Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos

Detalhes bibliográficos
Autor(a) principal: TELES, José Andreey Almeida
Data de Publicação: 2012
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UFRPE
Texto Completo: http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4636
Resumo: Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37ºC; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.
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spelling MOTA, Rinaldo AparecidoPORTO, Ana Lúcia FigueiredoSÁ, Fabrício Bezerra deSILVA, Karla Patrícia Chaves daTELES, José Andreey Almeida2016-06-08T14:30:49Z2012-02-13TELES, José Andreey Almeida. Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos. 2012. 62 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4636Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37ºC; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.O mormo é uma enfermidade infecto-contagiosa de caráter agudo ou crônico que acomete principalmente os equídeos, causando enormes prejuízos na cadeia produtiva do cavalo. Para controlar a enfermidade, o Ministério da Agricultura, Pecuária e Abastecimento instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico oficial pela fixação do complemento (FC), maleinização e sacrifício dos animais positivos. Os kits atuais utilizados no diagnóstico da doença são importados, dificultando e encarecendo sua aplicação na rotina. Objetivou-se com este estudo padronizar um teste de ELISA indireto utilizando o extrato protéico de Burkholderia mallei isolada a partir de equídeo portador no estado de Pernambuco. As amostras foram cultivadas em ágar sangue 10%, incubada por 48h a 37ºC; posteriormente caracterizou-se fenotípica e genotipicamente uma das colônias isoladas, e em seguida a cultivou em BHI para enriquecimento; logo após, esta foi repicada para o meio Dor-set Henley o qual foi incubado a 37ºC sob 60rpm por oito semanas. Para padronização do teste utilizouse o conjugado Proteína G Peroxidase Sigma na diluição de 1:90.000, com soros diluídos em 1:100 e o antígeno em 1:400. Utilizou-se 60 soros como controle negativo testados frente à FC para determinação do ponto de corte o qual ficou em 0,042nm. Feitas as padronizações, foram testadas 300 amostras, onde 99% (297) foram concordantes com os resultados obtidos na FC. Ao final, o ensaio apresentou 100% de sensibilidade e 98,2% de especificidade, com valores preditivo positivo e negativo de 97,7% e 100% respectivamente. O teste de concordância kappa foi 0,98 e a repetibilidade intra e interplaca ficaram em 8,8% e 10,3% respectivamente. Diante dos resultados obtidos durante os ensaios, atesta-se que o teste de ELISA indireto pode ser utilizado como uma ferramenta de diagnóstico eficiente. Entretanto, mais ensaios devem ser realizados visando consolidar a confiabilidade do referido teste.Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-08T14:30:49Z No. of bitstreams: 1 Jose Andreey Almeida Teles.pdf: 1056555 bytes, checksum: 1cd9d8325b9ce830ca450c9fea318bad (MD5)Made available in DSpace on 2016-06-08T14:30:49Z (GMT). No. of bitstreams: 1 Jose Andreey Almeida Teles.pdf: 1056555 bytes, checksum: 1cd9d8325b9ce830ca450c9fea318bad (MD5) Previous issue date: 2012-02-13Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPqapplication/pdfporUniversidade Federal Rural de PernambucoPrograma de Pós-Graduação em Biociência AnimalUFRPEBrasilDepartamento de Morfologia e Fisiologia AnimalBurkholderia malleiSorologiaDiagnósticoSorologyDiagnosisCIENCIAS AGRARIAS::MEDICINA VETERINARIADesenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeosinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-1510757014399315592600600600600-8922364187987396204453670264235017319-2555911436985713659info:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UFRPEinstname:Universidade Federal Rural de Pernambuco (UFRPE)instacron:UFRPELICENSElicense.txtlicense.txttext/plain; charset=utf-82165http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/4636/1/license.txtbd3efa91386c1718a7f26a329fdcb468MD51ORIGINALJose Andreey Almeida Teles.pdfJose Andreey Almeida Teles.pdfapplication/pdf1056555http://www.tede2.ufrpe.br:8080/tede2/bitstream/tede2/4636/2/Jose+Andreey+Almeida+Teles.pdf1cd9d8325b9ce830ca450c9fea318badMD52tede2/46362024-02-23 12:31:13.2oai:tede2: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Biblioteca Digital de Teses e Dissertaçõeshttp://www.tede2.ufrpe.br:8080/tede/PUBhttp://www.tede2.ufrpe.br:8080/oai/requestbdtd@ufrpe.br ||bdtd@ufrpe.bropendoar:2024-05-28T12:31:59.541888Biblioteca Digital de Teses e Dissertações da UFRPE - Universidade Federal Rural de Pernambuco (UFRPE)false
