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

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 allonamers 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 TCID50/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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/22280297v1_ufig1.gif" ALT="Figure 1"> View larger version (86K): org.highwire.dtl.DTLVardef@1250f56org.highwire.dtl.DTLVardef@11eb2dorg.highwire.dtl.DTLVardef@6711dcorg.highwire.dtl.DTLVardef@c5bf3e_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIAllonamers are allosterically-regulated DNA aptamers with multiple binding pockets C_LIO_LIQ-LAAD uses allonamers to detect SARS-CoV-2 spike protein in clinical samples C_LIO_LIQ-LAAD has high sensitivity and specificity and a low limit of detection C_LIO_LIQ-LAAD can detect spike proteins from multiple SARS-CoV-2 variants C_LIO_LIQ-LAAD is a dynamic, cost-effective rapid antigen test for detection of SARS-CoV-2 C_LI