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Registro Completo |
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Biblioteca(s): |
Embrapa Agrobiologia. |
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Data corrente: |
25/04/2008 |
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Data da última atualização: |
17/11/2015 |
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Tipo da produção científica: |
Orientação de Tese de Pós-Graduação |
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Autoria: |
GALVÃO, P. G. |
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Título: |
Ajuste metodológico para sequenciamento de plasmídeos de Bacillus thuringiensis subsp. kurstaki estirpe S76. |
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Ano de publicação: |
2008 |
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Fonte/Imprenta: |
2008. 136 p. Dissertação (Mestrado) - Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ. |
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Idioma: |
Português |
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Notas: |
Parceria: UFRJ.
Orientador: José Ivo Baldani;
Co-orientador: Jean Luiz Simões Araújo. |
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Conteúdo: |
0 Bacillus thuringiensis deposita, no citoplasma da celula em esporula~ao, inclus5es cristalinas de natureza proteica com atividade para larvas de insetos de diversas ordens, nemat6ides, acaros e protozoarios. Os genes cry, que codificam as delta-endotoxinas contidas nesses cristais, estao localizados em plasmideos conjugativos de alto peso molecular, que se replicam com baixo numero de c6pias e possuem notavel estabilidade segregacional. Diversos aspectos do complexo perfil de elementos extracromossomais de B. thuringiensis, que pode representar ate 20% do genoma da bacteria, tern sido estudados desde a decada de 1970. Entretanto, 0 seqiienciamento se restringe a regi5es contendo os genes de delta-endotoxinas e ffagmentos adjacentes. Estudos recentes utilizando a estirpe brasileira 876 de B. thuringiensis kurstaki, indicaram uma alta atividade entomopatogenica contra 0 genero Diatraea. 8abe-se que nas estirpes da subespecie kurstaki os genes responsaveis pel a produ~ao das delta-endotoxinas com atividade para Lepidoptera estao localizados em plasmideos de aproximadamente 44 e 110 MDa. Entretanto, pouco e conhecido a respeito dos outros plasmideos de Btk 876. 0 seqiienciamento de plasmideos desta estirpe podera fomecer uma serie de novas informa~5es a respeito do DNA extracromossomal desta bacteria, explicando, inclusive, a alta mortalidade causada por esta estirpe. Ate 0 momento, existem poucos estudos caracterizando os plasmideos de B. thuringiensis, principalmente os maiores. Por isso, a otimiza~ao da metodologia utilizada para extra~ao e purifica~ao dessas moIeculas tomou-se extremamente necessaria. Os plasmideos de BtK 876 foram extraidos e purificados atraves de ultracentrifuga~ao em gradiente de CsCI e com 0 kit comercial QIAGE~, sendo que ambas as tecnicas apresentaram urn perfil plasmidial semelhante. Foi selecionado 0 plasmideo denominado pBt20 de BtK 876, que foi extraido do gel e posteriormente eletroeluido e amplificado com 0 kit comercial GenomiPhiTM DNA Amplification, visando aumentar a mBSSB de DNA plasmidial para as futuras etapas do trabalho. 0 DNA plasmidial do pBt20 foi ffagmentado atraves de nebuliza~ao e os ftagmentos obtidos (100 a 2000 pb) geraram uma bibiioteca com aproximadamente 6.600 clones. Foram seqiienciados 1.440 clones, que possibilitaram a montagem de 56 contigs, totalizando 33,7 kb. A compara~ao desses contigs com 0 banco de dados revelou 41% com valores de e-value nao-significativos, 7% que codificam para proteinas que nao apresentaram similaridade com 0 banco de dados, 18% que codificam para proteinas hipoteticas conservadas e 34% que possuem fun~5es putativas. Dentre as proteinas similares ao banco de dados destacam-se HxlR, Doc, Mob, Rep, alem das enzimas pertencentes as familias beta-Iactamases, exoribonuclease e fosfotransferases. A ocorrencia de redundancia observada na biblioteca, e consequentemente, na montagem das sequencias, nao permitiu 0 fechamento do sequenciamento do pBt20.
