Detection of citrus exocortis viroid in crude extracts by dot-blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the technique

Abstract

Dot-blot assays to detect citrus exocortis viroid (CEV), in clarified sap and unfractionated total nucleic acid preparations of CEV-infected Gynura aurantiaca and chrysanthemum, were impaired by the non-specific binding of the radioactive probe shown by the healthy controls. This non-specific background was considerably reduced by the addition to the hybridization mixture, of the fraction of nucleic acids from healthy plants which are insoluble in 2 M LiCl (containing mainly the large ribosomal RNAs). Sample denaturation with formaldehyde was found to provide a high increase of hybridization, when compared with samples either denatured with formamide or directly spotted. Nitrocellulose was observed to be a better solid support than charge-modified nylon, in terms of the sensitivity of viroid detection by spot hybridization.