Development of a duplex quantitative polymerase chain reaction assay for detection of bovine herpesvirus 1 and bovine viral diarrhea virus in bovine follicular fluid

The objective of this study was to develop a duplex quantitative polymerase chain reaction (qPCR) assay for simultaneous detection of bovine herpesvirus 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) type I and type II. Follicular fluid was collected from a BoHV-1 acutely infected heifer, a BVDV I persistently infected heifer, and from 10 ovaries recovered from an abattoir. Both the BoHV-1 and BVDV contaminated follicular fluid were diluted 1:5 to 1:107 using the pooled, abattoir-origin follicular fluid. Each dilution sample was analyzed using the duplex qPCR, virus isolation, reverse transcription-nested PCR (RT-nPCR), and BoHV-1 qPCR. The duplex qPCR was able to simultaneously detect BoHV-1 and BVDV I in the fluid diluted to 1:100 and 1:1000, respectively. These results corresponded with the reverse transcription-nested PCR and BoHV-1 qPCR. Therefore, the duplex qPCR might be used for quality assurance testing to identify these two viruses in cells, fluids and tissues collected from donor animals and used in reproductive technologies.