Bacillus thuringiensis deposits in the sporulation cell cytoplasm, crystalline protein inclusions with activity for larvae of various insect orders, nematodes, mites and protozoa. The cry genes, which encode the delta-endotoxins are located in high molecular weight plasmids that replicate with low copy number and show remarkable segregational stability. Different aspects of the B. thuringiensis plasmid complex profile, which can represent up to 20% of the bacteria genome, have been studied since the 1970 decade. However, the sequencing is restricted to the regions containing the delta-endotoxins genes and adjacent fTagments. Recent studies using the Brazilian 576 strain of B. thuriflgieflsis kurstaki, showed high activity against the entomopatogenic genera Diatraea. It is known that in kurstaki strains the genes responsible for the production of delta-endotoxins with activity for Lepidoptera are located in plasmids of approximately 44 and 110 MDa. However, little is known about the other plasmids of Btk S76. The sequencing of these plasmids can provide a range of new information about the extracromossomal DNA of this bacterium, even explaining the high mortality caused by this strain. So far, there are few studies characterizing the plasmids of B. thuringiens/s, especially the larger ones. Therefore, the optimization of the extraction and purification methodology for these molecules has become extremely necessary. The plasmids were extracted fTom BtK S76 and purified through ultracentrifugation in CsCI gradient and the commercial kit QIAGEN@, and both techniques showed a similar plasmid profile. It was selected the plasmid called pBt20 of BtK S76, which was extracted fTom the gel and then electroeluted and amplified through the commercial GenomiPhiTM DNA Amplification kit, to increase the plasmid DNA mass for next steps of this work. The plasmid DNA of pBt20 was fragmented through nebulization and a library of approximately 6.600 clones was generated with fragments between 100 to 2000 bp. It was sequenced 1.440 clones, which allowed to assembly 56 contigs, totaling 33.7 Kb. The comparison of these contigs with the database revealed 7% that showed no similarity with the database, 18% that encode for conserved hypothetical proteins and 34% that show putative functions, 41 % with non-significant e-value. Among the proteins similar to the data base there are HxIR, Doc, Mob, Rep, in addition to the enzymes belonging to the beta-Iactamase, exoribonuclease and fosfotransferases families. The occurrence of redundancy found in the library and consequently the assembling of the sequences did not allow to close the sequencing of the pBt20 plasmid. Menos0 Bacillus thuringiensis deposita, no citoplasma da celula em esporula~ao, inclus5es cristalinas de natureza proteica com atividade para larvas de insetos de diversas ordens, nemat6ides, acaros e protozoarios. Os genes cry, que codificam as delta-endotoxinas contidas nesses cristais, estao localizados em plasmideos conjugativos de alto peso molecular, que se replicam com baixo numero de c6pias e possuem notavel estabilidade segregacional. Diversos aspectos do complexo perfil de elementos extracromossomais de B. thuringiensis, que pode representar ate 20% do genoma da bacteria, tern sido estudados desde a decada de 1970. Entretanto, 0 seqiienciamento se restringe a regi5es contendo os genes de delta-endotoxinas e ffagmentos adjacentes. Estudos recentes utilizando a estirpe brasileira 876 de B. thuringiensis kurstaki, indicaram uma alta atividade entomopatogenica contra 0 genero Diatraea. 8abe-se que nas estirpes da subespecie kurstaki os genes responsaveis pel a produ~ao das delta-endotoxinas com atividade para Lepidoptera estao localizados em plasmideos de aproximadamente 44 e 110 MDa. Entretanto, pouco e conhecido a respeito dos outros plasmideos de Btk 876. 0 seqiienciamento de plasmideos desta estirpe podera fomecer uma serie de novas informa~5es a respeito do DNA extracromossomal desta bacteria, explicando, inclusive, a alta mortalidade causada por esta estirpe. Ate 0 momento, existem poucos estudos caracterizando os plasmideos de B. thuringiensis, principalmente os maio... Mostrar Tudo |
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Thesagro: |
Bacillus Thuringiensis; Plasmídeo. |
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Categoria do assunto: |
-- |
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Marc: |
LEADER 06246nam a2200145 a 4500
001 1629856
005 2015-11-17
008 2008 bl uuuu m 00u1 u #d
100 1 $aGALVÃO, P. G.
