Genome-Informed Loop-Mediated Isothermal Amplification (LAMP) Assays for Detection and Differentiation of Ralstonia...

The Ralstonia solanacearum species complex (RSSC) causes bacterial wilt in a broad range of economically important hosts. The RSSC has been reclassified into three species, of which R. solanacearum sequevars 1 and 2 in phylotype IIB are designated as Select Agents in the U.S. Many rapid tests for "R. solanacearum" target the RSSC and thus cross-react with R. pseudosolanacearum, which is endemic to the U.S. To avoid false positive identifications and unnecessary triggering of Select Agent measures, four portable LAMP-based screening assay variations were developed and validated for specific detection of R. pseudosolanacearum: real-time LAMP assays for detection of R. pseudosolanacearum (1) and differentiation of R. pseudosolanacearum phylotypes I and III in the AmpliFire^® Isothermal Fluorometer (2) or Genie^® III (3), and a colorimetric LAMP for detection of R. pseudosolanacearum (4). All assays demonstrated high sensitivity (10-50 colony-forming units (CFU)/reaction) and specificity; 100% analytical specificity and selectivity based on 177 RSSC isolates (81 phylotype I, 70 phylotype II, 15 phylotype III and 11 phylotype IV) and 53 non-RSSC bacterial pathogens on relevant hosts and soil, and 100% diagnostic specificity and sensitivity based on testing inoculated plants and a Euphresco sample panel. The assays presented here represent useful diagnostic tools that can be deployed on-site to aid domestic survey and detection efforts.