Multiplex real-time RT-PCR detection of three viruses associated with the bovine respiratory disease complex

Bovine respiratory disease complex (BRDC) is considered the most significant illness associated with feedlot cattle in North America and possibly worldwide. BRDC is a multi-factorial disease with environmental conditions interacting with multiple viral and bacterial pathogens to produce severe respiratory illness. Bovine herpesvirus 1, bovine viral diarrhoea virus and bovine parainfluenza virus 3 are three of the major viruses associated with BRDC. In this study, a multiplex real-time RT-PCR using Taqman primers and probes was developed to detect simultaneously all three of these important BRDC viruses. The assay was optimised and validated using cell-culture infected material and bovine clinical samples from BRDC cases. The sensitivity of the assay was analysed by comparing the multiplex with the individual singleplex assays, which demonstrated that viral detection was not inhibited, by multiplexing the real-time RT-PCRs. This technique is the first reported real-time PCR method for BPIV-3 and the first multiplex designed to detect three BRDC viral pathogens.