title: 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.
abstract: Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The 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-131) 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, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV 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 and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.
authors.affiliation: Auburn University, Alabama, USA. givenmd@vetmed.auburn.edu
authors.forename: M D
authors.initials: MD
authors.lastname: Givens
authors.forename: P K
authors.initials: PK
authors.lastname: Galik
authors.forename: K P
authors.initials: KP
authors.lastname: Riddell
authors.forename: D A
authors.initials: DA
authors.lastname: Stringfellow
authors.forename: K V
authors.initials: KV
authors.lastname: Brock
authors.forename: M D
authors.initials: MD
authors.lastname: Bishop
authors.forename: K J
authors.initials: KJ
authors.lastname: Eilertsen
authors.forename: N M
authors.initials: NM
authors.lastname: Loskutoff
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology* / Q000821:virology*; D018920:Coculture Techniques; D015925:Cryopreservation; D046508:Culture Techniques; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics* / Q000302:isolation & purification*; D005307:Fertilization in Vitro; D020133:Reverse Transcriptase Polymerase Chain Reaction