A high sensitivity RT-PCR assay for the diagnosis of grapevine viroids in field and tissue culture samples.

An RNA extraction procedure, modified from published methods, and a high sensitivity reverse transcription-polymerase chain reaction (RT-PCR) assay have been developed for the detection of the five viroids in grapevines. All five viroids have been found in the 10 different grape varieties tested so far. This assay, specially optimised for viroids in low copy number by careful selection of DNA primers, has been used in conjunction with dot blot hybridization assay for the study of viroids in vines regenerated by shoot apical meristem culture (SAMC) and fragmented shoot apex culture (FSAC). The data indicate a differential reduction of viroids, rather than viroid elimination, in the regenerated vines. Transmission of viroids via grape seeds was also observed.