African horsesickness virus serotyping and identification of multiple co-infecting serotypes with a single genome segment 2 RT-PCR amplification and reverse line blot hybridization

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

“…The serotype of an AHSV can be determined indirectly using techniques that characterize segment 2 of the virus genome. One such a method, which is sensitive enough to be used directly on the dsRNA isolated from clinical specimens, has been described (Koekemoer and Van Dijk, 2004). It entails RT-PCR amplification of cDNA of the 5 -end of genome segment 2 which is then characterized by hybridization with probes on a solid support membrane.…”

“…Viral RNA was extracted from infected cells with a commercial acid-phenol/guanidinium thiocyanate based reagent (TRI-reagent, Molecular Research Centre, Inc.) as described before (Koekemoer and Van Dijk, 2004). dsRNA was isolated by means of differential LiCl-precipitation.…”

“…A universal set of RT-PCR primers (Koekemoer and Van Dijk, 2004) was used to amplify the first 521-553 base pairs of genome segment 2 of all the reference and field isolate viruses. Nine sets of hybridization probes were designed to each hybridize only with target cDNA from the complementary AHSV serotype and to have a unique peak T m that could be identified by a melting curve analysis.…”

“…The dsRNA was denatured with MMOH and RT was initiated with genome segment 2 universal primers as described before (Koekemoer and Van Dijk, 2004). One microliter of the cDNA mixture was used to perform the realtime PCR in a final volume of 20 l. The real-time PCR mixture consisted of the LightCycler®FastStart DNA Master PLUS HybProbe master mix (Roche Diagnostics), 0.1 M of each of the universal segment 2 primers (Koekemoer and Van Dijk, 2004) and 0.2 M each of the anchor and sensor probes ( Table 1). The hybridization probes were manufactured by TIB MOLBIOL GmbH.…”

“…A logical conclusion was to develop serotype-specific molecular tests that identify nucleotide sequences on genome segment 2. These have been reported in the form of serotype-specific RT-PCR primers (Sailleau et al, 2000) and genome segment 2 serotype-specific probe hybridization (Koekemoer et al, 2000;Koekemoer and Van Dijk, 2004). With the latter method it was shown that there is enough variance between the nine serotypes for probes, consisting of partial genome segment 2 amplification products, to hybridize in a serotype-specific manner.…”