PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen

Detalhes bibliográficos
Autor(a) principal: Dias, Francisca Elda Ferreira
Data de Publicação: 2013
Outros Autores: Nunes, Caris Maroni [UNESP], Cavalcante, Tânia Vasconcelos, Castro, Andréa Azevedo Pires De, Ferreira, Jorge Luis, Garcia, José Fernando [UNESP]
Tipo de documento: Artigo
Idioma: por
Título da fonte: Repositório Institucional da UNESP
Texto Completo: http://dx.doi.org/10.5216/cab.v14i3.7638
http://hdl.handle.net/11449/109899
Resumo: This study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Brucella abortus diagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with B. abortus (10(0) to 10(7) bacteria/mL) and DNA was extracted by phenol/chloroform protocol. DNA was amplified by PCR with oligonucleotides previously described BF-5'gcgctcaggctgccgacgcaa3' (6-FAM labeled) and BR-5'accagccattgcggtcggta3' for B. abortus. Oligonucleotides generated DNA fragments of 193 bp. DNA fragments visualization was done under UV light at silver stained 8% poliacrylamide gel, and fluorescent capillary electrophoresis performed in an automatic DNA fragment analyzer. The detection limit of capillary electrophoresis for B. abortus was 10³ bacteria/mL, while for silver stained 8% poliacrylamide gel it was 10(5) bacteria/mL. PCR with fluorescent capillary electrophoresis is fast, efficient and highly sensitive test for DNA detection of Brucella in bovine semen, and itcan be an important tool for health evaluation of the herd and semen sanitary control in artificial insemination centers.
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spelling PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semenFluorescent PCR associated with capillary electrophoresis as a diagnostic tool of bacteria in semenBrucella abortusdiagnósticoeletroforese capilarPCR fluorescentesêmen bovinobovine semenBrucella abortuscapillary electrophoresisdiagnostic fluorescent PCRThis study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Brucella abortus diagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with B. abortus (10(0) to 10(7) bacteria/mL) and DNA was extracted by phenol/chloroform protocol. DNA was amplified by PCR with oligonucleotides previously described BF-5'gcgctcaggctgccgacgcaa3' (6-FAM labeled) and BR-5'accagccattgcggtcggta3' for B. abortus. Oligonucleotides generated DNA fragments of 193 bp. DNA fragments visualization was done under UV light at silver stained 8% poliacrylamide gel, and fluorescent capillary electrophoresis performed in an automatic DNA fragment analyzer. The detection limit of capillary electrophoresis for B. abortus was 10³ bacteria/mL, while for silver stained 8% poliacrylamide gel it was 10(5) bacteria/mL. PCR with fluorescent capillary electrophoresis is fast, efficient and highly sensitive test for DNA detection of Brucella in bovine semen, and itcan be an important tool for health evaluation of the herd and semen sanitary control in artificial insemination centers.Este estudo avaliou o limiar de detecção da técnica de PCR aliada à eletroforese capilar para diagnóstico da Brucella abortus em sêmen bovino. Doses inseminantes livres de patógenos foram contaminadas experimentalmente com B. abortus em escalas que variavam de 10(0) a 10(7) bactérias/mL e submetidas à extração de DNA pelo método de fenol/clorofórmio. A amplificação por PCR foi realizada utilizando-se oligonucleotídeos iniciadores, previamente descritos na literatura, BF-5'gcgctcaggctgccgacgcaa3' (cromóforo FAM) e BR-5'accagccattgcggtcggta3' para B. abortus.) Os pares de oligonucleotídeos geraram fragmentos de 193 pb. Após PCR, a visualização dos fragmentos foi realizada em gel de acrilamida 8% corada pela prata e por eletroforese capilar fluorescente em equipamento automático de análise de fragmentos de DNA. A detecção de DNA de B. abortus em sêmen bovino através de eletroforese capilar fluorescente foi possível a partir de concentração de 10³ bactérias/mL, enquanto que em gel de poliacrilamida 8% o limite de detecção foi de 10(5) bactérias/mL. A eletroforese capilar demonstrou ser uma alternativa rápida, eficaz e de alta sensibilidade na detecção de DNA de Brucella em sêmen bovino, podendo ser uma valiosa ferramenta para a avaliação da sanidade do rebanho e para o controle de qualidade do sêmen produzido em centrais de inseminação artificial.Universidade Federal do TocantinsUniversidade Estadual PaulistaUniversidade Estadual PaulistaUniversidade Federal de GoiásUniversidade Federal do Tocantins (UFT)Universidade Estadual Paulista (Unesp)Dias, Francisca Elda FerreiraNunes, Caris Maroni [UNESP]Cavalcante, Tânia VasconcelosCastro, Andréa Azevedo Pires DeFerreira, Jorge LuisGarcia, José Fernando [UNESP]2014-10-01T13:08:39Z2014-10-01T13:08:39Z2013-09-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/article366-372application/pdfhttp://dx.doi.org/10.5216/cab.v14i3.7638Ciência Animal Brasileira. Universidade Federal de Goiás, v. 14, n. 3, p. 366-372, 2013.1809-6891http://hdl.handle.net/11449/10989910.5216/cab.v14i3.7638S1809-689120130003000142-s2.0-84885362065S1809-68912013000300014.pdf18923598712074089991374083045897SciELOreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPporCiência Animal Brasileira0,216info:eu-repo/semantics/openAccess2023-10-26T06:11:28Zoai:repositorio.unesp.br:11449/109899Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-10-26T06:11:28Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false
