Thesis (MSc)--University of Stellenbosch, 2005. / ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructive
potato viruses capable of drastically diminishing crop yields by up to 80%. The presence of
these viruses in planting material namely seed potato stocks are routinely diagnosed by
enzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes South
Africa are obtained from Europe. These kits have produced false positive and false negative
results in the past. Potatoes South Africa required an ELISA that was reliable, cheap and
specific for the detection of South African strains of the two respective viruses.
In this study the viral coat protein genes were amplified by RT-PCR from a South African
source of infected plant material. The PVY and PLRV coat protein genes were subsequently
cloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses were
aligned and compared to corresponding viral coat protein gene sequences obtained from
Genbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRV
were sub-cloned into an expression system (pET-14b) to induce and express the respective
recombinant viral coat proteins. The induction of the cloned coat protein genes yielded
successful production of the recombinant PVY coat protein but the induction and expression
of the recombinant PLRV coat protein was unsuccessful.
The isolated recombinant PVY CP was then used to immunize a rabbit to produce highly
specific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used to
develop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. The
ELISA kit was subsequently used in preliminary trials to determine if the kit could detect
PVY infected plant material. The initial results of the ELISA trials using PVY infected
material obtained from Potatoes South Africa yielded positive results. / AFRIKAANSE OPSOMMING: Aartappel Virus Y (PVY) en Aartappel Rolblad Virus (PLRV) is twee van die mees
vernietigende aartappel virusse wat ‘n oes tot 80% kan verlaag. Virus infeksie van plant
materiaal tewete aartappelmoere word deur “enzyme-linked immunosorbent assay” (ELISA)
toetsstelle bevestig. Die toetsstelle wat op die oomblik gebruik word deur Aartappels Suid-
Afrika word in Europa vervaardig. Hierdie toetsstelle het vals positiewe en vals negatiewe
resultate in die verlede gegee. Aartappels Suid-Afrika benodig toetsstelle wat betroubaar,
goedkoop en spesifiek vir Suid-Afrikaanse virus stamme is.
In hierdie studie is besmette plantmateriaal vanuit Suid-Afrika gebruik vir die amplifisering
van virale mantel proteïen gene met behulp van RT-PCR. Die PVY en PLRV mantel proteïen
gene was daarna in die pGEM-T Easy vektor gekloneer en nukleotied volgordes is bepaal.
Die nukleotied volgordes is met ander PVY en PLRV gene vanaf Genbank vergelyk. Die
twee ge-amplifiseerde en gekloneerde mantel proteïen gene van PVY en PLRV is uitgesny en
gekloneer in ‘n ekspressie sisteem (pET-14b) om die mantel proteïen te produseer. Induksie
van die gekloneerde mantel proteïen gene het gelei tot die suksesvolle produksie van ‘n PVY
mantel proteïen, maar produksie van die PLRV mantel proteïen was onsuksesvol.
Die geïsoleerde PVY mantel proteïen is vervolgens gebruik vir die immunisering van ‘n
konyn vir die produksie van konyn anti-PVY antiliggame. Die antiserum verkry vanaf die
konyn is gebruik vir die ontwikkeling van ‘n ELISA vir die identifisering van PVY infeksies
in aartappelmoere. Voorlopige proewe is deurgevoer om te bepaal of hierdie ELISA PVY
infeksies in plantmateriaal sou kon opspoor. Aanvanklike resultate toon dat die ELISA
suksesvol PVY infeksies in plantmateriaal verkry vanaf Aartappels Suid-Afrika kan opspoor.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/16616 |
| Date | 04 1900 |
| Creators | Matzopoulos, Mark |
| Contributors | Bellstedt, D.U. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | 79 leaves : ill. |
| Rights | University of Stellenbosch |
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