Dovas C I, Katis N I
Plant Pathology Laboratory, Faculty of Agriculture, Aristotle University of Thessaloniki, 54 124 Thessaloniki, Greece.
J Virol Methods. 2003 Jan;107(1):99-106. doi: 10.1016/s0166-0934(02)00197-0.
A reverse transcription (RT) polymerase chain reaction (PCR) assay was developed to allow rapid, and simultaneous detection of Vitivirus and Foveavirus sequences in two steps. The method involved a one-step RT-PCR, in which the combination of degenerate deoxyinosine-substituted primers amplified part of the polymerase region of both genera, followed by a nested PCR amplification that increased specificity and sensitivity of detection. The increase in sensitivity also permitted the use of a simple and rapid template preparation protocol, involving the spotting of plant sap extract on a nylon membrane. Consistent amplification with infected grapevine plants was possible after inclusion of additives for inhibiting polyphenolic compounds during template preparation. This spot nested RT-PCR method can reliably detect virus species of both genera in grapevine allowing simple, fast, and cost-effective analysis of a large number of samples in certification schemes.