245 $aAjuste metodológico para sequenciamento de plasmídeos de Bacillus thuringiensis subsp. kurstaki estirpe S76.
260 $a2008. 136 p. Dissertação (Mestrado) - Universidade Federal do Rio de Janeiro, Rio de Janeiro$c2008
500 $aParceria: UFRJ. Orientador: José Ivo Baldani; Co-orientador: Jean Luiz Simões Araújo.
520 $a0 Bacillus thuringiensis deposita, no citoplasma da celula em esporula~ao, inclus5es cristalinas de natureza proteica com atividade para larvas de insetos de diversas ordens, nemat6ides, acaros e protozoarios. Os genes cry, que codificam as delta-endotoxinas contidas nesses cristais, estao localizados em plasmideos conjugativos de alto peso molecular, que se replicam com baixo numero de c6pias e possuem notavel estabilidade segregacional. Diversos aspectos do complexo perfil de elementos extracromossomais de B. thuringiensis, que pode representar ate 20% do genoma da bacteria, tern sido estudados desde a decada de 1970. Entretanto, 0 seqiienciamento se restringe a regi5es contendo os genes de delta-endotoxinas e ffagmentos adjacentes. Estudos recentes utilizando a estirpe brasileira 876 de B. thuringiensis kurstaki, indicaram uma alta atividade entomopatogenica contra 0 genero Diatraea. 8abe-se que nas estirpes da subespecie kurstaki os genes responsaveis pel a produ~ao das delta-endotoxinas com atividade para Lepidoptera estao localizados em plasmideos de aproximadamente 44 e 110 MDa. Entretanto, pouco e conhecido a respeito dos outros plasmideos de Btk 876. 0 seqiienciamento de plasmideos desta estirpe podera fomecer uma serie de novas informa~5es a respeito do DNA extracromossomal desta bacteria, explicando, inclusive, a alta mortalidade causada por esta estirpe. Ate 0 momento, existem poucos estudos caracterizando os plasmideos de B. thuringiensis, principalmente os maiores. Por isso, a otimiza~ao da metodologia utilizada para extra~ao e purifica~ao dessas moIeculas tomou-se extremamente necessaria. Os plasmideos de BtK 876 foram extraidos e purificados atraves de ultracentrifuga~ao em gradiente de CsCI e com 0 kit comercial QIAGE~, sendo que ambas as tecnicas apresentaram urn perfil plasmidial semelhante. Foi selecionado 0 plasmideo denominado pBt20 de BtK 876, que foi extraido do gel e posteriormente eletroeluido e amplificado com 0 kit comercial GenomiPhiTM DNA Amplification, visando aumentar a mBSSB de DNA plasmidial para as futuras etapas do trabalho. 0 DNA plasmidial do pBt20 foi ffagmentado atraves de nebuliza~ao e os ftagmentos obtidos (100 a 2000 pb) geraram uma bibiioteca com aproximadamente 6.600 clones. Foram seqiienciados 1.440 clones, que possibilitaram a montagem de 56 contigs, totalizando 33,7 kb. A compara~ao desses contigs com 0 banco de dados revelou 41% com valores de e-value nao-significativos, 7% que codificam para proteinas que nao apresentaram similaridade com 0 banco de dados, 18% que codificam para proteinas hipoteticas conservadas e 34% que possuem fun~5es putativas. Dentre as proteinas similares ao banco de dados destacam-se HxlR, Doc, Mob, Rep, alem das enzimas pertencentes as familias beta-Iactamases, exoribonuclease e fosfotransferases. A ocorrencia de redundancia observada na biblioteca, e consequentemente, na montagem das sequencias, nao permitiu 0 fechamento do sequenciamento do pBt20. Bacillus thuringiensis deposits in the sporulation cell cytoplasm, crystalline protein inclusions with activity for larvae of various insect orders, nematodes, mites and protozoa. The cry genes, which encode the delta-endotoxins are located in high molecular weight plasmids that replicate with low copy number and show remarkable segregational stability. Different aspects of the B. thuringiensis plasmid complex profile, which can represent up to 20% of the bacteria genome, have been studied since the 1970 decade. However, the sequencing is restricted to the regions containing the delta-endotoxins genes and adjacent fTagments. Recent studies