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J Virol Methods. 2010 May;165(2):325-9. doi: 10.1016/j.jviromet.2010.02.027. Epub 2010 Feb 26.

Detection of Toggenburg Orbivirus by a segment 2-specific quantitative RT-PCR.

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  • 1Institute of Virology and Immunoprophylaxis, CH-3147 Mittelhaeusern, Switzerland. martin.hofmann@ivi.admin.ch

Abstract

Toggenburg Orbivirus (TOV) has been detected recently in healthy goats in Switzerland. The virus is related closely to bluetongue virus (BTV) and is considered tentatively as a 25th serotype of BTV. Upon detection of additional TOV-positive goats in Switzerland, Germany, and Italy, these TOV isolates were characterized genetically by partial sequencing of the viral genome segment 2 which encodes VP2, the major outer capsid protein of orbiviruses. A TOV-specific RT-qPCR was developed, targeting conserved areas within segment 2. Since TOV cannot be propagated up to now outside its natural host, a synthetic positive control for the RT-qPCR was constructed by cloning the entire coding region of segment 2 and subsequent in vitro transcription of RNA from both ends to obtain double-stranded RNA. The TOV-specific RT-qPCR was able to detect as few as 30 dsRNA copies and proved to be equally sensitive as a pan BTV assay that was shown previously to have a detection limit of 0.001 TCID(50).

Copyright 2010 Elsevier B.V. All rights reserved.

PMID:
20219538
[PubMed - indexed for MEDLINE]
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