Development of a rapid, sensitive and non-lethal diagnostic assay for the detection of viral haemorrhagic septicaemia virus.
A non-lethal diagnostic procedure based on polymerase chain reaction (PCR) technology was developed to detect viral haemorrhagic septicaemia virus (VHSV). Sensitivity of the assay was tested using purified viral RNA and seeded tissues. Detection limits of the reverse transcriptase-polymerase chain reaction (RT-PCR) assay were estimated to be 10 fg of purified RNA and 0.97 x 10(3) or 10(0) TCID(50)/g of seeded tissue, depending on the experimental approach employed (viral adsorption allowed for 1 or 24h). Addition of nested PCR increased sensitivity up to 100-fold when cDNA excised from the agarose gel was used as template. Both, RT-PCR and nested RT-PCR, as well as Southern blot were applied to RNA extracted from blood of experimentally infected brown trout and the results were compared with those obtained by applying the same techniques to tissues and also with those of conventional viral isolation in cell culture. The superiority of the nested RT-PCR applied to blood samples has been clearly demonstrated in terms of sensitivity, obtaining positive results in 85% of fish tested, as against 40% obtained by RT-PCR and Southern blot, and only 5% viral isolations in cell culture. This procedure could turn into an important tool for screening of wild stocks as well as valuable individuals in commercial fish farms, since it makes to kill the fish unnecessary.
López-Vázquez
C
C
Unidad de Ictiopatología, Instituto de Acuicultura, Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, Spain.
Dopazo
C P
CP
Olveira
J G
JG
Barja
J L
JL
Bandín
I
I
D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D003588:Cytopathogenic Effect, Viral; D004847:Epithelial Cells / Q000166:cytology / Q000821:virology; D005069:Evaluation Studies as Topic; D005393:Fish Diseases / Q000175:diagnosis* / Q000821:virology; D007962:Leukocytes / Q000821:virology; D029141:Novirhabdovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D012680:Sensitivity and Specificity; D014337:Trout / Q000821:virology
D012367:RNA, Viral