3.3.69
DEVELOPMENT OF A PCR-BASED ASSAY FOR THE DETECTION OF POTATO VIRUS A IN PRIMARY INFECTED TUBER MATERIAL
D DARLING, KJ O'DONNELL and E CANNING Scottish Agricultural Science Agency, East Craigs, Edinburgh EH12 8NJ, UK Background and objectives
The Scottish Agricultural Science Agency carries out work towards the Scottish Seed Potato Classification Scheme (SPCS) [1], which specifies strict tolerances for the level of virus permitted in seed potatoes. The majority of this testing is carried out by ELISA. However, the presence of virus-transmitting aphids late in the growing season may lead to the development of primary infection, where the low virus titre is restricted to the tubers and ELISA is not sensitive enough to detect primary infection in tuber material. Therefore, tuber dormancy must be broken to allow the resulting leaf material, exhibiting secondary viral infection, to be tested by ELISA in a process which takes approximately 8 weeks. We report here on the development of a PCR-based assay, sensitive enough to detect low virus titres directly from tubers material which significantly reduces the testing time required, thereby increasing testing efficiency. Materials and methods
Immunocapture was employed using monocional antibodies against PVA to isolate the virus particles, followed by RT-PCR using specific PVA primers and then positive samples were detected using a PCR ELISA microtitre plate system, based on the method of Tavernarakis et al [1], minimising the time required to process results. Results and conclusions
This methodology was found to be 100 times more sensitve than ELISA detecting the presence of 5pg viral RNA and therefore could successfully detect low virus titres in tuber material, Following further testing on actual SPCS tuber samples, this assay may have the potential to replace the current protocol of growing on tubers suspected of having primary viral infection. References
1. Jeffries CJ, 1986. BCPC Monograph No.33, 239-247. 2. Tavernarakis N, Hatzidakis G, Viatakis G and Krambovitis E, 1995. Serodiagn. Immunother. Infect. Disease 7, 202-206.