Developing and Validating a CRISPR-Cas12a Point-of-Care Diagnostic Platform for Tuberculosis and Rifampicin Resistance Detection in Resource-Limited Settings in Nigeria

Current nucleic acid amplification tests for tuberculosis, including Xpert MTB/RIF, require expensive instrumentation and reliable electricity, limiting their utility in peripheral Nigerian health facilities. This research develops and validates a CRISPR-Cas12a-based lateral flow assay platform for simultaneous detection of Mycobacterium tuberculosis and rifampicin resistance mutations suitable for implementation in resource-limited settings across Nigeria. A technology development and validation design will guide guide RNA design targeting conserved regions of IS6110 and the rpoB hot-spot region. Recombinase polymerase amplification will be optimised as the isothermal preamplification step compatible with crude clinical lysates. Cas12a cleavage and lateral flow readout will be optimised on sputum, urine, and stool matrices from 50 clinical isolates. Analytical sensitivity and specificity will be established on reference strains and clinical samples. Prospective diagnostic accuracy evaluation will be conducted in 300 presumptive TB patients at peripheral health centres in Kaduna and Gombe States using liquid culture and Xpert as reference standards. Feasibility studies will assess user acceptability and workflow compatibility by healthcare workers without laboratory training. Original contributions include the first CRISPR-Cas12a TB diagnostic platform designed and validated specifically for Nigerian clinical matrices and operational conditions, provision of a new theoretical model for multi-target CRISPR-based infectious disease diagnostics, and a field-implementation evidence base for regulatory submission to NAFDAC. Keywords: CRISPR-Cas12a, tuberculosis, point-of-care diagnostics, rifampicin resistance, resource-limited settings