Adaptation of a filter paper method for RNA template preparation for the detection of avocado sunblotch viroid by reverse transcription qPCR

Abstract

An easy, rapid and inexpensive method of preparing RNA template for a reverse transcription qPCR assay for avocado sunblotch viroid (ASBVd) is described. This method depends on the principle of reversible binding of viroid RNA to filter paper under different concentrations of monovalent cation. Lysis buffers containing either sodium or lithium chloride were compared, and 1.5 M lithium chloride was shown to be optimal for the adsorption of the viroid RNA to the filter paper. The extraction method was validated using field samples and equivalent yields of viroid RNA were obtained using this method and either a commercial RNA extraction kit or a dsRNA chromatography method. The filter paper method of RNA extraction is ideally suited for the large-scale surveillance for ASBVd.