Genome-Informed Recombinase Polymerase Amplification Assay Coupled with a Lateral Flow Device for In-Field Detection...

Dickeya spp. cause blackleg and soft rot diseases of potato and several other plant species worldwide, resulting in high economic losses. Rapid detection and identification of the pathogen is essential for facilitating efficient disease management. Our aim in this research was to develop a rapid and field-deployable recombinase polymerase amplification (RPA) assay coupled with a lateral flow device (LFD) that will accurately detect Dickeya spp. in infected plant tissues without the need for DNA isolation. A unique genomic region (mglA/mglC genes) conserved among Dickeya spp. was used to design highly specific robust primers and probes for an RPA assay. Assay specificity was validated with 34 representative strains from all Dickeya spp. and 24 strains from other genera and species; no false positives or negatives were detected. An RPA assay targeting the internal transcribed spacer region of the host genome was included to enhance the reliability and accuracy of the Dickeya assay. The detection limit of 1 fg was determined by both sensitivity and spiked sensitivity assays; no inhibitory effects were observed when 1 µl of host sap, macerated in Tris-EDTA buffer, was added to each reaction in the sensitivity tests. 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.