Development of a Multiplexed LAMP-CRISPR Assay for Simultaneous Detection of SARS-CoV-2 Variants and Co-Circulating Respiratory Pathogens in Oyo State, South West Nigeria

The co-circulation of SARS-CoV-2 variants with influenza, respiratory syncytial virus, and other respiratory pathogens creates diagnostic complexity in syndromic respiratory illness management, and the molecular tools required for simultaneous discrimination are beyond the reach of most Nigerian health facilities. This research develops and validates a multiplexed RT-LAMP-CRISPR diagnostic platform for simultaneous detection of SARS-CoV-2 and co-circulating respiratory pathogens adapted for deployment at peripheral health facilities in Oyo State, South West Nigeria. In the development phase, specific crRNAs targeting conserved regions of SARS-CoV-2 N gene, influenza A H1 and H3 genes, RSV F gene, and human metapneumovirus will be designed and optimised. LAMP primer sets will be validated for isothermal amplification at 65 degrees Celsius within 30 minutes. Cas12a cleavage and lateral flow readout will be integrated for each target in a colour-coded multiplexed cassette. Analytical sensitivity, specificity, and cross-reactivity testing will be conducted using reference stocks and 200 archived respiratory specimens. Clinical validation will be performed prospectively in 400 patients with acute respiratory illness at three health facilities in Ibadan, assessed against RT-PCR as reference. Operational validation including shelf life, cold chain independence, and performance by lay users will follow. Original contributions include the first multiplexed respiratory pathogen LAMP-CRISPR platform validated for Nigerian clinical samples, a novel biosensor architecture combining isothermal amplification with visual CRISPR readout, and a field implementation framework for syndromic respiratory diagnostics in low-resource settings. Keywords: LAMP-CRISPR, SARS-CoV-2, respiratory pathogens, multiplexed diagnostics, Oyo State