Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. Th e objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-1 31) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, gr oups of 5 washed NFD and groups of 5 developed, washed embryos were harvest ed. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with t rypsin and cryopreservation in ethylene glycol. All washes were performed a ccording to International Embryo Transfer Society standards. Developed embr yos and NFD were sonicated prior to assay. All samples were assayed for BVD V using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation a nd RT-nPCR. Results from viral assays of other exposed samples indicate enh anced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, s pecificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system. (C) 2001 b y Elsevier Science Inc.