dc.title.por.fl_str_mv Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
title Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
spellingShingle Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
TELES, José Andreey Almeida
Burkholderia mallei
Sorologia
Diagnóstico
Sorology
Diagnosis
CIENCIAS AGRARIAS::MEDICINA VETERINARIA
title_short Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
title_full Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
title_fullStr Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
title_full_unstemmed Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
title_sort Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos
author TELES, José Andreey Almeida
author_facet TELES, José Andreey Almeida
author_role author
dc.contributor.advisor1.fl_str_mv MOTA, Rinaldo Aparecido
dc.contributor.referee1.fl_str_mv PORTO, Ana Lúcia Figueiredo
dc.contributor.referee2.fl_str_mv SÁ, Fabrício Bezerra de
dc.contributor.referee3.fl_str_mv SILVA, Karla Patrícia Chaves da
dc.contributor.author.fl_str_mv TELES, José Andreey Almeida
contributor_str_mv MOTA, Rinaldo Aparecido
PORTO, Ana Lúcia Figueiredo
SÁ, Fabrício Bezerra de
SILVA, Karla Patrícia Chaves da
dc.subject.por.fl_str_mv Burkholderia mallei
Sorologia
Diagnóstico
topic Burkholderia mallei
Sorologia
Diagnóstico
Sorology
Diagnosis
CIENCIAS AGRARIAS::MEDICINA VETERINARIA
dc.subject.eng.fl_str_mv Sorology
Diagnosis
dc.subject.cnpq.fl_str_mv CIENCIAS AGRARIAS::MEDICINA VETERINARIA
description Glanders is an infectious-contagious disease of acute or chronic character which principally affects horses, causing enormous losses in the productive chain of this animal. To control the disease, the Ministry of Agriculture, Husbandry and Supply instituted mandatory sanitation measures in the entire national territory which include an official diagnosis through the complement fixation (CF) test, maleinization and sacrifice of the animals that are positive. Nowadays the kits used for the diagnosis of the disease are imported, making their routine application difficult and more expensive. The objective of this study was to standardize an indirect ELISA test, using the proteic extract of Burkholderia mallei isolated from a carrier horse in the state of Pernambuco. The samples were cultivated in 10% blood agar and incubated for 48h at 37ºC; later, one of the isolated colonies was characterized phenotypically and genotypically and immediately cultivated in brain heart infusion (BHI) for enrichment; then it was peaked (repicada) for the Dor-set Henley medium which was incubated at 37ºC under 60rpm for eight weeks. To standardize the test the Protein G Peroxidase Sigma Conjugate was used in the dilution of 1:90.000, with serums diluted in 1:100 and the antigen in 1:400. Sixty serums were used as negative controls, tested before the CF to determine the cutting point which was 0.042nm. After establishing the standardization, 300 samples were tested, of which 99% (297) were in agreement with the results obtained in the CF. At the end, of assay presented 100% sensibility and 98.2% specificity, with predictive (preditivo) positive and negative values of 97.7% and 100% respectively. The Kappa concordance test was 0.98 and the intra and interplac repeatability were 8.8% and 10.3% respectively. From the results obtained, it is possible to affirm that the indirect ELISA test can be used as an efficient diagnosis tool. However, more essays must be carried out to consolidate the reliability of this test.
publishDate 2012
dc.date.issued.fl_str_mv 2012-02-13
dc.date.accessioned.fl_str_mv 2016-06-08T14:30:49Z
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dc.identifier.citation.fl_str_mv TELES, José Andreey Almeida. Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos. 2012. 62 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.
dc.identifier.uri.fl_str_mv http://www.tede2.ufrpe.br:8080/tede2/handle/tede2/4636
identifier_str_mv TELES, José Andreey Almeida. Desenvolvimento e avaliação de um teste ELISA indireto para o diagnóstico sorológico do mormo em equídeos. 2012. 62 f. Dissertação (Programa de Pós-Graduação em Biociência Animal) - Universidade Federal Rural de Pernambuco, Recife.
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