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POSTERS: Pathogen detection, quantification and diagnosis

Improved detection of Clavibacter michiganensis subsp. sepedonicus using droplet digital PCR
Amy Charkowski - Colorado State University. Frida Zink- Colorado State University, Shaista Karim- Colorado State University, Luke Tembrock- Colorado State University

The Gram-positive bacteriumClavibacter michiganensis subsp. sepedonicus(Cms) is the causal agent of bacterial ring rot (BRR) in potato. In the United States, Cms is a zero-tolerance pathogen in seed potato production, and the presence of Cms in a single plant or tuber on a farm can result in rejection of whole seed lot. Assay results near threshold levels for common Cms detection assays, including enzyme-linked immunosorbent assay (ELISA) and quantitative real-time PCR (qPCR) assay, are common and this makes it difficult to decide whether the pathogen is present. We developed a droplet digital PCR (ddPCR) assay for Cms and compared its accuracy and sensitivity to currently used assays. We found that ddPCR is more sensitive than qPCR and that unlike qPCR, it could reliably detect one infected potato tuber core mixed with 800 uninfected tuber cores. We also tested field samples that gave ambiguous results with ELISA and qPCR, presumably due to low levels of Cms and found that ddPCR provided unambiguous test results for these field samples. A limited sampling of tubers from a local grocery store showed that Cms is still present in the potato system. Our results provide evidence that ddPCR offers a robust alternative to PCR and ELISA for CMS testing.