Browsing by Author "Matzopoulos, Mark"

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    The development of enzyme-linked immunosorbent assays to detect potato virus Y and potato leaf roll virus using recombinant viral coat proteins as antigens
    (Stellenbosch : University of Stellenbosch, 2005-04) Matzopoulos, Mark; Bellstedt, D. U.
    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.