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A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae)
Dissertation (MSc (Entomology))--University of Pretoria, 2008.
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| Format: | Thesis |
| Language: | English |
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University of Pretoria
2013
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| _version_ | 1867613502010556416 |
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| access_status_str | Open Access |
| author2 | Kruger, Kerstin |
| author_browse | Kruger, Kerstin |
| author_facet | Kruger, Kerstin |
| collection | Thesis |
| dc_rights_str_mv | © 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
| description | Dissertation (MSc (Entomology))--University of Pretoria, 2008. |
| format | Thesis |
| id | oai:repository.up.ac.za:2263/31373 |
| institution | University of Pretoria (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:37:09.691Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from UPSpace — University of Pretoria Institutional Repository |
| publishDate | 2013 |
| publishDateRange | 2013 |
| publishDateSort | 2013 |
| publisher | University of Pretoria |
| publisherStr | University of Pretoria |
| record_format | dspace |
| source_str | UPSpace — University of Pretoria Institutional Repository |
| spelling | oai:repository.up.ac.za:2263/31373 A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) Kruger, Kerstin ndouglas@zoology.up.ac.za Smit, Nicoleen UCTD Glrav-3 Planococcus ficus Diseases Grapevine leafroll-associated virus 3 Dissertation (MSc (Entomology))--University of Pretoria, 2008. Viral diseases represent a major obstacle to commercial growing of grapevines. Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most economically important viral diseases in South African vineyards. The most important vector of GLRaV-3 in South Africa is the vine mealybug Planococcus ficus. Studies show that single first instar-nymphs can successfully transmit GLRaV-3 to healthy grapevines. Quantifying the amount of virus acquired in relation to feeding time of mealybugs may lead to a better understanding of GLRaV-3 mealybug transmission. In this study, a real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify GLRaV-3 in grapevines and mealybugs. Real-time qRT-PCR is a highly sensitive technique. The quality of purified RNA and the accuracy of the quantification technique are essential for reliable quantitative use. To develop a real-time qRT-PCR assay two tasks were undertaken. Firstly, four different RNA isolation techniques, namely phenolchloroform, Gentra Purescript® RNA Isolation kit, Qiagen QIAzol™ and Qiagen RNeasy® Plant Mini kit, were assessed for use in qRT-PCR. Phenol-chloroform RNA extraction method was superior to the other RNA extraction methods tested, with regard to purity of RNA, RT-PCR efficiency, reproducibility and number of GLRaV-3 positive samples detected. Secondly, DNA and cRNA external standard models were designed for quantifying GLRaV-3 in grapevines and mealybugs. External standards have defined concentrations of a target nucleic acid and are used to generate a standard curve. The DNA standard model had a wider detection and quantification range than the cRNA standard model. However, the DNA standard model, unlike the cRNA standard model is not subjected to the RT step. Therefore, using the DNA standard model, GLRaV-3 is quantified based on the starting cDNA concentration and not the initial RNA concentration. The cRNA standard model provides information on the initial RNA concentration. The DNA standard model is best applied as a quantification method where initial RNA concentration is not relevant. The cRNA standard model can be applied for accurate quantification of GLRaV-3 in grapevines and mealybugs. The real-time qRT-PCR assay designed in this study can be applied in other laboratories. Copyright Zoology and Entomology Restricted Natural and Agricultural Sciences 2013-09-09T12:13:12Z 2009-11-04 2013-09-09T12:13:12Z 2009-09-02 2008 2009-10-21 Dissertation Smit, N 2008, A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococus ficus (Signoret) (Hemiptera : Pseudococcidae), MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-10212009-164856/ > E1521/gm http://hdl.handle.net/2263/31373 http://upetd.up.ac.za/thesis/available/etd-10212009-164856/ en © 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. application/pdf University of Pretoria |
| spellingShingle | UCTD Glrav-3 Planococcus ficus Diseases Grapevine leafroll-associated virus 3 A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title | A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title_full | A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title_fullStr | A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title_full_unstemmed | A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title_short | A real-time RT-PCR assay for the detection and quantification of grapevine leafroll-associated virus 3 (GLRaV-3) in Vitis vinifera (Vitaceae) and Planococcus ficus (Signoret) (Hemiptera : Pseudococcidae) |
| title_sort | real time rt pcr assay for the detection and quantification of grapevine leafroll associated virus 3 glrav 3 in vitis vinifera vitaceae and planococcus ficus signoret hemiptera pseudococcidae |
| topic | UCTD Glrav-3 Planococcus ficus Diseases Grapevine leafroll-associated virus 3 |
| url | http://hdl.handle.net/2263/31373 http://upetd.up.ac.za/thesis/available/etd-10212009-164856/ |