Differential diagnosis of bluetongue virus using a reverse transcriptase-polymerase chain reaction for genome segment...

Bluetongue virus (BTV) has persisted within Europe for the past five years, highlighting a need for rapid and reliable virus detection and identification methods. Various RT-PCR protocols and strategies, which target genome segment 7, were evaluated for their ability to detect all members of the BTV species (serogroup), with the aim of developing a fully validated reverse transcriptase-polymerase chain reaction- (RT-PCR) based diagnostic assay. A nested PCR strategy, using near terminal and internal segment 7 primers, detected all 24 BTV serotypes, but also cross-reacted with some other related Orbivirus species. In an attempt to circumvent these problems, conventional PCR and touch-down PCR methods, using similar primers were also investigated. Both methods were able to amplify cDNA from only 21 of the 24 BTV types. Further sequence analyses of the VP7 gene from the remaining isolates (types 7, 15 and 19) will permit the design of additional and more effective virus-species specific primers and RT-PCR-based assays. This may include the introduction of a multiplex PCR system.