Real-time RT-PCR high resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI

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dc.contributor.authorBester, Rachelle
dc.contributor.authorJooste, Anna E. C.
dc.contributor.authorMaree, Hans J.
dc.contributor.authorBurger, Johan T.
dc.date.accessioned2013-05-07T10:23:56Z
dc.date.available2013-05-07T10:23:56Z
dc.date.issued2012-09
dc.date.updated2012-12-10T20:05:11Z
dc.descriptionThe original publication is available at http://www.virologyj.com/content/9/1/219en_ZA
dc.descriptionPublication of this article was funded by the Stellenbosch University Open Access Fund.
dc.description.abstractAbstract Background Grapevine leafroll-associated virus 3 (GLRaV-3) is the main contributing agent of leafroll disease worldwide. Four of the six GLRaV-3 variant groups known have been found in South Africa, but their individual contribution to leafroll disease is unknown. In order to study the pathogenesis of leafroll disease, a sensitive and accurate diagnostic assay is required that can detect different variant groups of GLRaV-3. Methods In this study, a one-step real-time RT-PCR, followed by high-resolution melting (HRM) curve analysis for the simultaneous detection and identification of GLRaV-3 variants of groups I, II, III and VI, was developed. A melting point confidence interval for each variant group was calculated to include at least 90% of all melting points observed. A multiplex RT-PCR protocol was developed to these four variant groups in order to assess the efficacy of the real-time RT-PCR HRM assay. Results A universal primer set for GLRaV-3 targeting the heat shock protein 70 homologue (Hsp70h) gene of GLRaV-3 was designed that is able to detect GLRaV-3 variant groups I, II, III and VI and differentiate between them with high-resolution melting curve analysis. The real-time RT-PCR HRM and the multiplex RT-PCR were optimized using 121 GLRaV-3 positive samples. Due to a considerable variation in melting profile observed within each GLRaV-3 group, a confidence interval of above 90% was calculated for each variant group, based on the range and distribution of melting points. The intervals of groups I and II could not be distinguished and a 95% joint confidence interval was calculated for simultaneous detection of group I and II variants. An additional primer pair targeting GLRaV-3 ORF1a was developed that can be used in a subsequent real-time RT-PCR HRM to differentiate between variants of groups I and II. Additionally, the multiplex RT-PCR successfully validated 94.64% of the infections detected with the real-time RT-PCR HRM. Conclusion The real-time RT-PCR HRM provides a sensitive, automated and rapid tool to detect and differentiate different variant groups in order to study the epidemiology of leafroll disease.en_ZA
dc.description.versionPublishers' Versionen_ZA
dc.format.extent11 p. : ill.
dc.identifier.citationBester, R., Jooste, A. E. C., Maree, H. & Burger, J. T. 2012. Real-time RT-PCR high resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI. Virology Journal, 9(1):219, doi.org/10.1186/1743-422X-9-219.en_ZA
dc.identifier.issn1743-422X (online)
dc.identifier.otherdoi:10.1186/1743-422X-9-219
dc.identifier.urihttp://hdl.handle.net/10019.1/80726
dc.language.isoen_ZAen_ZA
dc.language.rfc3066en
dc.publisherBioMed Centralen_ZA
dc.rights.holderRachelle Bester et al.; licensee BioMed Central Ltd.en_ZA
dc.subjectGrapevine leafroll virusen_ZA
dc.subjectGrapes -- Diseases and pestsen_ZA
dc.subjectMolecular variantsen_ZA
dc.subjectLeafroll disease -- Epidemiologyen_ZA
dc.titleReal-time RT-PCR high resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VIen_ZA
dc.typeArticleen_ZA
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