AbstractBackgroundDue to their prevalence worldwide, the β-lactamases CTX-M and plasmid-mediated CMY-2 are important antimicrobial resistance enzymes in a clinical setting. While culture- and PCR-based detection methods exist for these targets, they are time consuming and require specialist equipment and trained personnel to carry out.MethodsIn this study, three rapid diagnostic single-plex and a prototype triplex assay were developed, using recombinase polymerase amplification with lateral flow detection (RPA-LF), and tested for their sensitivity and specificity using two isolate DNA panels (n = 90 and n = 120 isolates). In addition, the RPA-LF assays were also tested with a small number of faecal extract samples (n = 18).ResultsThe RPA-LF assays were able to detect blaCXT-M-group-1, blaCTX-M-group-9 and blaCMY-2-type variants with high sensitivity (82.1%–100%) and specificity (100%) within a short turnaround time (15–20 min for amplification and detection).ConclusionsRPA-LF assays developed in this study have the potential to be used at or close to the point of care, as well as in low-resource settings, producing rapid results to support healthcare professionals in their treatment decisions.