using the Brazilian 576 strain of B. thuriflgieflsis kurstaki, showed high activity against the entomopatogenic genera Diatraea. It is known that in kurstaki strains the genes responsible for the production of delta-endotoxins with activity for Lepidoptera are located in plasmids of approximately 44 and 110 MDa. However, little is known about the other plasmids of Btk S76. The sequencing of these plasmids can provide a range of new information about the extracromossomal DNA of this bacterium, even explaining the high mortality caused by this strain. So far, there are few studies characterizing the plasmids of B. thuringiens/s, especially the larger ones. Therefore, the optimization of the extraction and purification methodology for these molecules has become extremely necessary. The plasmids were extracted fTom BtK S76 and purified through ultracentrifugation in CsCI gradient and the commercial kit QIAGEN@, and both techniques showed a similar plasmid profile. It was selected the plasmid called pBt20 of BtK S76, which was extracted fTom the gel and then electroeluted and amplified through the commercial GenomiPhiTM DNA Amplification kit, to increase the plasmid DNA mass for next steps of this work. The plasmid DNA of pBt20 was fragmented through nebulization and a library of approximately 6.600 clones was generated with fragments between 100 to 2000 bp. It was sequenced 1.440 clones, which allowed to assembly 56 contigs, totaling 33.7 Kb. The comparison of these contigs with the database revealed 7% that showed no similarity with the database, 18% that encode for conserved hypothetical proteins and 34% that show putative functions, 41 % with non-significant e-value. Among the proteins similar to the data base there are HxIR, Doc, Mob, Rep, in addition to the enzymes belonging to the beta-Iactamase, exoribonuclease and fosfotransferases families. The occurrence of redundancy found in the library and consequently the assembling of the sequences did not allow to close the sequencing of the pBt20 plasmid.
650 $aBacillus Thuringiensis
650 $aPlasmídeo
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Registro original: |
Embrapa Agrobiologia (CNPAB) |
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Registro Completo
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Biblioteca(s): |
Embrapa Gado de Corte; Embrapa Unidades Centrais. |
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Data corrente: |
21/03/2018 |
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Data da última atualização: |
23/03/2018 |
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Tipo da produção científica: |
Artigo em Periódico Indexado |
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Circulação/Nível: |
A - 2 |
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Autoria: |
RODRIGUES, R. A.; MENESES, I. I. F. S.; JORGE, K. S. G.; SILVA, M. R.; SANTOS, L. R. dos; LILENBAUM, W.; ETGES, R. N.; ARAUJO, F. R. |
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Afiliação: |
Rudielle A. Rodrigues, Programa de Pós-Graduação em Ciências Veterinárias/Faculdade de Medicina Veterinária e Zootecnia - FAMEZ/Universidade Federal de Mato Grosso do Sul - UFMS; Ingrid I. F. S. Meneses, Programa de Pós-Graduação em Biotecnologia e Biodiversidade da Região Centro-Oeste/Universidade Federal de Mato Grosso do Sul - UFMS; Klaudia S. G. Jorge, Programa de Pós-Graduação em Ciências Veterinárias/Faculdade de Medicina Veterinária e Zootecnia - FAMEZ/Universidade Federal de Mato Grosso do Sul - UFMS; MARCIO ROBERTO SILVA, CNPGL; LENITA RAMIRES DOS SANTOS, CNPGC; Walter Lilenbaum, Laboratório de Bacteriologia Veterinária/Universidade Federal Fluminense - UFF; Rodrigo N. Etges, Secretaria da Agricultura/Pecuária e Irrigação; FLABIO RIBEIRO ARAUJO, CNPGC. |
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Título: |
False-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis. |
