Fast template preparation for molecular detection of Ralstonia solanacearum and Clavibacter m. sub sepedonicus in different samples

Member states of EU conduct systematic official surveys to confirm in the potato production system the absence of Cms, casual agent of potato ring rot, and of Rs, casual agent of potato brown rot. The PCR test on DNA extracted from potato and processing water samples is the most frequently first screening test used among those regulated by EU Directives. The considerable amount of samples lots and the necessity of quick diagnosis results opens the need for a more rapid detection of these two bacteria with sensitivity comparable to the one of the official methods. Different type of samples artificially contaminated by 107 to 100 cfu/ml bacteria suspensions were processed by a preliminary additional centrifugation, to obtain crude extracts. Three methods also were then tested: i) lyses with 10% 0.5 N NaOH, ii) lyses plus purification on FTA TMCard (Whatman) and iii) digestion with amylase enzyme. The sensitivity of PCR, added with 2% blotto, on crude and on extracts treated with the above three methods was compared to PCR on DNA extracted by a commercial kit. The PCR detection limit on DNA extracted from potato and potato processing water by a commercial kit was respectively 103 cfu/ml and 104 cfu/ml. The PCR assays performed on crude extract of potato processing water showed a sensitivity increase to 102cfu/ml, whereas the PCR assays on potato samples lysated and purified on FTA TM card ii) showed the same sensitivity of official DNA extraction, permitting also to detect latently infected tubers.