title: Validation of two real-time PCRs targeting the PE-PGRS 20 gene and the region of difference 4 for the characterization of Mycobacterium bovis isolates.
abstract: This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg (diagnostic sensitivity and specificity were 100 and 100%. The qPCR tests were performed using 4 extraction sets, 3 qPCR kits, and with a range of equipment; yet, all combinations produced similar results in a diagnostic test, demonstrating the robustness of this method. The techniques proved to be efficient, robust, sensitive, and specific for the diagnosis of M. bovis.
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: M L
authors.initials: ML
authors.lastname: Sales
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: A A
authors.initials: AA
authors.lastname: Fonseca
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: L
authors.initials: L
authors.lastname: Orzil
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: A P
authors.initials: AP
authors.lastname: Alencar
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: M A
authors.initials: MA
authors.lastname: Hodon
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: M A
authors.initials: MA
authors.lastname: Issa
authors.affiliation: Lanagro/MG, Ministério da Agricultura, Pecuária e Abastecimento, Pedro Leopoldo, MG, Brasil.
authors.forename: P M
authors.initials: PM
authors.lastname: Soares Filho
authors.affiliation: EMBRAPA Gado de Leite, Juiz de Fora, MG, Brasil.
authors.forename: M R
authors.initials: MR
authors.lastname: Silva
authors.affiliation: Escola de Veterinária da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil.
authors.forename: A P
authors.initials: AP
authors.lastname: Lage
authors.affiliation: Escola de Veterinária da Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil mabryan@vet.ufmg.br.
authors.forename: M B
authors.initials: MB
authors.lastname: Heinemann
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D002417:Cattle; D004269:DNA, Bacterial; D007364:Intercalating Agents; D009163:Mycobacterium bovis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D014380:Tuberculosis, Bovine / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007364:Intercalating Agents