title: A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes.
abstract: Since 1998, six distinct serotypes of Bluetongue virus (BTV) have invaded Southern and Central Europe, persisting in some regions for up to 6 years and resulting in the deaths of >1.8 million sheep. Rapid and reliable methods of virus detection and identification play an essential part in our fight against bluetongue disease (BT). We have therefore developed and evaluated a duplex, one-step RT-PCR assay that detects genome segment 7 (encoding the major serogroup (virus-species) specific antigen and outer-core-protein VP7) from any of the 24 BTV serotypes. Although Seg-7 is highly conserved, there are sequence differences in the near terminal regions that identify two distinct phylogenetic groups. Two sets of primers (targeting Seg-7 terminal regions of viruses from these two groups) were included in a duplex RT-PCR assay system. Assay sensitivity was evaluated using tissue culture derived virus, infected vector insects and clinical samples (blood and other tissues). The assay reliably amplified Seg-7 from any of the BTV strains tested, including isolates of the 24 BTV serotypes and isolates from different geographic origins. No cross-reactions were detected with members of closely related Orbivirus species (African horsesickness virus (AHSV), Epizootic haemorrhagic disease virus (EHDV), Equine encephalosis virus (EEV) and Palyam virus (PALV)).
authors.affiliation: Arbovirology Department, Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK. simon.anthony@bbsrc.ac.uk <simon.anthony@bbsrc.ac.uk>
authors.forename: S
authors.initials: S
authors.lastname: Anthony
authors.forename: H
authors.initials: H
authors.lastname: Jones
authors.forename: K E
authors.initials: KE
authors.lastname: Darpel
authors.forename: H
authors.initials: H
authors.lastname: Elliott
authors.forename: S
authors.initials: S
authors.lastname: Maan
authors.forename: A
authors.initials: A
authors.lastname: Samuel
authors.forename: P S
authors.initials: PS
authors.lastname: Mellor
authors.forename: P P C
authors.initials: PP
authors.lastname: Mertens
mesh_terms: D000818:Animals; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D002519:Ceratopogonidae / Q000821:virology; D017931:DNA Primers; D005814:Genes, Viral; D007303:Insect Vectors / Q000821:virology; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping; D012756:Sheep; D014758:Viral Core Proteins / Q000235:genetics*; D014766:Viremia / Q000175:diagnosis
chemical_list: D017931:DNA Primers; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus