Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle

Detalhes bibliográficos
Autor(a) principal: Takiuchi, Elisabete
Data de Publicação: 2003
Outros Autores: Médici, Kerlei Cristina, Alfieri, Fernandes, Alfieri, Amauri Alcindo
Tipo de documento: Artigo
Idioma: por
Título da fonte: Semina. Ciências Agrárias (Online)
Texto Completo: https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2115
Resumo: The glycoprotein D gene of bovine herpesvirus type 1 (BHV-1) was detected in clinical samples from naturally infected cattle by semi-nested polymerase chain reaction (SN-PCR). Different protocols were tested to increase the sensitivity and specificity of the technique. An association of DNA extraction methods using phenol/chloroform/isoamyl alcohol followed by silica/guanidine isothiocyanate yield greater concentration and quality of amplified DNA. After optimization of primers and reaction conditions, the genome of BHV-1 (Los Angeles strain) was detected by SN-PCR in tissue culture supernatant and artificially infected semen at the 1 and 0.1 TCID50 limit, respectively. When used on clinical specimens from naturally infected cattle, the SN-PCR yield positive results in semen of seropositive bull and in organ fragments of aborted cattle fetus. The SN-PCR was a viable alternative, which was faster, sensitive, specific and less laborious to be used in the routine diagnosis of BHV-1 infection and semen health monitoring.
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spelling Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattleOtimização da reação em cadeia pela polimerase (Semi Nested-PCR) para a detecção do herpesvírus bovino tipo 1 em fragmentos de órgãos fetais e em sêmen de bovinos naturalmente infectadosCattleBovine herpesvirus type 1DiagnosisPCRAbortionSemen.BovinoHerpesvírus bovino tipo 1DiagnósticoPCRAbortoSêmen.The glycoprotein D gene of bovine herpesvirus type 1 (BHV-1) was detected in clinical samples from naturally infected cattle by semi-nested polymerase chain reaction (SN-PCR). Different protocols were tested to increase the sensitivity and specificity of the technique. An association of DNA extraction methods using phenol/chloroform/isoamyl alcohol followed by silica/guanidine isothiocyanate yield greater concentration and quality of amplified DNA. After optimization of primers and reaction conditions, the genome of BHV-1 (Los Angeles strain) was detected by SN-PCR in tissue culture supernatant and artificially infected semen at the 1 and 0.1 TCID50 limit, respectively. When used on clinical specimens from naturally infected cattle, the SN-PCR yield positive results in semen of seropositive bull and in organ fragments of aborted cattle fetus. The SN-PCR was a viable alternative, which was faster, sensitive, specific and less laborious to be used in the routine diagnosis of BHV-1 infection and semen health monitoring.A reação em cadeia pela polimerase (PCR) foi empregada para a detecção parcial do gene da glicoproteína D do herpesvírus bovino tipo 1 (BHV-1) em material biológico proveniente de bovinos naturalmente infectados. Para o aumento da sensibilidade e da especificidade da PCR, foram avaliados diferentes protocolos. Para a extração do DNA, a associação dos métodos fenol/clorofórmio/álcool isoamílico e sílica/tiocianato de guanidina, proporcionou a amplificação do DNA em maior concentração e qualidade. Após a otimização dos primers e condições da reação foi possível detectar, por meio da Semi Nested-PCR (SN-PCR), o genoma da estirpe Los Angeles do BHV-1 em sobrenadante de cultura celular, sem processamento prévio, até o limite de 1 TCID50. Em sêmen artificialmente infectado o limite de detecção do BHV-1 foi de 0,1 TCID50. Quando utilizada em material biológico, proveniente de bovinos naturalmente infectados, a SN-PCR apresentou resultados positivos tanto em fragmentos de órgãos de fetos bovinos abortados quanto em sêmen. Essa técnica revelou-se uma alternativa viável, rápida, sensível e específica para aplicação na rotina de diagnóstico da infecção pelo BHV-1, bem como, para o monitoramento sanitário do sêmen.UEL2003-05-10info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/211510.5433/1679-0359.2003v24n1p43Semina: Ciências Agrárias; Vol. 24 No. 1 (2003); 43-56Semina: Ciências Agrárias; v. 24 n. 1 (2003); 43-561679-03591676-546Xreponame:Semina. Ciências Agrárias (Online)instname:Universidade Estadual de Londrina (UEL)instacron:UELporhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2115/1816Takiuchi, ElisabeteMédici, Kerlei CristinaAlfieri, FernandesAlfieri, Amauri Alcindoinfo:eu-repo/semantics/openAccess2009-05-22T20:17:15Zoai:ojs.pkp.sfu.ca:article/2115Revistahttp://www.uel.br/revistas/uel/index.php/semagrariasPUBhttps://ojs.uel.br/revistas/uel/index.php/semagrarias/oaisemina.agrarias@uel.br1679-03591676-546Xopendoar:2009-05-22T20:17:15Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL)false
dc.title.none.fl_str_mv Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
Otimização da reação em cadeia pela polimerase (Semi Nested-PCR) para a detecção do herpesvírus bovino tipo 1 em fragmentos de órgãos fetais e em sêmen de bovinos naturalmente infectados
title Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
spellingShingle Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
Takiuchi, Elisabete
Cattle
Bovine herpesvirus type 1
Diagnosis
PCR
Abortion
Semen.
