title: Validation of a real time PCR for classical Swine Fever diagnosis.
abstract: The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5'NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5'NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Natanael Lamas
authors.initials: NL
authors.lastname: Dias
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Antônio Augusto
authors.initials: AA
authors.lastname: Fonseca Júnior
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Anapolino Macedo
authors.initials: AM
authors.lastname: Oliveira
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Erica Bravo
authors.initials: EB
authors.lastname: Sales
authors.affiliation: Embrapa Dairy Cattle, Rua Eugênio do Nascimento 610, 36038-330 Juiz de Fora, MG, Brazil.
authors.forename: Bruna Rios Coelho
authors.initials: BR
authors.lastname: Alves
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Fernanda Alves
authors.initials: FA
authors.lastname: Dorella
authors.affiliation: Laboratório de Biologia Molecular e Laboratório de Diagnóstico de Doenás Virais, Laboratório Nacional Agropecuário-Minas Gerais (LANAGRO-MG), Ministério da Agricultura, Pecuária e Abastecimento, Avenida Rômulo Joviano s/n, Fazenda Modelo, 33600-000 Pedro Leopoldo, MG, Brazil.
authors.forename: Marcelo Fernandes
authors.identifier: 0000-0001-8457-7875
authors.initials: MF
authors.lastname: Camargos