Viral Capsid Protein Architecture Informed Selection of ssDNA Aptamers for Specific Detection of FMDV

Foot-and-mouth disease virus (FMDV) is a highly contagious pathogen that poses a serious threat to the global livestock industry, highlighting the need for stable and serotype-specific diagnostic tools. In this study, single-stranded DNA (ssDNA) aptamers were developed against a full-length recombinant FMDV P1 polyprotein (FLRPp), which preserves native-like capsid conformational epitopes. Following 11 rounds of Systematic Evolution of Ligands by Exponential Enrichment (SELEX) with negative selection, aptamer candidates were identified and experimentally characterized. Binding to surface-accessible capsid regions was validated using fluorescence-based assays, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assays (ELISA). An aptamer-based sandwich ELISA further demonstrated the functional applicability of the selected aptamers, enabling sensitive detection of FLRPp and inactivated FMDV capsid with a subnanomolar limit of detection. Among the candidates, FMD_1 exhibited the highest affinity toward the O-type capsid precursor ( K D = 3.46 × 10 –10 M) and strong serotype specificity relative to A and Asia1 serotypes. These results establish a structure-guided aptamer selection strategy that provides analytically functional recognition elements for serotype-specific detection of FMDV.