title: Detection of infectious pancreatic necrosis virus in subclinically infected Atlantic salmon by virus isolation in cell culture or real-time reverse transcription polymerase chain reaction: influence of sample preservation and storage.
abstract: Infectious pancreatic necrosis, an important problem of the salmon industry worldwide, is caused by Infectious pancreatic necrosis virus (IPNV). Fish surviving an IPNV infection become virus carriers, and the identification of infected fish is highly relevant to disease control. The target organ for IPNV diagnosis is the kidney, where the virus persists, usually with low virus loads. The current study documents a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay that proved 100 times more sensitive than a conventional RT-PCR. Cell culture and real-time RT-PCR were compared for their ability to detect IPNV in carrier Atlantic salmon kidney samples after different preservation and storage procedures. Storage of whole tissue at -80°C for 1 month and storage of tissue homogenized in transport medium (TM) at +4°C for 1 week before investigation in cell cultures resulted in a marked reduction of virus infectivity. For detection by real-time RT-PCR, storage of whole tissue was suboptimal, whereas storage of tissue homogenized in TM did not affect virus detection. The results of the present study demonstrate that both cell culture and real-time RT-PCR are reliable tests for the detection of low amounts of IPNV in kidneys of carrier Atlantic salmon, and both methods are relatively robust against minor preservation and storage deviations, or both. Preservation of tissues in RNA stabilization solution seems only necessary when samples are to be shipped at ambient temperatures or when laboratory testing might be delayed. Independent of detection method, these results indicate that for long-term storage, samples are best kept at -80°C after homogenization in TM.
authors.affiliation: National Veterinary Institute, PB 750 Sentrum, 0106 Oslo, Norway. irene.orpetveit@vetinst.no
authors.forename: Irene
authors.initials: I
authors.lastname: Orpetveit
authors.forename: Aase B
authors.initials: AB
authors.lastname: Mikalsen
authors.forename: Hilde
authors.initials: H
authors.lastname: Sindre
authors.forename: Oystein
authors.initials: O
authors.lastname: Evensen
authors.forename: Birgit H
authors.initials: BH
authors.lastname: Dannevig
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Midtlyng
mesh_terms: D000818:Animals; D018175:Birnaviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D005393:Fish Diseases / Q000175:diagnosis / Q000821:virology*; D018061:Infectious pancreatic necrosis virus / Q000302:isolation & purification*; D007668:Kidney / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D020317:Salmo salar; D013048:Specimen Handling / Q000662:veterinary*