FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN

Detalhes bibliográficos
Autor(a) principal: Ferreira Dias, Francisca E.
Data de Publicação: 2013
Outros Autores: Nunes, Cáris Marone, Cavalcante, Tânia Vasconcelos, Castro, Andréa A. Pires de, Ferreira, Jorge Luis, Garcia, José Fernando
Tipo de documento: Artigo
Idioma: por
Título da fonte: Ciência animal brasileira (Online)
Texto Completo: https://revistas.ufg.br/vet/article/view/7638
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 (100 to 107 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 105 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.KEYWORDS: bovine semen; Brucella abortus; capillary electrophoresis; diagnostic fluorescent PCR.
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spelling FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMENPCR FLUORESCENTE ASSOCIADA À ELETROFORESE CAPILAR COMO FERRAMENTA DE DIAGNÓSTICO DE BACTÉRIAS NO SEMENAnimal reproductionsêmen bovinoBrucella abortusPCR fluorescenteEletroforese capilardiagnóstico.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 (100 to 107 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 105 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.KEYWORDS: bovine semen; Brucella abortus; capillary electrophoresis; diagnostic fluorescent PCR.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 100 a 107 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 105 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.ALAVRAS-CHAVE: Brucella abortus; diagnóstico; eletroforese capilar; PCR fluorescente; sêmen bovino. Universidade Federal de Goiás2013-09-29info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionResearchAvaliado por paresPesquisa Cientificaapplication/pdftext/htmlapplication/pdfhttps://revistas.ufg.br/vet/article/view/763810.5216/cab.v14i3.7638Brazilian Animal Science/ Ciência Animal Brasileira; Vol. 14 No. 3 (2013); 366-372Ciência Animal Brasileira / Brazilian Animal Science; v. 14 n. 3 (2013); 366-3721809-68911518-2797reponame:Ciência animal brasileira (Online)instname:Universidade Federal de Goiás (UFG)instacron:UFGporhttps://revistas.ufg.br/vet/article/view/7638/15219https://revistas.ufg.br/vet/article/view/7638/15220https://revistas.ufg.br/vet/article/view/7638/16860Ferreira Dias, Francisca E.Nunes, Cáris MaroneCavalcante, Tânia VasconcelosCastro, Andréa A. Pires deFerreira, Jorge LuisGarcia, José Fernandoinfo:eu-repo/semantics/openAccess2013-10-14T12:20:03Zoai:ojs.revistas.ufg.br:article/7638Revistahttps://revistas.ufg.br/vetPUBhttps://revistas.ufg.br/vet/oai||revistacab@gmail.com1809-68911518-2797opendoar:2024-05-21T19:55:26.727893Ciência animal brasileira (Online) - Universidade Federal de Goiás (UFG)true
dc.title.none.fl_str_mv FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
PCR FLUORESCENTE ASSOCIADA À ELETROFORESE CAPILAR COMO FERRAMENTA DE DIAGNÓSTICO DE BACTÉRIAS NO SEMEN
title FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
spellingShingle FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
Ferreira Dias, Francisca E.
Animal reproduction
sêmen bovino
Brucella abortus
PCR fluorescente
Eletroforese capilar
diagnóstico.
title_short FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
title_full FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
title_fullStr FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
title_full_unstemmed FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
title_sort FLUORESCENT PCR ASSOCIATED WITH CAPILLARY ELECTROPHORESIS AS A DIAGNOSTIC TOOL OF BACTERIA IN SEMEN
author Ferreira Dias, Francisca E.
author_facet Ferreira Dias, Francisca E.
Nunes, Cáris Marone
Cavalcante, Tânia Vasconcelos
Castro, Andréa A. Pires de
Ferreira, Jorge Luis
Garcia, José Fernando
author_role author
author2 Nunes, Cáris Marone
Cavalcante, Tânia Vasconcelos
Castro, Andréa A. Pires de
Ferreira, Jorge Luis
Garcia, José Fernando
author2_role author
author
author
author
author
dc.contributor.author.fl_str_mv Ferreira Dias, Francisca E.
Nunes, Cáris Marone
Cavalcante, Tânia Vasconcelos
Castro, Andréa A. Pires de
Ferreira, Jorge Luis
Garcia, José Fernando
dc.subject.por.fl_str_mv Animal reproduction
sêmen bovino
Brucella abortus
PCR fluorescente
Eletroforese capilar
diagnóstico.
topic Animal reproduction
sêmen bovino
Brucella abortus
PCR fluorescente
Eletroforese capilar
diagnóstico.
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 (100 to 107 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 105 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.KEYWORDS: bovine semen; Brucella abortus; capillary electrophoresis; diagnostic fluorescent PCR.
publishDate 2013
dc.date.none.fl_str_mv 2013-09-29
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
Research
Avaliado por pares
Pesquisa Cientifica
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://revistas.ufg.br/vet/article/view/7638
10.5216/cab.v14i3.7638
url https://revistas.ufg.br/vet/article/view/7638
identifier_str_mv 10.5216/cab.v14i3.7638
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv https://revistas.ufg.br/vet/article/view/7638/15219
https://revistas.ufg.br/vet/article/view/7638/15220
https://revistas.ufg.br/vet/article/view/7638/16860
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
text/html
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 Brazilian Animal Science/ Ciência Animal Brasileira; Vol. 14 No. 3 (2013); 366-372
Ciência Animal Brasileira / Brazilian Animal Science; v. 14 n. 3 (2013); 366-372
1809-6891
1518-2797
reponame:Ciência animal brasileira (Online)
instname:Universidade Federal de Goiás (UFG)
instacron:UFG
instname_str Universidade Federal de Goiás (UFG)
instacron_str UFG
institution UFG
reponame_str Ciência animal brasileira (Online)
collection Ciência animal brasileira (Online)
repository.name.fl_str_mv Ciência animal brasileira (Online) - Universidade Federal de Goiás (UFG)
repository.mail.fl_str_mv ||revistacab@gmail.com
_version_ 1799874785811890176

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