|O Hearn, Emily|
|NARAGHI-ARANI, PEJMAN - Lawrence Livermore National Laboratory|
|LENHOFF, RAYMOND - Lawrence Livermore National Laboratory|
Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/16/2009
Publication Date: 11/1/2009
Citation: Wilson, W.C., Hindson, B., O Hearn, E.S., Hall, S., Tellgren-Roth, C., Torres, C., Naraghi-Arani, P., Mecham, J.O., Lenhoff, R. 2009. A Multiplex Real-time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation of Bluetongue Virus and Epizootic Hemorrhagic Disease Virus Serogroups. Journal of Veterinary Diagnostic Investigation, Vol 21:6, 760-770.
Interpretive Summary: This manuscript assay describes a new genetic test for bluetongue and Epizootic hemorrhagic disease viruses that detects and distinguishes between these viruses. This assay is advantageous to previous assays in that it is less labor intensive, less prone to contamination problems, detects and distinguishes these viruses in a single tube. Differentiation of BTV and EHDV is necessary, since diagnosis of infection caused by these viruses is often confused and EHDV that has been associated with bluetongue-like disease in cattle. Although U.S. EHDV strains have not been experimentally proven to cause disease in cattle, there is serologic evidence of infection in cattle.
Technical Abstract: Bluetongue virus (BTV) causes disease in domestic and wild ruminants resulting in significant economic loss. The closely related Epizootic hemorrhagic diseases virus (EHDV) has been associated with bluetongue-like disease in cattle. Although US EHDV strains have not been experimentally proven to cause disease in cattle there is serologic evidence of infection in cattle. Therefore rapid diagnosis and differentiation of BTV and EHDV is required. The genetic sequence information and bioinformatic analysis necessary to design a real-time reverse transcriptase polymerase chain reaction assay for the early detection of indigenous and exotic BTV and EHDV is described. This foundation was accomplished by phylogenetic analysis of two conserved target genes; one that is highly expressed in infected mammalian cells, the other is highly expressed in infected insect cells. The analysis of all BTV and EHDV prototype strains indicated that a complex primer design was necessary for a comprehensive gene amplification diagnostic test. This information has been used as the basis for the development of rapid multiplex BTV/EHDV real-time PCR that detects all known serotypes and distinguishes between BTV and EHDV. The sensitivity of the assay is sufficient for a rapid single tube diagnostic application without the contamination problems associated with standard PCR and especially nested-PCR tests.