PCR AND NONISOTOPIC LABELING TECHNIQUES FOR PLANT-VIRUS DETECTION

PCR technology permits the detection of viruses at levels several orde rs of magnitude lower than is possible by other methods. This high sen sitivity facilitates detection of virus sequences during the early sta ges of infection of plants and in soil and vector samples. Early detec tion of beet necrotic yellow vein virus (BNYVV) in Beta vulgaris is an important part of the strategy for prevention of the spread of rhizom ania, a commercially significant disease of sugar beet. A diagnostic t est for BNYVV has been developed. This test involves amplification of the viral genome by PCR coupled with non-isotopic labeling and detecti on of specific sequences. The PCR amplification of BNYVV sequences has been optimized with respect to primer design, sample preparation and reaction conditions. Several non-isotopic labeling strategies for sign al amplification have been compared. Hybridization with digoxigenin-la belled cDNA permits the most sensitive detection of PCR products and i s the most appropriate method for routine diagnosis. These observation s are discussed in the context of the. application of PCR for detectin g a wide range of viruses.