Abstract
When Diener discovered Potato spindle tuber viroid in 1971 (Diener, Virology 45:411–428, 1971), only a limited number of techniques were available for plant virus detection and purification. Biological assays using indicator hosts showing characteristic symptoms of infection and able to support high levels of viroid replication played a critical role in viroid detection and characterization. Polyacrylamide gel electrophoresis (PAGE) was the first molecular technique to be used for the rapid (2–3 days) identification of viroid-infected plants. Because it is the only diagnostic method that is sequence-independent, PAGE under denaturing conditions continues to play a key role in the identification of new viroids. Starting in the early 1980s, dot blot hybridization began to replace PAGE for routine viroid diagnosis. The first diagnostic protocols based on reverse transcription-polymerase chain reaction (RT-PCR) appeared approximately 10 years later, and much effort has subsequently been devoted to simplifying the sample preparation procedure and identifying group-specific primer pairs. This chapter describes four simple, easy-to-follow protocols—two involving PAGE and two others based on enzymatic amplification of viroid cDNAs—that currently play key roles in viroid discovery and characterization.
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Additional References
Flores R, Owens RA (2008) Viroids. In: Mahy BWJ, Van Regenmortel MHV (eds) Encyclopedia of virology, 3rd edn. Elsevier/Academic, London, UK (The largest single reference source of current virological knowledge for both specialist and general readers)
Hadidi A, Flores R, Randles JW, Semancik JS (eds) (2003) Viroids. CSIRO, Collingwood, Australia (This monograph provides a comprehensive overview of the biological and molecular properties of viroids. The most complete single source of information about viroids)
Singh RP (2007) Molecular detection of plant viroids. In: Punja ZK, De Boer SH, Sanfaçon H (eds) Biotechnology and plant disease management. CAB International, Wallingford, UK, pp 277–300 (R-PAGE and other electrophoretic methods are compared with other detection techniques such as molecular hybridization and RT-PCR)
Internet Resources
Subviral RNA Database (http://subviral.med.uottawa.ca/) (Maintained by Martin Pelchat (RNA Group/Groupe ARN, Département de biochimie, Université de Sherbrooke, Sherbrooke (Québec) J1H 5N4, Canada), this website contains the sequences of nearly 3000 viroids and related RNAs)
The Universal Database of the International Committee on the Taxonomy of Viruses (http://www.ncbi.nlm.nih.gov/ICTVdb) (A convenient source of current information on viroid (and virus) taxonomy)
Invitrogen Detection Technologies (http://www.probes.com/handbook/) (Contains an informative discussion of nucleic acid staining using SYBR Gold, SYBR Green I, and SYBR Green II)
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Owens, R.A., Sano, T., Duran-Vila, N. (2012). Plant Viroids: Isolation, Characterization/Detection, and Analysis. In: Watson, J., Wang, MB. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 894. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-882-5_17
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DOI: https://doi.org/10.1007/978-1-61779-882-5_17
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