dc.title.none.fl_str_mv PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
Fluorescent PCR associated with capillary electrophoresis as a diagnostic tool of bacteria in semen
title PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
spellingShingle PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
Dias, Francisca Elda Ferreira
Brucella abortus
diagnóstico
eletroforese capilar
PCR fluorescente
sêmen bovino
bovine semen
Brucella abortus
capillary electrophoresis
diagnostic fluorescent PCR
title_short PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
title_full PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
title_fullStr PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
title_full_unstemmed PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
title_sort PCR fluorescente associada à eletroforese capilar como ferramenta de diagnóstico de bactérias no semen
author Dias, Francisca Elda Ferreira
author_facet Dias, Francisca Elda Ferreira
Nunes, Caris Maroni [UNESP]
Cavalcante, Tânia Vasconcelos
Castro, Andréa Azevedo Pires De
Ferreira, Jorge Luis
Garcia, José Fernando [UNESP]
author_role author
author2 Nunes, Caris Maroni [UNESP]
Cavalcante, Tânia Vasconcelos
Castro, Andréa Azevedo Pires De
Ferreira, Jorge Luis
Garcia, José Fernando [UNESP]
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade Federal do Tocantins (UFT)
Universidade Estadual Paulista (Unesp)
dc.contributor.author.fl_str_mv Dias, Francisca Elda Ferreira
Nunes, Caris Maroni [UNESP]
Cavalcante, Tânia Vasconcelos
Castro, Andréa Azevedo Pires De
Ferreira, Jorge Luis
Garcia, José Fernando [UNESP]
dc.subject.por.fl_str_mv Brucella abortus
diagnóstico
eletroforese capilar
PCR fluorescente
sêmen bovino
bovine semen
Brucella abortus
capillary electrophoresis
diagnostic fluorescent PCR
topic Brucella abortus
diagnóstico
eletroforese capilar
PCR fluorescente
sêmen bovino
bovine semen
Brucella abortus
capillary electrophoresis
diagnostic fluorescent PCR
description This study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Brucella abortus diagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with B. abortus (10(0) to 10(7) bacteria/mL) and DNA was extracted by phenol/chloroform protocol. DNA was amplified by PCR with oligonucleotides previously described BF-5'gcgctcaggctgccgacgcaa3' (6-FAM labeled) and BR-5'accagccattgcggtcggta3' for B. abortus. Oligonucleotides generated DNA fragments of 193 bp. DNA fragments visualization was done under UV light at silver stained 8% poliacrylamide gel, and fluorescent capillary electrophoresis performed in an automatic DNA fragment analyzer. The detection limit of capillary electrophoresis for B. abortus was 10³ bacteria/mL, while for silver stained 8% poliacrylamide gel it was 10(5) bacteria/mL. PCR with fluorescent capillary electrophoresis is fast, efficient and highly sensitive test for DNA detection of Brucella in bovine semen, and itcan be an important tool for health evaluation of the herd and semen sanitary control in artificial insemination centers.
publishDate 2013
dc.date.none.fl_str_mv 2013-09-01
2014-10-01T13:08:39Z
2014-10-01T13:08:39Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.5216/cab.v14i3.7638
Ciência Animal Brasileira. Universidade Federal de Goiás, v. 14, n. 3, p. 366-372, 2013.
1809-6891
http://hdl.handle.net/11449/109899
10.5216/cab.v14i3.7638
S1809-68912013000300014
2-s2.0-84885362065
S1809-68912013000300014.pdf
1892359871207408
9991374083045897
url http://dx.doi.org/10.5216/cab.v14i3.7638
http://hdl.handle.net/11449/109899
identifier_str_mv Ciência Animal Brasileira. Universidade Federal de Goiás, v. 14, n. 3, p. 366-372, 2013.
1809-6891
10.5216/cab.v14i3.7638
S1809-68912013000300014
2-s2.0-84885362065
S1809-68912013000300014.pdf
1892359871207408
9991374083045897
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv Ciência Animal Brasileira
0,216
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 366-372
application/pdf
dc.publisher.none.fl_str_mv Universidade Federal de Goiás
publisher.none.fl_str_mv Universidade Federal de Goiás
dc.source.none.fl_str_mv SciELO
reponame:Repositório Institucional da UNESP
instname:Universidade Estadual Paulista (UNESP)
instacron:UNESP
instname_str Universidade Estadual Paulista (UNESP)
instacron_str UNESP
institution UNESP
reponame_str Repositório Institucional da UNESP
collection Repositório Institucional da UNESP
repository.name.fl_str_mv Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)
repository.mail.fl_str_mv
_version_ 1799964710366347264

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