Rapid Gel-Based Recombinase Polymerase Amplification Assay for Detection and Sequence Confirmation of Ehrlichia canis

Canine monocytic ehrlichiosis (CME), caused by Ehrlichia canis, requires rapid, field-deployable diagnostics in endemic regions. This study developed and evaluated a recombinase polymerase amplification (RPA) assay for rapid isothermal detection of E. canis DNA using a dual-format gel-based approach. The diagnostic agreement of the RPA assay was evaluated by comparison with TaqMan qPCR and droplet digital PCR (ddPCR) using 41 well-characterized canine blood samples. The RPA assay yielded detectable amplification following an optimized incubation time of 30 min at 39 °C, combining agarose gel electrophoresis for visualization with amplicon recovery for sequencing. RPA detected E. canis in 25/41 samples (60.9%), identical to qPCR (60.9%) but lower than ddPCR (82.9%). Diagnostic evaluation showed moderate agreement between RPA and ddPCR (κ = 0.487, 95% CI: 0.233-0.741) and perfect agreement between RPA and qPCR (κ = 1.00). RPA demonstrated the same detection rate as qPCR in acute CME cases while using a low-cost heating device instead of specialized thermocyclers and identified positive cases without false-positive results. Sequencing of RPA amplicons confirmed 100% identity with the E. canis dsb gene reference sequence (AF403710.1). This RPA-based framework offers a flexible, dual-format approach that balances speed, accessibility, and diagnostic reliability, making it highly suitable for CME management in resource-limited settings.