A spot-PCR technique for the detection of phloem-limited grapevine viruses.

Specific amplification of genomic fragments of grapevine trichovirus A (GVA), grapevine trichovirus B (GVB) and grapevine leafroll-associated closterovirus 3 (GLRaV3) was obtained by reverse transcription-PCR on total nucleic acid solubilized from small pieces of charged nylon membrane, on which a drop of crude infected grapevine sap was spotted (spot-PCR). A thermal treatment (95 degrees for 10 degrees) of spotted sap in a buffered solution improved the release of viral template. Consistent amplification was obtained with three viruses from fragments of the same respective blots up to 1 month after spotting, while a detection threshold limit comparable with standard PCR techniques was found for this method. Duplex PCR (i.e. amplification of different viruses from a mixed-infected grapevine source) was also found to be effective, since GVA and GLRaV3 were amplified by a mixture of specific primers in the same reaction. This rapid and easy sampling technique, using leaf petioles to express crude sap, may have a wide field application for screening grapevine viruses.