. 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]
MooneyJ.,
PowellE.,
ClabbyC.,
PowellR.1995; Detection of Aeromonas salmonicida in wild Atlantic salmon using a specific DNA probe test. Dis Aquat Org 21:131–135[CrossRef]
NazarenkoI.,
LoweB.,
DarflerM.,
IkonomiP.,
SchusterD.,
RashtchianA.2002a; Multiplex quantitative PCR using self-quenched primers labeled with a single fluorophore. Nucleic Acids Res 30:e37[CrossRef]
NazarenkoI.,
PiresR.,
LoweB.,
ObaidyM.,
RashtchianA.2002b; Effect of primary and secondary structure of oligodeoxyribonucleotides on the fluorescent properties of conjugated dyes. Nucleic Acids Res 30:2089–2195[CrossRef]
O'BrienD.,
MooneyJ.,
RyanD.,
PowellE.,
HineyM.,
SmithP. R.,
PowellR.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
SharkeyF. H.,
BanatI. M.,
MarchantR.2004; Detection and quantification of gene expression in environmental bacteriology. Appl Environ Microbiol 70:3795–3806[CrossRef]
SørumH.,
KvelloJ. H.,
HasteinT.1993; Occurrence and stability of plasmids in Aeromonas salmonicida ss salmonicida isolated from salmonids with furunculosis. Dis Aquat Org 16:199–206[CrossRef]
SuzukiK.,
SasakiJ.,
UramotoM.,
NakaseT.,
KomagataK.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]
ThompsonJ. D.,
HigginsD. G.,
GibsonT. 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]
XiC.,
BalbergM.,
BoppartS. A.,
RaskinL.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]