Development of Q-LAAD, an allonamer-based antigen test for the rapid detection of SARS-CoV-2

ABSTRACT The SARS-CoV-2 virus has spread globally causing coronavirus disease 2019 (COVID-19). Rapidly and accurately identifying viral infections is an ongoing necessity. We used the systematic evolution of ligands by exponential enrichment (SELEX) technique to produce a DNA allonamer with two distinct binding domains made allosteric through a linker section; one domain binds SARS-CoV-2 spike (S) protein, inducing a conformational change that allows the reporter domain to bind a fluorescent reporter molecule. We used bead-based fluorescence and immunofluorescence assays to confirm the allonamer’s affinity and specificity for S-protein and confirmed that the allonamer can bind to S-proteins with mutations corresponding to those of the alpha, beta, gamma, and delta variants. We then developed the allonamer-based Quantum-Logic Aptamer Analyte Detection (Q-LAAD) test, a rapid, high-throughput antigen test for qualitative detection of SARS-CoV-2 in clinical settings. We validated Q-LAAD against retrospective and prospective clinical anterior nasal swab samples collected from symptomatic patients suspected of having COVID-19. Q-LAAD showed 97% sensitivity and 100% specificity compared to the RT-qPCR assay. Q-LAAD has a limit of detection (LOD) of 1.88 TCID 50 /mL, is cost-effective and convenient, and requires only a common fluorescence plate reader. Q-LAAD may be a useful clinical diagnostic tool in the fight against SARS-CoV-2. HIGHLIGHTS Allonamers are allosterically-regulated DNA aptamers with multiple binding pockets Q-LAAD uses allonamers to detect SARS-CoV-2 spike protein in clinical samples Q-LAAD has high sensitivity and specificity and a low limit of detection Q-LAAD can detect spike proteins from multiple SARS-CoV-2 variants Q-LAAD is a dynamic, cost-effective rapid antigen test for detection of SARS-CoV-2