Spot Hybridization for Detection of Viroids and Viruses

Publisher Summary Viroids are not detectable in healthy individuals of the same species, but after introduction into such individuals, they are replicated autonomously and cause the appearance of the characteristic disease syndrome. Although at least one viroid, that is, the potato spindle tuber viroid (PSTV), can be eliminated from infected plants. Two types of diagnostic test have been used to detect PSTV: bioassay on suitable tomato cultivars and polyacrylamide gel electrophoresis (PAGE) of extracted nucleic acids. Neither of these methods is suitable for the rapid screening of thousands of seed potato tubers. Nucleic acid hybridization in solution is extremely sensitive and has been used to quantitate viroid concentrations in purified RNA preparations and to index avocados for the presence of avocado sunblotch viroid, but even partial purification of nucleic acids from multiple samples is laborious. One possible alternative is hybridization of highly radioactive DNA complementary to PSTV (PSTV cDNA) with PSTV bound to a solid support, followed by autoradiographic detection of the resulting DNA–RNA hybrids. Two conditions had to be fulfilled before hybridization with PSTV cDNA could be developed into a practical diagnostic test for the detection of PSTV in numerous samples: (1) PSTV cDNA had to be available in unlimited quantities and at high specific radioactivity, requiring molecular cloning of the cDNA by recombinant DNA technology and in vitro labeling and (2) to expedite and simplify sample preparation, clarified sap rather than purified nucleic acid had to be suitable as the viroid source. Both conditions have been fulfilled and a practical diagnostic test for the detection of PSTV based on nucleic acid hybridization has been developed. It is also at least 10 times more sensitive than PAGE. The chapter describes existing nucleic acid spot hybridization methodology and modifications that may be required for its use with other viroids or plant viruses.