Abstract
Acinetobacter baumannii is a critical WHO priority pathogen due to its multidrug resistance and high mortality in carbapenem-resistant infections. Resistance is predominantly mediated by class D carbapenemase genes blaOXA-23 and blaOXA-40, which spread rapidly via horizontal gene transfer in healthcare settings. To address the lack of a rapid assay capable of detecting both blaOXA-23 and blaOXA-40 in a single analytical workflow, we developed a multiplex two-step RPA–CRISPR/Cas12a assay. Since infections caused by strains harboring either gene require identical therapeutic management, their co-detection in a single reaction is clinically justified. Although simultaneous use of two crRNAs within a single CRISPR/Cas12a reaction is often considered technically challenging due to potential inter-crRNA competition, here it advantageously enables dual-target coverage without compromising sensitivity. The assay demonstrated high specificity with no cross-reactivity against a panel of clinically relevant bacterial species, including closely related Acinetobacter spp. Evaluation using genomic DNA extracted from 63 cultured clinical A. baumannii isolates revealed blaOXA-23 in 19 isolates (30.2%), blaOXA-40 in 28 (44.4%), and co-carriage of both genes in 9 (14.3%), with at least one resistance gene detected in 60.3% of isolates. The complete workflow was accomplished within 45 min without specialized equipment, offering a rapid, sensitive, and cost-effective solution for point-of-care molecular surveillance of carbapenem-resistant A. baumannii in clinical and resource-limited settings.