A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

摘要

differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-1 1) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes I and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790 bp specific BTV PCR product. Likewise, the EHDV primers produced a 387 bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls.