A RAPID AND SENSITIVE DOT-BLOT HYBRIDIZATION ASSAY FOR THE DETECTION OF CITRUS-EXOCORTIS VIROID IN CITRUS MEDICA WITH DIGOXIGENIN-LABELED RNA PROBES

A rapid and sensitive dot-blot hybridization assay using in vitro-tran scribed digoxigenin-labelled RNA probes (riboprobes) was developed aim ing at detection of citrus exocortis viroid (CEVd) in crude sap of inf ected Citrus medica plants. The protocol includes a very quick and sim ple preparation of RNA extracts from samples using a denaturation step with formaldehyde. From our results, the employment of this step is h ighly recommended because the hybridization signals in formaldehyde-de natured samples were significantly stronger when compared with that of extracts without formaldehyde treatment. The assay was found to be se nsitive enough to detect 0.1 ng of purified CEVd RNA and was able to d etect viroid in 0.2 mg of symptomatic Citrus medica leaves. The use of riboprobes also allowed hybridization under high temperature conditio ns, avoiding non-specific background.