Serotype-specific detection of African horsesickness virus by real-time PCR and the influence of genetic variations

Real-time PCR hybridization probe sets were tested for the specific detection of amplified genome segment 2 cDNA from all nine serotypes of African horsesickness virus (AHSV). The hybridization probes were derived from the sequences of genome segments 2 of the nine reference strains of the virus and were designed to have clearly distinguishable peak melting temperatures. Viral dsRNA from each of the serotypes was specifically detected after reverse transcription, real-time PCR and melting curve analysis. The method was used to successfully serotype a range of field isolates, although most of the these showed peak melting temperature shifts. These shifts could be related to nucleotide substitutions in the regions that are targeted by the probes. Sensitivity was demonstrated to be sufficient for use with dsRNA isolated directly from infected organ samples, making it potentially useful as a rapid diagnostic tool.