Development and Validation of a CRISPR-Cas12a-Based Rapid Diagnostic Platform for Simultaneous Detection of MRSA and ESBL-Producing Enterobacteriaceae in Nigerian Clinical Specimens

Rapid, accurate, and low-cost diagnostics for antibiotic-resistant pathogens are a critical unmet need in Nigerian healthcare, where culture-based methods are slow and molecular platforms are often unaffordable. CRISPR-Cas12a-based nucleic acid detection offers exceptional sensitivity and specificity combined with potential for low-cost lateral flow readout, representing a transformative diagnostic paradigm. This study developed and validated a CRISPR-Cas12a-based multiplexed assay for simultaneous detection of MRSA (targeting mecA and spa genes) and ESBL-producing Enterobacteriaceae (targeting blaCTX-M-15 and blaTEM genes) from clinical specimens. Guide RNAs were designed and optimized using in silico tools and validated against reference strains. Assay performance was evaluated on 300 prospective clinical samples from teaching hospitals in Lagos, Kano, and Enugu States. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated against conventional culture and PCR as composite reference standards. The developed assay achieved analytical sensitivity of 10 copies per reaction for all four targets. Clinical sensitivity was 97.3 percent and specificity 98.6 percent for mecA detection. blaCTX-M-15 clinical sensitivity was 95.8 percent with 97.4 percent specificity. Total assay time was 65 minutes from DNA extraction to readout. Cost per reaction was estimated at 60 percent lower than commercial real-time PCR kits. A lateral flow dipstick readout was successfully integrated for instrument-free results interpretation. This platform represents an original technological contribution to AMR diagnostics suitable for implementation in resource-limited Nigerian laboratory settings. Keywords: CRISPR-Cas12a, MRSA, ESBL, rapid diagnostics, Nigeria.