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

Autor(es)

Cardoso, Tereza Cristina ^{[1, 2]} ; Silva-Frade, Camila ^{[1, 2]} ; Taparo, Cilene Vidovix ^{[1, 2]} ; Okamura, Lucas Hidenori ^{[1, 2]} ; Flores, Eduardo Furtado ^[3]

Afiliação do(s) autor(es)

^[1] UNESP Sao Paulo State Univ, Lab Anim Virol, BR-16050680 Sao Paulo - Brazil

^[2] Coll Vet Med, BR-16050680 Sao Paulo - Brazil

^[3] Univ Fed Santa Maria, Coll Vet Med, Dept Vet Prevent Med, BR-97115900 Santa Maria, RS - Brazil

• Tipo de documento: Artigo Científico
• Fonte: MOLECULAR AND CELLULAR PROBES; v. 27, n. 5-6, p. 237-242, OCT-DEC 2013.
• Citações Web of Science: 5

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 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. (C) 2013 Elsevier Ltd. All rights reserved. (AU)

• Processo FAPESP: 10/52465-9 - Isolamento e caracterização genética do reovirus de perus (TReoV) provenientes de quadros de enterite associados a PEMS (Poult Enteritis Mortality Syndrome) no Brasil
• Beneficiário: Tereza Cristina Cardoso da Silva
• Modalidade de apoio: Auxílio à Pesquisa - Regular