Detection of Chrysanthemum stunt viroid and Potato spindle tuber viroid by return‐polyacrylamide gel electrophoresis*

Abstract

Return-polyacrylamide gel electrophoresis (R-PAGE) is one of the methods used for the detection of viroids, in particular Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd). The method is based on temperature-related conformational changes of the circular single-stranded RNA molecule. This results in a characteristic electrophoretic behaviour, providing a clear separation between the viroid and nucleic acids of the plant. Since the description of R-PAGE, several modifications of the procedure have been published. In addition, several molecular techniques have been applied for the detection of viroids. However, R-PAGE is still particularly suitable for phytosanitary purposes because detection is not influenced by variations in the nucleotide sequence. This means that any Pospiviroid will be detected, provided the amount is above the detection threshold. In addition, relatively large amounts of leaf material can be handled in one test. This paper presents a recently developed R-PAGE procedure, based on the use of a horizontal electrophoresis system and paper wicks as buffer reservoirs. In comparison with the existing procedures, the new procedure is easy to perform and the use of chemicals is decreased. Results of the new procedure are shown and discussed in relation to those obtained by the previous procedure.

Natural infections of tomato by Citrus exocortis viroid, Columnea latent viroid, Potato spindle tuber viroid and Tomato chlorotic dwarf viroid

“…After detection by r-PAGE, the isolates were propagated on tomato (see below). Four to five weeks after inoculation young leaves of the inoculated plants were collected and nucleic acids, including viroids, were isolated by a phenol extraction and subsequently stored under ethanol at )20°C (Roenhorst et al, 2000).…”

“…The presence of the viroids in these symptomatic tomatoes was established by return-polyacrylamide gel electrophoresis (r-PAGE, Huttinga et al, 1987;Roenhorst et al, 2000). However, their identity could not be established by electrophoresis alone.…”

Diagnostic techniques for viroids

“…The disadvantage of PAGE is its susceptibility to impurities; the most critical step of this method is therefore sample preparation (Hadidi et al ., ). Nevertheless, PAGE is still widely used, especially in combination with other methods (Roenhorst et al ., ).…”

“…Return PAGE (R-PAGE) (Schumacher et al, 1983(Schumacher et al, , 1986), on the other hand, has enabled the separation and identification of severe and mild strains (Singh & Boucher, 1987). This method is based on temperature-related conformational changes of the circular viroid RNA and has been mainly used for the detection of chrysanthemum stunt viroid (CSVd) and PSTVd (Roenhorst et al, 2000). Identification of linear and circular RNAs is possible by using two-dimensional PAGE (2D-PAGE), which can be implemented in several different ways (Schumacher et al, 1983;Feldstein et al, 1997;Vadamalai et al, 2006;Hou et al, 2009).…”

“…The latest modifications of the different PAGE protocols have shown that this technique is easy to perform, versatile, suitable for large-scale testing and, in combination with silver staining, even highly sensitive and specific (Roenhorst et al, 2000;Hadidi et al, 2003). The disadvantage of PAGE is its susceptibility to impurities; the most critical step of this method is therefore sample preparation (Hadidi et al, 2003).…”