Abstract:
Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during
2004–2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex
real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes
of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected
horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for
RT, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was
efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay
was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more
sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect
related orbiviruses, such as bluetongue and equine encephalosis viruses.
