Feasibility of a diagnostic kit for multiplex DIG-chemiluminescent detection of tomato viruses.

Six specific DIG-labeled riboprobes have been synthesized to the following viruses: alfalfa mosaic alfamovirus (AMV), cucumber mosaic cucumovirus (CMV), potato Y potyvirus (PVY), tomato mosaic tobamovirus (ToMV), tomato spotted wilt tospovirus (TSWV), and tomato yellow leaf curl geminivirus (TYLCV). The probes were stored for several months as dried pellet at 4°C or in ethanol at -70°C and used separately or in mixture in nucleic acid hybridization tests. Samples were applied to Nylon membranes either as spots of crude plant extract prepared in 50 mM NaOH-2.5 mM EDTA or as tissue imprints made in the field on a dried, alkali-treated membrane. The different storage conditions did not affect the limit of sensitivity. The DIG-cRNA probes stored at 4°C were used also to detect virus infection in the imprints of different virus-host combinations, including woody and herbaceous plants. The dried pellet stored at 4 °C seems a suitable format for supplying ready-to-use DIG-labeled riboprobes in a kit which could also contain a number of ready-to-use alkali-treated Nylon membranes already spotted with control samples.