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
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Data
2013-10-01
Autores
Cardoso, Tereza Cristina
Silva-Frade, Camila [UNESP]
Táparo, Cilene Vidovix [UNESP]
Okamura, Lucas Hidenori [UNESP]
Flores, Eduardo Furtado
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Resumo
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 invitro 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 invitro 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 invitro 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. © 2013 Elsevier Ltd.
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BoHV-5, Bovine, Embryos, Quantitative PCR, Bovine herpes virus, computer program, controlled study, cow, embryo, gene, histone 2a gene, nonhuman, oocyte, priority journal, real time polymerase chain reaction, sybr green fluorescence assay, zygote, Bovinae, Bovine herpesvirus 5
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Molecular and Cellular Probes, v. 27, n. 5-6, p. 237-242, 2013.