A duplex RT-PCR assay for detection of genome segment 7 (VP7 gene) from 24 BTV serotypes

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 vir-us 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)). Published by Elsevier B.V.