Development and Validation of a Low-Cost CRISPR-Cas12a Diagnostic Platform for Simultaneous Detection of SARS-CoV-2, Influenza A, and Respiratory Syncytial Virus in Nigerian Healthcare Settings

Rapid and multiplex point-of-care diagnostics for respiratory viral pathogens are urgently needed in low-resource settings, where distinguishing between SARS-CoV-2, influenza A, and respiratory syncytial virus clinically is unreliable and PCR platforms are inaccessible. This translational study developed and validated a CRISPR-Cas12a-based lateral flow biosensor for simultaneous detection of all three targets from a single nasopharyngeal swab, calibrated and field-validated across three tertiary hospitals in Abuja (North Central), Port Harcourt (South South), and Kano (North West). Diagnostic platform development included guide RNA (gRNA) design targeting conserved genomic regions, optimization of isothermal RT-RPA pre-amplification, and crRNA-guided Cas12a cleavage with lateral flow readout. Analytical validation using reference standards was followed by clinical validation against WHO-approved RT-PCR as the gold standard in 600 patient samples. Sensitivity and specificity for SARS-CoV-2 detection were 96.8 and 98.4 percent respectively. Influenza A sensitivity was 94.2 percent and specificity 97.6 percent. RSV sensitivity reached 92.4 percent. Turnaround time was 45 minutes per test, and reagent cost was estimated at 1,800 naira per reaction. The CRISPR platform demonstrated robust field performance across all three geopolitical zones tested. This study contributes an original, locally validated respiratory viral diagnostic tool with direct applicability to Nigerian and broader African health systems. Keywords: CRISPR-Cas12a, point-of-care diagnostics, respiratory viruses, SARS-CoV-2, Nigeria.