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Chapitre D'ouvrage Année : 2015

Detection and characterization of viral species/subspecies using isothermal Recombinase Polymerase Amplification (RPA) assays

Résumé

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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Dates et versions

hal-01208781 , version 1 (02-10-2015)

Identifiants

Citer

Laurent Glais, Emmanuel Jacquot. Detection and characterization of viral species/subspecies using isothermal Recombinase Polymerase Amplification (RPA) assays. Plant Pathology, 1302, Editions Springer; Humana Press/Springer Imprint, 2015, Methods in Molecular Biology, 978-1-4939-2619-0. ⟨10.1007/978-1-4939-2620-6_16⟩. ⟨hal-01208781⟩
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