title: Validation of a reference control for an SYBR-Green fluorescence assay-based real-time PCR for detection of bovine herpesvirus 5 in experimentally exposed bovine embryos.
abstract: The objective of this study was to optimize an internal control to improve SYBR-Green-based qPCR to amplify/detect the BoHV-5 US9 gene in bovine embryos produced in vitro and experimentally exposed to the virus. We designed an SYBR-Green-based binding assay that is quick to perform, reliable, easily optimized and compares well with the published assay. Herein we demonstrated its general applicability to detect BoHV-5 US9 gene in bovine embryos produced in vitro experimentally exposed to BoHV-5. In order to validate the assay, three different reference genes were tested; and the histone 2a gene was shown to be the most adequate for normalizing the qPCR reaction, by considering melting and standard curves (p < 0.05). On the other hand, no differences were found in the development of bovine embryos in vitro whether they were exposed to BoHV-5 reference and field strains comparing to unexposed embryos. The developed qPCR assay may have important field applications as it provides an accurate BoHV-5 US9 gene detection using a proven reference gene and is considerably less expensive than the TaqMan qPCR currently employed in sanitary programs.
authors.affiliation: UNESP, São Paulo State University, Laboratory of Animal Virology and College of Veterinary Medicine, São Paulo 16050-680, Brazil. Electronic address: tcardoso@fmva.unesp.br.
authors.forename: Tereza Cristina
authors.initials: TC
authors.lastname: Cardoso
authors.forename: Camila
authors.initials: C
authors.lastname: Silva-Frade
authors.forename: Cilene Vidovix
authors.initials: CV
authors.lastname: Táparo
authors.forename: Lucas Hidenori
authors.initials: LH
authors.lastname: Okamura
authors.forename: Eduardo Furtado
authors.initials: EF
authors.lastname: Flores
mesh_terms: D000818:Animals; D002417:Cattle / Q000196:embryology* / Q000821:virology; D002418:Cattle Diseases / Q000821:virology*; D004622:Embryo, Mammalian / Q000821:virology*; D005453:Fluorescence; D005456:Fluorescent Dyes; D005814:Genes, Viral; D006566:Herpesviridae Infections / Q000662:veterinary* / Q000821:virology; D025881:Herpesvirus 5, Bovine / Q000235:genetics* / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D005456:Fluorescent Dyes
keywords: BoHV-5; Bovine; Embryos; Quantitative PCR