Full Text Available
Note: Clicking the button above will open the full text document at the original institutional repository in a new window.
The development of a diagnostic assay for nepoviruses in grapevine
Thesis (MSc)--Stellenbosch University, 2015.
Saved in:
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Thesis |
| Language: | en_ZA |
| Published: |
Stellenbosch : Stellenbosch University
2015
|
| Subjects: | |
| Tags: |
No Tags, Be the first to tag this record!
|
| _version_ | 1867613954394554368 |
|---|---|
| access_status_str | Open Access |
| author | Frazenburg, Lolita |
| author2 | Burger, Johan T. |
| author_browse | Burger, Johan T. Frazenburg, Lolita |
| author_facet | Burger, Johan T. Frazenburg, Lolita |
| author_sort | Frazenburg, Lolita |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2015. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/96964 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:21.236Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2015 |
| publishDateRange | 2015 |
| publishDateSort | 2015 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/96964 The development of a diagnostic assay for nepoviruses in grapevine Frazenburg, Lolita Burger, Johan T. Souza Richards, Rosineide Stellenbosch University. Faculty of Agrisciences. Dept. of Genetics. Grapevine Nepoviruses GFLV (Grapevine fanleaf virus) ToRSV (Tomato ringspot virus) UCTD Thesis (MSc)--Stellenbosch University, 2015. ENGLISH ABSTRACT: The nepoviruses are a group of nematode-transmitted plant viruses that are distributed worldwide and infect a wide range of plant species, including grapevine. Most of the nepoviruses are foreign to South Africa and to date, only Grapevine fanleaf virus (GFLV) is present. The Department of Agriculture, Forestry and Fisheries (DAFF), as the official National Plant Protection Organisation (NPPO) of South Africa, is committed to prevent the importation and spread of plant pathogens by administering the Agricultural Pests Act, 1983 (Act No. 36 of 1983). Effective measures are implemented by which the introduction of agricultural pests may be prohibited to safeguard the agricultural environment. One of the core functions of DAFF is to render a routine plant health diagnostic service for imported plants and plant products to prevent exotic pathogens from entering the country. The objective of this study was to develop a diagnostic assay for the detection of nepoviruses in grapevine. The project aimed to produce antibodies by recombinant DNA technology against bacterially expressed viral coat protein of a specific nepovirus [Tomato ringspot virus (ToRSV)] and subsequently develop a DAS-ELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) assay for the detection of the virus. The coat protein (CP) was successfully isolated from imported ToRSV-infected grapevine material. Two expression systems were utilised for expression of the ToRSV-CP, the GST gene fusion system and an Agrobacterium-mediated expression system. The GST gene fusion system was unsuccessful as insufficient soluble protein expression prevented the production of antibodies and thus the development of the DAS-ELISA assay. Tissue print immunoassay (TPIA) initially showed positive results for transient expression of the fusion protein in tobacco plants, but further confirmation proved to be inconclusive. The project also aimed to develop a real-time PCR assay for the specific detection and relative quantification of GFLV, based on a conserved region of the RNA-2 genome. A partial GFLV-RNA-2 from a South African isolate of grapevine was sequenced and used for the design of specific primers. The quantitative real-time PCR assay based on SYBR green technology proved to be sensitive in detecting levels as low as 0.11ng/reaction in infected plants, making it a highly effective diagnostic tool for