Detection of United States Orbivirus serogroup using a multiplex RT-PCR

A multiplex RT-PCR assay, for simultaneous detection and differentiation of United States serogroup of Orbiviruses, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) in cell culture, was developed. Sets of primers were designed to hybridize to genome segment six of EHDV-2 and to genome segment 10 of BTV-10. The RT-PCR assay utilized a single-tube PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The EHDV primers produced a 387 base pair (bp) specific PCR product from RNA samples of cell culture-adapted EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17; or from total nucleic acid extract of baby hamster kidney (BHK) cells controls. Likewise, the BTV primers generated a 251-bp amplicon from RNA samples of BTV serotypes 2, 10, 11, 13, and 17, whereas EHDV-1 and EHDV-2; and BHK21 cells total nucleic acid extract failed to demonstrate the 251-bp specific BTV PCR product. EHDV and BTV PCR amplification products were easily identified on the basis of size differences on ethidium bromide-stained agarose gels. This multiplex RT-PCR assay provides supportive diagnostic method for rapid detection of BTV and/or EHDV-infections among susceptible ruminants.