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Título: | Development of an in situ hybridisation procedure for the detection of sole aquabirnavirus in infected fish cell cultures |
Autor: | Alonso, M. Carmen; Cano Cejas, Irene; Castro, Dolores; Pérez Prieto, Sara I. CSIC ; Borrego, Juan J. | Palabras clave: | Immunofluorescence In situ hybridisation Sole aquabirnavirus Tissue culture |
Fecha de publicación: | 15-mar-2004 | Editor: | Elsevier BV | Citación: | Journal of Virological Methods 116(2): 133-138 (2004) | Resumen: | An in situ hybridisation (ISH) technique has been developed to detect sole aquabirnavirus in infected fish cell lines bluegill fibroblast (BF-2), EPC, and chinook salmon embryo cells (CHSE-214). A 613bp cDNA probe for viral RNA coding for a fragment of VP2 protein was generated by reverse transcription polymerase chain reaction (RT-PCR) using infectious pancreatic necrosis virus (IPNV) specific DNA primers. Infected cells were strongly labelled, and no non-specific reaction was observed in non-infected cells used as negative controls. The specificity of the probe was examined by testing it against a range of IPNV serotypes such as Ab, Sp and VR-299. The ISH technique was compared with the immunofluorescence procedure to determine the sensitivity of detection of sole aquabirnavirus in BF-2 cells. The probe used in the ISH technique detected weak positivity at 8h post-inoculation (p.i.) in the cytoplasm of infected BF-2 cells inoculated with 103 TCID 50/ml, whilst the labelling appears at 24h p.i. when the immunofluorescence technique was applied. At all other time intervals the results were equivalent. | Versión del editor: | http://dx.doi.org/10.1016/j.jviromet.2003.11.003 | URI: | http://hdl.handle.net/10261/251186 | DOI: | 10.1016/j.jviromet.2003.11.003 | Identificadores: | doi: 10.1016/j.jviromet.2003.11.003 issn: 0166-0934 |
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