the detection of GFLV. AFRIKAANSE OPSOMMING: Die nepovirusse is 'n groep van nematode-oordraagbare plant virusse wat wêreldwyd versprei word en 'n wye verskeidenheid van plantspesies infekteer, insluitend wingerd. Die meeste van die nepovirusse is uitheems aan Suid-Afrika en tot op datum is net Wingerd netelblaar virus (GFLV) teenwoordig. Die Departement van Landbou, Bosbou en Visserye (DAFF), as die amptelike Nasionale Plant Beskermings Organisasie (NPBO) van Suid-Afrika, is daartoe verbind om die invoer en verspreiding van plantpatogene te voorkom deur administrasie van die Wet op Landbouplae, 1983 (Wet No. 36 van 1983). Doeltreffende maatreëls word geïmplementeer waardeur die invoer van landbouplae verbied word om sodoende die landbou-omgewing te beskerm. Een van die kernfunksies van DAFF is om 'n roetine plant gesondheid diagnostiese diens vir ingevoerde plante en plantprodukte te lewer om te verhoed dat eksotiese patogene die land binnedring. Die doel van hierdie studie was om 'n diagnostiese toets vir die opsporing van nepovirusse in wingerd te ontwikkel. Die projek was daarop gemik om antiliggame te vervaardig deur rekombinante DNA-tegnologie teen bakterieël-uitgedrukte virale mantelproteïen van 'n spesifieke nepovirus [Tomato ringspot virus (ToRSV)] en vervolgens ‘n DASELISA (Double Antibody Sandwich Enzyme-linked Immunosorbent) toets vir die opsporing van die virus te ontwikkel. Die mantelproteïen (CP) is met sukses geïsoleer vanaf ingevoerde ToRSV-besmette wingerdmateriaal. Twee uitdrukking stelsels is gebruik vir uitdrukking van die ToRSV-CP, die “GST gene fusion” stelsel en 'n Agrobacterium-bemiddelde uitdrukking stelsel. Die “GST gene fusion” stelsel was egter onsuksesvol aangesien onvoldoende oplosbare proteïen uitdrukking die produksie van antiliggame en dus die ontwikkeling van die DAS-ELISA toets verhoed het. “Tissue print immunoassay” (TPIA) het aanvanklik positiewe resultate getoon vir tydelike uitdrukking van die fusie proteïen in tabakplante, maar verdere bevestiging was onoortuigend. Die projek was ook daarop gemik om ‘n in-tyd polimerase ketting reaksie (PKR) toets vir die spesifieke opsporing en relatiewe kwantifisering van GFLV, gebaseer op 'n gekonserveerde volgorde van die RNA-2 genoom, te ontwikkel. 'n Gedeeltelike GFLV-RNA-2 nukleïensuurvolgorde van 'n Suid-Afrikaanse wingerd isolaat is bepaal en gebruik vir die ontwerp van spesifieke inleiers. Die kwantitatiewe in-tyd PKR toets gebaseer op SYBR groen tegnologie was sensitief genoeg om vlakke van so laag as 0.11ng/reaksie in geïnfekteerde plante op te spoor, wat dit 'n hoogs effektiewe diagnostiese hulpmiddel vir die opsporing van GFLV maak. Masters 2015-05-20T09:28:46Z 2015-05-20T09:28:46Z 2015-12 Thesis http://hdl.handle.net/10019.1/96964 en_ZA Stellenbosch University xvi, 121 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Grapevine Nepoviruses GFLV (Grapevine fanleaf virus) ToRSV (Tomato ringspot virus) UCTD Frazenburg, Lolita The development of a diagnostic assay for nepoviruses in grapevine |
| title | The development of a diagnostic assay for nepoviruses in grapevine |
| title_full | The development of a diagnostic assay for nepoviruses in grapevine |
| title_fullStr | The development of a diagnostic assay for nepoviruses in grapevine |
| title_full_unstemmed | The development of a diagnostic assay for nepoviruses in grapevine |
| title_short | The development of a diagnostic assay for nepoviruses in grapevine |
| title_sort | development of a diagnostic assay for nepoviruses in grapevine |
| topic | Grapevine Nepoviruses GFLV (Grapevine fanleaf virus) ToRSV (Tomato ringspot virus) UCTD |
| url | http://hdl.handle.net/10019.1/96964 |
| work_keys_str_mv | AT frazenburglolita thedevelopmentofadiagnosticassayfornepovirusesingrapevine AT frazenburglolita developmentofadiagnosticassayfornepovirusesingrapevine |