An improved in situ hybridization method for the detection of fish pathogens.
A fluorescent in situ hybridization (FISH) method was developed for detection of infectious pancreatic necrosis virus (IPNV) in paraffin-embedded tissues of Atlantic salmon, Salmo salar L. Several methods of probe labelling and detection were evaluated and found unsuitable for FISH because of tissue autofluorescence. Likewise, the use of avidin to detect biotin-labelled probe was obviated by the presence of endogenous biotin. An existing approach, using digoxigenin (DIG)-labelled probes and detection by anti-DIG antibody-labelled with alkaline phosphatase, was modified to use a fluorescent substrate, 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate/4-chloro-2-methylbenzene diazonium hemi-zinc chloride salt (HNPP/Fast Red TR). This improved method allowed sensitive detection of IPNV target, without interference from autofluorescence or endogenous alkaline phosphatase. Furthermore, the reporter produces a discrete, non-fading signal, which is particularly suitable for analysis by confocal microscopy.
McCarthy
U M
UM
FRS Marine Laboratory, Aberdeen, UK. una_mccarthy@umit.maine.edu
Urquhart
K L
KL
Bricknell
I R
IR
D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D003979:Diazonium Compounds; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology*; D005456:Fluorescent Dyes / Q000378:metabolism; D017403:In Situ Hybridization / Q000379:methods; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018061:Infectious pancreatic necrosis virus / Q000235:genetics / Q000302:isolation & purification*; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000821:virology; D009281:Naphthalenes; D016612:Paraffin Embedding; D020317:Salmo salar / Q000821:virology*
D003979:Diazonium Compounds; D005456:Fluorescent Dyes; D009281:Naphthalenes; C094602:3-hydroxy-N-(2'-biphenyl)-2-naphthalenecarboxamide phosphate; C027766:Fast Red TA