Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1
In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA sa...
| Autores principales: | , , , , , , , , , , , , , , |
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| Formato: | Artículo |
| Lenguaje: | Inglés |
| Publicado: |
PLOS
2019
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| Materias: | |
| Acceso en línea: | http://hdl.handle.net/20.500.12123/4855 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091023 https://doi.org/10.1371/journal.pone.0091023 |
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| author | Araújo, Cristina Pires de Osório, Ana Luiza Alves Rosa Jorge, Klaudia dos Santos Goncalves Ramos, Carlos Alberto N. Filho, Antonio Francisco S. Vidal, Carlos Eugênio Soto Roxo, Eliana Nishibe, Christiane Almeida Junior, Nalvo Franco de Júnior, Antônio A. F. Silva, Marcio Roberto Neto, Jose Diomedes Barbosa Cerqueira, Valíria Duarte Zumarraga, Martin Jose Araujo, Flabio Ribeiro de |
| author_browse | Almeida Junior, Nalvo Franco de Araujo, Flabio Ribeiro de Araújo, Cristina Pires de Cerqueira, Valíria Duarte Filho, Antonio Francisco S. Jorge, Klaudia dos Santos Goncalves Júnior, Antônio A. F. Neto, Jose Diomedes Barbosa Nishibe, Christiane Osório, Ana Luiza Alves Rosa Ramos, Carlos Alberto N. Roxo, Eliana Silva, Marcio Roberto Vidal, Carlos Eugênio Soto Zumarraga, Martin Jose |
| author_facet | Araújo, Cristina Pires de Osório, Ana Luiza Alves Rosa Jorge, Klaudia dos Santos Goncalves Ramos, Carlos Alberto N. Filho, Antonio Francisco S. Vidal, Carlos Eugênio Soto Roxo, Eliana Nishibe, Christiane Almeida Junior, Nalvo Franco de Júnior, Antônio A. F. Silva, Marcio Roberto Neto, Jose Diomedes Barbosa Cerqueira, Valíria Duarte Zumarraga, Martin Jose Araujo, Flabio Ribeiro de |
| author_sort | Araújo, Cristina Pires de |
| collection | INTA Digital |
| description | In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. |
| format | Artículo |
| id | INTA4855 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | PLOS |
| publisherStr | PLOS |
| record_format | dspace |
| spelling | INTA48552019-04-09T13:26:38Z Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 Araújo, Cristina Pires de Osório, Ana Luiza Alves Rosa Jorge, Klaudia dos Santos Goncalves Ramos, Carlos Alberto N. Filho, Antonio Francisco S. Vidal, Carlos Eugênio Soto Roxo, Eliana Nishibe, Christiane Almeida Junior, Nalvo Franco de Júnior, Antônio A. F. Silva, Marcio Roberto Neto, Jose Diomedes Barbosa Cerqueira, Valíria Duarte Zumarraga, Martin Jose Araujo, Flabio Ribeiro de Mycobacterium Bovis Tejidos Animales Ganado Bovino ADN Tuberculosis PCR Animal Tissues Cattle DNA TbD1 Polymerase Chain Reaction In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. Instituto de Biotecnología Fil: Araújo, Cristina Pires de. Universidade Federal de Mato Grosso do Sul. Faculdade de Medicina Veterinária e Zootecnia. Programa de Pós-graduação em Ciência Animal; Brasil Fil: Osório, Ana Luiza Alves Rosa. Universidade Federal de Mato Grosso do Sul. Faculdade de Medicina Veterinária e Zootecnia. Programa de Pós-graduação em Ciência Animal; Brasil Fil: Jorge, Klaudia dos Santos Goncalves. Universidade Federal de Mato Grosso do Sul. Faculdade de Medicina Veterinária e Zootecnia. Programa de Pós-graduação em Ciência Animal; Brasil Fil: Ramos, Carlos Alberto N. Bolsista DTI, CNPq; Brasil Fil: Filho, Antonio Francisco S. Universidade Federal de Mato Grosso do Sul. Faculdade de Medicina Veterinária e Zootecnia. Programa de Pós-graduação em Ciência Animal; Brasil Fil: Vidal, Carlos Eugênio Soto. Universidade Federal de Santa Maria. Programa de Pos-Graduación en Medicina Veterinaria; Brasil Fil: Roxo, Eliana. Instituto Biológico de São Paulo; Brasil Fil: Nishibe, Christiane. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; Brasil Fil: Almeida Junior, Nalvo Franco de. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; Brasil Fil: Júnior, Antônio A. F. Laboratório Nacional Agropecuário, Pedro Leopoldo; Brasil Fil: Silva, Marcio Roberto. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Leite; Brasil Fil: Neto, Jose Diomedes Barbosa. Universidade Federal do Pará; Brasil Fil: Cerqueira, Valíria Duarte. Universidade Federal do Pará; Brasil Fil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; Brasil 2019-04-09T13:15:57Z 2019-04-09T13:15:57Z 2014-03 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://hdl.handle.net/20.500.12123/4855 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091023 1932-6203 https://doi.org/10.1371/journal.pone.0091023 eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-sa/4.0/ Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) application/pdf PLOS PLoS ONE 9 (3) : e91023. (2014) |
| spellingShingle | Mycobacterium Bovis Tejidos Animales Ganado Bovino ADN Tuberculosis PCR Animal Tissues Cattle DNA TbD1 Polymerase Chain Reaction Araújo, Cristina Pires de Osório, Ana Luiza Alves Rosa Jorge, Klaudia dos Santos Goncalves Ramos, Carlos Alberto N. Filho, Antonio Francisco S. Vidal, Carlos Eugênio Soto Roxo, Eliana Nishibe, Christiane Almeida Junior, Nalvo Franco de Júnior, Antônio A. F. Silva, Marcio Roberto Neto, Jose Diomedes Barbosa Cerqueira, Valíria Duarte Zumarraga, Martin Jose Araujo, Flabio Ribeiro de Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title | Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title_full | Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title_fullStr | Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title_full_unstemmed | Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title_short | Detection of Mycobacterium bovis in Bovine and Bubaline tissues using Nested-PCR for TbD1 |
| title_sort | detection of mycobacterium bovis in bovine and bubaline tissues using nested pcr for tbd1 |
| topic | Mycobacterium Bovis Tejidos Animales Ganado Bovino ADN Tuberculosis PCR Animal Tissues Cattle DNA TbD1 Polymerase Chain Reaction |
| url | http://hdl.handle.net/20.500.12123/4855 https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0091023 https://doi.org/10.1371/journal.pone.0091023 |
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