Digoxigenin-labeled riboprobes applied to phytosanitary certification of tomato in Italy

A dot-blot hybridization system using digoxigenin-labeled riboprobes and chemiluminescent detection was developed for the diagnosis of infections by cucumber mosaic cucumovirus (CMV), tomato spotted wilt tospovirus (TSWV), potato Y potyvirus (PVY), tomato yellow leaf curl geminivirus (TYLCV), and, occasionally, alfalfa mosaic alfamovirus (AMV) and tomato mosaic tobamovirus (ToMV) in tomato seedlings. This system was successfully applied to sanitary certification purposes. Tomato samples were collected using a systematic sampling method. The optimal ratio of tissue sample to extraction solution was 1.2 g to 6 ml. Hybridization reactions were done using a riboprobe mixture. This procedure saved time and cut costs without reducing sensitivity. TSWV was detected up to a ratio of 21 mu g of infected tissue per spot; whereas the other viruses were detectable at a ratio of 17 mu g of infected tissue per spot. The method allowed the analysis of 400 to 500 samples (representative of approximately 1.15 million tomato seedlings) per day and fulfilled the requirements for virus detection in routine diagnosis.