title: A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses.
abstract: The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5' and parCoat3' was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.
authors.affiliation: Laboratório de Virologia Vegetal, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal. Electronic address: carlavaranda@uevora.pt.
authors.forename: C M R
authors.initials: CM
authors.lastname: Varanda
authors.affiliation: IMAR-CMA, Departamento de Ciências da Vida, Universidade de Coimbra, 3004-517 Coimbra, Portugal.
authors.forename: J M S
authors.initials: JM
authors.lastname: Cardoso
authors.affiliation: Laboratório de Virologia Vegetal, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal.
authors.forename: M D M
authors.initials: MD
authors.lastname: Oliveira
authors.affiliation: Laboratório de Microbiologia do Solo, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal.
authors.forename: S
authors.initials: S
authors.lastname: Oliveira
authors.affiliation: Laboratório de Virologia Vegetal, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal.
authors.forename: M I E
authors.initials: MI
authors.lastname: Clara
authors.affiliation: Laboratório de Virologia Vegetal, Instituto de Ciências Agrárias e Ambientais Mediterrânicas, Universidade de Évora, Apartado 94, 7002-554 Évora, Portugal.
authors.forename: M R F
authors.initials: MR
authors.lastname: Félix
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D031658:Olea / Q000821:virology*; D010935:Plant Diseases / Q000821:virology*; D011174:Portugal; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019183:Tombusviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D012324:RNA-Dependent RNA Polymerase
keywords: OLV-1; OMMV; Olive trees; TNV-A; TNV-D