Development of a polyriboprobe for detecting pospiviroids. In: International Workshop on Viroids and Satellite RNAs

Spread of viroids belonging to the genus Pospiviroid (family Pospiviroidae) has been recently recorded in ornamentals and vegetables in several countries of the European Union (EU). Major concerns derive from the identification of Potato spindle tuber viroid (PSTVd) and Chrysanthemum stunt viroid (CSVd), which are quarantine pests in EU. Effectiveness of control measures against viroid spread, which are based mainly on the use of viroid-free propagation material and on the interception of infected plants, requires fast, reliable, sensitive and economic detection methods. Methods allowing simultaneous detection of several viroid species largely contribute to contain costs. In this context, a single molecular probe (polyprobe), composed of assembled sequence fragments from most viroid species belonging to Pospiviroids genus (family Pospiviroidae) appears very appropriate for detecting them by molecular hybridization because only one single transcription reaction is needed. Based on bioinformatics analyses of pospiviroid genomic RNAs, we have developed a polyprobe (POSPIprobe) that detects at least eight different pospiviroid species, including PSTVd and CSVd. Partial sequences from four different pospiviroids were selected and cloned directionally to generate a single vector that was used for synthesizing the POSPIprobe by in vitro-transcription. POSPIprobe specificity for eight pospiviroids was confirmed by Northern-blot, while its sensitivity was tested by dot-blot assays, providing similar results to that obtained using single probes separately. Dot-blot analyses with the POSPIprobe was validated by simultaneously testing 68 samples from tomato, chrysanthemum, Argyranthemum frutescens and pepper infected by different pospiviroids, confirming the high potential of this probe in quarantine, certification and survey programs. These data have been recently published in Journal of Virological Methods 2012.