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

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, Chrysa...

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Main Authors: EIRAS, MARCELO, RESENDE, RENATO O., MISSIAGGIA, ALEXANDRE A., ÁVILA, ANTÔNIO C. DE
Format: Artigo
Language: English
Published: Sociedade Brasileira de Fitopatologia 2017
Subjects:
diagnosis
plant viruses
Bunyaviridae
Online Access: http://repositorio.unb.br/handle/10482/25723
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spelling ir-10482-257232019-03-28T14:04:07Z 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 EIRAS, MARCELO RESENDE, RENATO O. MISSIAGGIA, ALEXANDRE A. ÁVILA, ANTÔNIO C. DE diagnosis plant viruses Bunyaviridae 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. 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. 2017-12-07T04:33:53Z 2017-12-07T04:33:53Z 2001 Artigo Fitopatol. bras.,v.26,n.2,p.170-175,2001 0100-4158 http://repositorio.unb.br/handle/10482/25723 10.1590/S0100-41582001000200009 en Acesso Aberto application/pdf Sociedade Brasileira de Fitopatologia http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-41582001000200009&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_abstract&pid=S0100-41582001000200009&lng=en&nrm=iso http://www.scielo.br/scielo.php?script=sci_pdf&pid=S0100-41582001000200009&lng=en&nrm=iso
institution REPOSITORIO UNB
collection REPOSITORIO UNB
language English
topic diagnosis
plant viruses
Bunyaviridae
spellingShingle diagnosis
plant viruses
Bunyaviridae
EIRAS, MARCELO
RESENDE, RENATO O.
MISSIAGGIA, ALEXANDRE A.
ÁVILA, ANTÔNIO C. DE
RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
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.
format Artigo
author EIRAS, MARCELO
RESENDE, RENATO O.
MISSIAGGIA, ALEXANDRE A.
ÁVILA, ANTÔNIO C. DE
author_sort EIRAS, MARCELO
title RT-PCR and Dot Blot hybridization methods for a universal detection of tospoviruses
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
publisher Sociedade Brasileira de Fitopatologia
publishDate 2017
url http://repositorio.unb.br/handle/10482/25723
_version_ 1641988586037313536
score 13.657419

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