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Ano de publicação: |
2017 |
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Fonte/Imprenta: |
Pesquisa Veterinária Brasileira, Brasília, DF, v. 37, n. 12, p. 1380-1384, dezembro. 2017. |
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Idioma: |
Inglês |
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Notas: |
Título em português: Reações falso-negativas ao teste cervical comparativo para tuberculose bovina. |
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Conteúdo: |
According to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success. MenosAccording to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary t... Mostrar Tudo |
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Palavras-Chave: |
Bacteriological culture; Cultura bacteriológica; Reações falso-negativas; Teste Cervical Comparativo; Tuberculose bovina. |
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Thesagro: |
Mycobacterium Bovis. |
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Thesaurus NAL: |
Tuberculosis. |
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Categoria do assunto: |
-- |
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URL: |
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/174398/1/False-negative-reactions-to-the-comparative.pdf
https://ainfo.cnptia.embrapa.br/digital/bitstream/item/174263/1/False-negative-reactions-to-the-comparative.pdf
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Marc: |
LEADER 02625naa a2200301 a 4500
001 2089602
005 2018-03-23
008 2017 bl uuuu u00u1 u #d
100 1 $aRODRIGUES, R. A.
245 $aFalse-negative reactions to the comparative intradermal tuberculin test for bovine tuberculosis.$h[electronic resource]
260 $c2017
500 $aTítulo em português: Reações falso-negativas ao teste cervical comparativo para tuberculose bovina.
520 $aAccording to the Brazilian National Program for the Control and Eradication of Animal Brucellosis and Tuberculosis (PNCEBT), the routine tests for the diagnosis of bovine tuberculosis in the country are the simple intradermal tuberculin test (SITT) of the Ministry of Agriculture, Livestock and Food Supply (MAPA), the caudal fold test and the comparative intradermal tuberculin test (CITT). The latter is also used as a confirmatory test. A group of 53 animals from three dairy herds in a focal area for bovine tuberculosis, that were submitted to depopulation in the state of Rio Grande do Sul, were submitted to the CITT. Tissues were cultured and the resulting colonies were confirmed by PCR and DNA sequencing. Among the 53 animals analyzed using the CITT, 32 (60.4%) were negative, 14 (26.4%) were positive and seven (13.2%) results were inconclusive. The CITT detected 11 of the 39 animals with culture-confirmed M. bovis infection as positive. Among the total of 14 uninfected animals based on cultures, the CBT detected eight as negative. Thus, the CITT demonstrated sensitivity of 28.2% and specificity of 57.1% for the population sampled. A total of 24/32 (75.0%) of the animals with negative CITT results were culture positive (confirmed by PCR) and were considered false negatives based on the CITT. The maintenance of these false-negative animals in herds has serious implications for the control of the disease, since they can be a source of infection. The addition of complementary tests could help identify such animals and increase the odds of diagnostic success.
650 $aTuberculosis
650 $aMycobacterium Bovis
653 $aBacteriological culture
653 $aCultura bacteriológica
653 $aReações falso-negativas
653 $aTeste Cervical Comparativo
653 $aTuberculose bovina
700 1 $aMENESES, I. I. F. S.
700 1 $aJORGE, K. S. G.
700 1 $aSILVA, M. R.
700 1 $aSANTOS, L. R. dos
700 1 $aLILENBAUM, W.
700 1 $aETGES, R. N.
700 1 $aARAUJO, F. R.
773 $tPesquisa Veterinária Brasileira, Brasília, DF$gv. 37, n. 12, p. 1380-1384, dezembro. 2017.
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