E-probes development for rapid, sensitive and specific pathogen detection in blueberries
Ana Maria Bocsanczy: University of Florida MREC
<div>In 2016 <em>Ralstonia solanacearum</em> was isolated and confirmed on multiple blueberry farms in Florida. The PCR-based detection process for this pathogen is expensive and time-consuming. Biosecurity policy requires rule-out R3B2 select agent strains presence before plant movement, thus tests are performed in only a handful of labs nationwide. A fast, sensitive and specific diagnostic protocol would allow earlier and more accurate detection. EDNA is a bioinformatics software platform used to design specific nucleic acid E-probes, and to detect and discriminate multiple pathogens in a metagenomics database in one test. Using MiProbe tool, we designed E-probes specific to <em>R. solanacearum</em> strains associated with blueberry and we used MiDetect tool for sensitivity and specificity analyses. Simulated metagenomes were created with available blueberry and Ralstonia genomes. A percentage range of pathogen abundance (0.001% to 10%) in the metagenome was used for the sensitivity analysis, while closely related strain genomes were added in the simulated metagenome (10%) for the specificity test. MiDetect could detect a 0.001% pathogen abundance, while it was specific at strain level. Currently we are developing E-probes for the select agent, and we are using MinION sequencing system for double blind validation tests with plant samples. We expect to prove that EDNA coupled with MinION is a fast, sensitive, specific, and cost-effective system to detect <em>R. solanacearum</em> in blueberry.</div>
View Presentation |
