title: Detection and differentiation of bovine herpesvirus 1 and 5 using a multiplex real-time polymerase chain reaction.
abstract: A multiplex real-time PCR was developed for the detection and differentiation of two closely related bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5). The multiplex real-time PCR combines a duplex real-time PCR that targets the DNA polymerase gene of BoHV-1 and BoHV-5 and a real-time PCR targeting mitochondrial DNA, as a house-keeping gene, described previously by Cawthraw et al. (2009). The assay correctly identified 22 BoHV-1 and six BoHV-5 isolates from the Biosecurity Sciences Laboratory virus collection. BoHV-1 and BoHV-5 were also correctly identified when incorporated in spiked semen and brain tissue samples. The detection limits of the duplex assay were 10 copies of BoHV-1 and 45 copies of BoHV-5. The multiplex real-time PCR had reaction efficiencies of 1.04 for BoHV-1 and 1.08 for BoHV-5. Standard curves relating Ct value to template copy number had correlation coefficients of 0.989 for BoHV-1 and 0.978 for BoHV-5. The assay specificity was demonstrated by testing bacterial and viral DNA from pathogens commonly isolated from bovine respiratory and reproductive tracts. The validated multiplex real-time PCR was used to detect and differentiate BoHV-1 and BoHV-5 in bovine clinical samples with known histories.
authors.affiliation: Biosecurity Sciences Laboratory, Health and Food Sciences Precinct, Biosecurity Queensland, Department of Employment, Economic Development and Innovation, PO Box 156, Brisbane, Archerfield, Qld 4108, Australia. ibrahim.diallo@deedi.qld.gov.au
authors.forename: Ibrahim S
authors.initials: IS
authors.lastname: Diallo
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Corney
authors.forename: Barry J
authors.initials: BJ
authors.lastname: Rodwell
mesh_terms: D000818:Animals; D002417:Cattle; D002418:Cattle Diseases / Q000175:diagnosis / Q000821:virology*; D004272:DNA, Mitochondrial / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D006566:Herpesviridae Infections / Q000175:diagnosis / Q000662:veterinary* / Q000821:virology; D007242:Herpesvirus 1, Bovine / Q000235:genetics / Q000302:isolation & purification*; D025881:Herpesvirus 5, Bovine / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial; D004279:DNA, Viral