Abstract
Numerous molecular-based detection protocols include an amplification step of the targeted nucleic acids. This step is important to reach the expected sensitive detection of pathogens in diagnostic procedures. Amplifications of nucleic acid sequences are generally performed, in the presence of appropriate primers, using thermocyclers. However, the time requested to amplify molecular targets and the cost of the thermocycler machines could impair the use of these methods in routine diagnostics. Recombinase polymerase amplification (RPA) technique allows rapid (short-term incubation of sample and primers in an enzymatic mixture) and simple (isothermal) amplification of molecular targets. RPA protocol requires only basic molecular steps such as extraction procedures and agarose gel electrophoresis. Thus, RPA can be considered as an interesting alternative to standard molecular-based diagnostic tools. In this paper, the complete procedures to set up an RPA assay, applied to detection of RNA (Potato virus Y, Potyvirus) and DNA (Wheat dwarf virus, Mastrevirus) viruses, are described. The proposed procedure allows developing species- or subspecies-specific detection assay.
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Acknowledgments
The authors thank Maxime Assous-Dupont (INRA, Montpellier, France) and Isabelle Naas (INRA, Rennes, France) for their help during the setup of the RPA; Isabelle Abt and Romain Mabon (INRA, Montpellier) for providing the leafhoppers; and Maryse Guillet, from the FN3PT, for providing the PVY polyclonal antibodies.
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Glais, L., Jacquot, E. (2015). Detection and Characterization of Viral Species/Subspecies Using Isothermal Recombinase Polymerase Amplification (RPA) Assays. In: Lacomme, C. (eds) Plant Pathology. Methods in Molecular Biology, vol 1302. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2620-6_16
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DOI: https://doi.org/10.1007/978-1-4939-2620-6_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2619-0
Online ISBN: 978-1-4939-2620-6
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