The development of enzyme-linked immunosorbent assays to detect potato virus Y and potato leaf roll virus using recombinant viral coat proteins as antigens

[摘要] ENGLISH ABSTRACT: Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV) are two of the most destructivepotato viruses capable of drastically diminishing crop yields by up to 80%. The presence ofthese viruses in planting material namely seed potato stocks are routinely diagnosed byenzyme-linked immunosorbent assay (ELISA) kits. The kits currently used by Potatoes SouthAfrica are obtained from Europe. These kits have produced false positive and false negativeresults in the past. Potatoes South Africa required an ELISA that was reliable, cheap andspecific 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 Africansource of infected plant material. The PVY and PLRV coat protein genes were subsequentlycloned into pGEM-T Easy vector and sequenced. The sequences of the two viruses werealigned and compared to corresponding viral coat protein gene sequences obtained fromGenbank. Subsequently the two amplified and cloned coat protein genes of PVY and PLRVwere sub-cloned into an expression system (pET-14b) to induce and express the respectiverecombinant viral coat proteins. The induction of the cloned coat protein genes yieldedsuccessful production of the recombinant PVY coat protein but the induction and expressionof the recombinant PLRV coat protein was unsuccessful.The isolated recombinant PVY CP was then used to immunize a rabbit to produce highlyspecific anti-PVY CP immunoglobulins. The antiserum obtained from the rabbit was used todevelop an ELISA to detect the presence of PVY in seed potato stocks in South Africa. TheELISA kit was subsequently used in preliminary trials to determine if the kit could detectPVY infected plant material. The initial results of the ELISA trials using PVY infectedmaterial obtained from Potatoes South Africa yielded positive results.

[发布机构] Stellenbosch University