A new duplex real-time RT-PCR assay for sensitive and specific detection of African horse sickness virus

A new real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for a simple and rapid diagnosis of African Horse Sickness (AHS) was developed. Primers and FAM-labeled TaqMan-MGB probes specific for African horse sickness virus (AHSV) were selected from the consensus sequence of the segment 8 of all 9 serotypes of AHSV reference strains. For the determination of the analytical sensitivity, an in vitro transcript (AHS_ns2 T7) of the target region was constructed and tested. Furthermore, the AHS_ns2 T7 transcript was used either as positive control or as a standard for quantifying target copies. A commercial heterologous Armored RNA was used as an internal positive control (IPC) for both RNA isolation and RT-PCR steps. The qRT-PCR AHS_ns2 was able to amplify the target sequence up to 0.71 copies/reaction. Its flexibility allowed to amplify a wide dynamic range of RNA copies from 1.5 to 0.001 fg. Within this range, the Ct values varied from 18 to 38 cycles with SD values always lower than 0.5 confirming their strong and constant linear correlation with the RNA target. Furthermore the newly designed duplex real-time RT-PCR proved to be strictly AHSV-specific as it did not amplify close related viruses.