Bovino
Herpesvírus bovino tipo 1
Diagnóstico
PCR
Aborto
Sêmen.
title_short Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
title_full Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
title_fullStr Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
title_full_unstemmed Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
title_sort Standardization of a polymerase chain reaction (Semi Nested–PCR) to detect bovine herpesvirus type 1 in aborted fetus and semen from naturally infected cattle
author Takiuchi, Elisabete
author_facet Takiuchi, Elisabete
Médici, Kerlei Cristina
Alfieri, Fernandes
Alfieri, Amauri Alcindo
author_role author
author2 Médici, Kerlei Cristina
Alfieri, Fernandes
Alfieri, Amauri Alcindo
author2_role author
author
author
dc.contributor.author.fl_str_mv Takiuchi, Elisabete
Médici, Kerlei Cristina
Alfieri, Fernandes
Alfieri, Amauri Alcindo
dc.subject.por.fl_str_mv Cattle
Bovine herpesvirus type 1
Diagnosis
PCR
Abortion
Semen.
Bovino
Herpesvírus bovino tipo 1
Diagnóstico
PCR
Aborto
Sêmen.
topic Cattle
Bovine herpesvirus type 1
Diagnosis
PCR
Abortion
Semen.
Bovino
Herpesvírus bovino tipo 1
Diagnóstico
PCR
Aborto
Sêmen.
description The glycoprotein D gene of bovine herpesvirus type 1 (BHV-1) was detected in clinical samples from naturally infected cattle by semi-nested polymerase chain reaction (SN-PCR). Different protocols were tested to increase the sensitivity and specificity of the technique. An association of DNA extraction methods using phenol/chloroform/isoamyl alcohol followed by silica/guanidine isothiocyanate yield greater concentration and quality of amplified DNA. After optimization of primers and reaction conditions, the genome of BHV-1 (Los Angeles strain) was detected by SN-PCR in tissue culture supernatant and artificially infected semen at the 1 and 0.1 TCID50 limit, respectively. When used on clinical specimens from naturally infected cattle, the SN-PCR yield positive results in semen of seropositive bull and in organ fragments of aborted cattle fetus. The SN-PCR was a viable alternative, which was faster, sensitive, specific and less laborious to be used in the routine diagnosis of BHV-1 infection and semen health monitoring.
publishDate 2003
dc.date.none.fl_str_mv 2003-05-10
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2115
10.5433/1679-0359.2003v24n1p43
url https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2115
identifier_str_mv 10.5433/1679-0359.2003v24n1p43
dc.language.iso.fl_str_mv por
language por
dc.relation.none.fl_str_mv https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/2115/1816
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv UEL
publisher.none.fl_str_mv UEL
dc.source.none.fl_str_mv Semina: Ciências Agrárias; Vol. 24 No. 1 (2003); 43-56
Semina: Ciências Agrárias; v. 24 n. 1 (2003); 43-56
1679-0359
1676-546X
reponame:Semina. Ciências Agrárias (Online)
instname:Universidade Estadual de Londrina (UEL)
instacron:UEL
instname_str Universidade Estadual de Londrina (UEL)
instacron_str UEL
institution UEL
reponame_str Semina. Ciências Agrárias (Online)
collection Semina. Ciências Agrárias (Online)
repository.name.fl_str_mv Semina. Ciências Agrárias (Online) - Universidade Estadual de Londrina (UEL)
repository.mail.fl_str_mv semina.agrarias@uel.br
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