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POSTERS: Pathogen detection, quantification and diagnosis

Detection of Clavibacter michiganensis and C. michiganensis ssp. nebraskensis using multiplex recombinant polymerase amplification coupled with LFD
Adriana Larrea-Sarmiento - University of Hawaii at Manoa. Mohammad Arif- University of Hawaii at Manoa, Anne Alvarez- University of Hawaii at Manoa

Clavibacter michiganensis (Cm) is an agriculturally important species comprising nine host-specific subspecies including C. m. subsp. nebraskensis (Cmn), which causes Goss's wilt and blight of maize. A robust, reliable, simple and field deployable method is required to specifically detect Cm and Cmn in infected plant tissue for timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Cm and Cmn directly from an infected host. Comparative genomic analyses were performed to identify unique and conserved genomic regions for long primer and probe design. The multiplex RPA assay was evaluated using 54 strains representing all nine subspecies of Cm and other closely related bacterial species. No false positives or false negatives were detected. The multiplex RPA reactions were also incubated in a closed hand at body temperature; results were again specific. The assay does not require DNA isolation and can be directly performed using host sap. The detection limit of 10 pg and 100 fg was determined for Cm- and Cmn-specific primers/probe, respectively. The developed multiplex assay is accurate and has applications at point-of-care diagnostics without the need for DNA isolation. This is the first multiplex RPA for any plant pathogen.