Novel gel-based and real-time PCR assays for the improved detection of African horse sickness virus

In order to improve, ensure and accelerate the diagnosis of African horse sickness, a highly devastating, transboundary animal disease listed by the World Animal Health Organisation, (OIE) three novel diagnostic PCR assays were developed and tested in this study. The reverse transcription-PCR (RT-PCR) tests were the following: (a) a conventional, gel-based RT-PCR, (b) a real-time PCR with SYBR-Green-named rRT-PCR SYBR-Green-, and (c) a real-time PCR rRT-PCR with TaqMan (R) probe (termed rRT-PCR TaqMan (R)). The same pair of primers-directed against African Horse Sickness Virus (AHSV) segment 5, encoding the nonstructural protein NS1, is used in the three tests listed above. The three PCR assays detected similarly the nine AHSV serotypes from cultivated viral suspensions of different origins. The RT-PCR assays provided high sensitivity ranging from 0.1 to 1.2 TCID50/ml. The specificity was also high, considering that related viruses, such as Bluetongue virus, and other equine viruses, such as West Nile Virus, remained negative for RT-PCR amplification. The detection of AHSV virus can be completed within 2-3 h. These results indicate that the novel PCR methods described in this paper provide robust and versatile tools that allow rapid and highly specific, simultaneous detection of all AHSV serotypes. (C) 2008 Elsevier B.V. All rights reserved.