RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses

Detalhes bibliográficos
Autor(a) principal: EIRAS, MARCELO
Data de Publicação: 2001
Outros Autores: RESENDE, RENATO O., MISSIAGGIA, ALEXANDRE A., ÁVILA, ANTÔNIO C. DE
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UnB
Texto Completo: http://repositorio.unb.br/handle/10482/25723
https://dx.doi.org/10.1590/S0100-41582001000200009
Resumo: Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.
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spelling RT-PCR and Dot Blot hybridization methods for a universal detection of tospovirusesMétodos de RT-PCR e de hibridização Dot Blot para detecção universal de tospovirusDiagnósticoPlantas - doenças e pragasBunyaviridaeTranscriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.Visando a um método para a detecção universal de tospovírus, utilizaram-se as técnicas de "Transcriptase reverse - polymerase chain reaction" (RT-PCR) e hibridização com sondas marcadas com digoxigenina. As espécies de tospovirus testadas foram: Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Os oligonucleotídeos foram sintetizados para anelar em regiões conservadas do genoma viral, sendo os produtos de PCR utilizados como sondas para hibridização através de "dot blot". Através de RT-PCR, utilizando-se diferentes combinações de oligonucleotídeos, somente foi possível a amplificação de todas as espécies quando se utilizou RNA de vírus purificado, sendo que, para a detecção a partir de RNA total, a RT-PCR apresentou problemas para a detecção das espécies ZLCV e IYSV. Sob condições de baixa adstringência, os testes de hibridização por "dot blot" com a sonda M (correspondente ao gene G1/G2) detectaram todas as espécies testadas, exceto IYSV. Com a sonda L, também sob condições de baixa adstringência, pôde-se detectar todas as espécies de tospovirus testadas simultaneamente em um único ensaio. Este método de detecção pode ser utilizado em serviços de quarentena e para indexação de germoplasma in vitro.Em processamentoSociedade Brasileira de Fitopatologia2017-12-07T04:33:53Z2017-12-07T04:33:53Z2001info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfFitopatol. bras.,v.26,n.2,p.170-175,2001http://repositorio.unb.br/handle/10482/25723https://dx.doi.org/10.1590/S0100-41582001000200009EIRAS, MARCELORESENDE, RENATO O.MISSIAGGIA, ALEXANDRE A.ÁVILA, ANTÔNIO C. DEinfo:eu-repo/semantics/openAccessengreponame:Repositório Institucional da UnBinstname:Universidade de Brasília (UnB)instacron:UNB2024-08-28T19:04:10Zoai:repositorio.unb.br:10482/25723Repositório InstitucionalPUBhttps://repositorio.unb.br/oai/requestrepositorio@unb.bropendoar:2024-08-28T19:04:10Repositório Institucional da UnB - Universidade de Brasília (UnB)false
dc.title.none.fl_str_mv RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
Métodos de RT-PCR e de hibridização Dot Blot para detecção universal de tospovirus
title RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
spellingShingle RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
EIRAS, MARCELO
Diagnóstico
Plantas - doenças e pragas
Bunyaviridae
title_short RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
title_full RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
title_fullStr RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
title_full_unstemmed RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
title_sort RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
author EIRAS, MARCELO
author_facet EIRAS, MARCELO
RESENDE, RENATO O.
MISSIAGGIA, ALEXANDRE A.
ÁVILA, ANTÔNIO C. DE
author_role author
author2 RESENDE, RENATO O.
MISSIAGGIA, ALEXANDRE A.
ÁVILA, ANTÔNIO C. DE
author2_role author
author
author
dc.contributor.author.fl_str_mv EIRAS, MARCELO
RESENDE, RENATO O.
MISSIAGGIA, ALEXANDRE A.
ÁVILA, ANTÔNIO C. DE
dc.subject.por.fl_str_mv Diagnóstico
Plantas - doenças e pragas
Bunyaviridae
topic Diagnóstico
Plantas - doenças e pragas
Bunyaviridae
description Transcriptase reverse - polymerase chain reaction (RT-PCR) and dot blot hybridization with digoxigenin-labeled probes were applied for the universal detection of Tospovirus species. The virus species tested were Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, Chrysanthemum stem necrosis virus, Impatiens necrotic spot virus, Zucchini lethal chlorosis virus, Iris yellow spot virus. Primers for PCR amplification were designed to match conserved regions of the tospovirus genome. RT-PCR using distinct primer combinations was unable to simultaneously amplify all tospovirus species and consistently failed to detect ZLCV and IYSV in total RNA extracts. However, all tospovirus species were detected by RT-PCR when viral RNA was used as template. RNA-specific PCR products were used as probes for dot hybridization. This assay with a M probe (directed to the G1/G2 gene) detected at low stringency conditions all Tospovirus species, except IYSV. At low stringency conditions, the L non-radioactive probe detected the seven Tospovirus species in a single assay. This method for broad spectrum detection can be potentially employed in quarantine services for indexing in vitro germplasm.
publishDate 2001
dc.date.none.fl_str_mv 2001
2017-12-07T04:33:53Z
2017-12-07T04:33:53Z
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 Fitopatol. bras.,v.26,n.2,p.170-175,2001
http://repositorio.unb.br/handle/10482/25723
https://dx.doi.org/10.1590/S0100-41582001000200009
identifier_str_mv Fitopatol. bras.,v.26,n.2,p.170-175,2001
url http://repositorio.unb.br/handle/10482/25723
https://dx.doi.org/10.1590/S0100-41582001000200009
dc.language.iso.fl_str_mv eng
language eng
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 Sociedade Brasileira de Fitopatologia
publisher.none.fl_str_mv Sociedade Brasileira de Fitopatologia
dc.source.none.fl_str_mv reponame:Repositório Institucional da UnB
instname:Universidade de Brasília (UnB)
instacron:UNB
instname_str Universidade de Brasília (UnB)
instacron_str UNB
institution UNB
reponame_str Repositório Institucional da UnB
collection Repositório Institucional da UnB
repository.name.fl_str_mv Repositório Institucional da UnB - Universidade de Brasília (UnB)
repository.mail.fl_str_mv repositorio@unb.br
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