Molecular Hybridization Techniques for Detecting and Studying Viroids

Serological methods cannot detect viroids because they do not code for any proteins. It is not surprising, therefore, that viroids were the first obligate plant pathogen for which molecular hybridization was used as a detection tool (Owens and Diener, 1981). Molecular hybridization has also facilitated considerable progress in identifying viroid replication intermediates, ribozyme activities, and RNA structure. Many laboratories have replaced molecular hybridization as a routine diagnostic tool with reverse transcription polymerase chain reaction (RT-PCR) (see Hadidi et al., 2011; James et al., 2006 for review). However, hybridization remains a useful technique because it balances sensitivity with ease of use, time, and cost. The basic principles of nucleic acid hybridization on solid supports are described in previous reviews (Hull, 1993; Mu¨ hlbach et al., 2003; Palla´s et al., 1998, 2011) and will not be repeated here. In this chapter we update the last two decades of progress using molecular hybridization methodologies (dot-blot, gel-blot, multiplex, tissue-printing, and in situ hybridization) to detect and study viroids.