TECHNICAL SESSION: Advanced detection and diagnosis of plant diseases
Genome-informed recombinase polymerase amplification assay for specific and sensitive detection of Dickeya species at point-of-care
Gamze Boluk - University of Hawaii at Manoa. Mohammad Arif- University of Hawaii at Manoa, Shefali Dobhal- University of Hawaii at Manoa, Anne Alvarez- University of Hawaii at Manoa, Alex Crockford- University of Wisconsin-Madison, Michael Melzer- University of Hawaii at Manoa
Dickeya species cause soft rot and on black leg diseases on potato, but also responsible for soft rot in many other plant species worldwide. Rapid pathogen detection is essential to facilitate efficient disease management. Our aim in this research was to develop a field deployable recombinase polymerase amplification (RPA) coupled with a lateral flow device (LFD) in order to detect Dickeya species from infected plant tissues without the need of DNA isolation. The comparative genomics approach was used to select a unique mglC gene for designing highly specific and robust primers and probe. The RPA assay specificity was validated with 35 strains from all Dickeya species and 15 strains from other neighbor genus and species; no false positives or negatives were detected. RPA primers and probe for internal control were also used to enhance the reliability and accuracy of the devloped assay. The detection limits of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effect was observed when 1µl host sap, macerated in TE buffer, was added in each sensitivity reaction. The developed RPA assay is rapid, highly accurate, sensitive and fully field-deployable. It has numerous applications in routine diagnostics, surveillance, biosecurity and disease management.