Multiplex recombinase polymerase amplification (RPA) assay developed using unique genomic regions and coupled with a lateral flow device for rapid on-site detection of genus Clavibacter and C. nebraskensis

Clavibacter is an agriculturally important bacterial genus comprising nine host-specific species/subspecies including C. nebraskensis (Cn), which causes Gosss wilt and blight of maize. A robust, simple, and field-deployable method is required to specifically detect Cn in infected plants and distinguish it from other Clavibacter species for quarantine purposes and timely disease management. A multiplex Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Device (LFD) was developed for sensitive and rapid detection of Clavibacter and Cn directly from infected host. Unique and conserved genomic regions, the ABC transporter ATP-binding protein CDS/ABC-transporter permease and the MFS transporter gene, were used to design primers/probes for specific detection of genus Clavibacter and Cn, respectively. The assay was evaluated using 52 strains, representing all nine species/subspecies of Clavibacter, other closely related bacterial species, and naturally- and artificially-infected plant samples; no false positives or negatives were detected. The 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 Clavibacter- and Cn-specific primers/probes, respectively. The detection limit for Cn-specific primer/probe set was decreased to 1,000 fg when 1 {micro}L of host sap was added into the RPA reaction containing 10-fold serially diluted genomic DNA; though no effect was observed on Clavibacter-specific primer/probe set. The assay is accurate and has applications at point-of-need diagnostics. This is the first multiplex RPA for any plant pathogen. IMPORTANCEClavibacter species are prevalent worldwide as have the potential to result in systemic infection. In the past, detection attempts have relied on both molecular- and immunological-based assays; however, current detection methods are time consuming and laborious. Field-deployable tests are desirable to identify potential samples infected with Clavibacter species. This study demonstrates that the field-deployable isothermal multi-target recombinase polymerase amplification can be performed for the simultaneous detection of the genus Clavibacter in general (all species), and C. nebraskensis, in particular, without specialized equipment. Additionally, the multiplex RPA coupled with a LFD may confer the benefits of faster detection and discrimination of Clavibacter species that affect critical regions susceptible to infection. This user-friendly format offers a flexible assay to complement both nucleic acid amplification and novel diagnosis methods without the need for DNA purification; this assay may serve as a point-of-reference for developing multiplex RPA assay for other plant pathogens.