Equine herpesvirus type 1 (EHV-1) is a globally prevalent equine pathogen responsible for severe respiratory, neurological, and reproductive disorders. Accurate and ultrasensitive detection of EHV-1 is critical for timely disease management. In this study, we report the development of the first G-quadruplex-forming aptamer specifically designed for EHV-1 detection. The aptamer was generated using an in silico approach, and its G-quadruplex conformation was confirmed using circular dichroism (CD) spectroscopy and crystal violet fluorescence assays. Binding affinity and specificity were assessed using a comprehensive panel of analytical techniques, including colorimetric assays, enzyme-linked apta-sorbent assay (ELASA), surface plasmon resonance (SPR), CD spectroscopy, and fluorescence analysis. The aptamer exhibited a high binding affinity in the picomolar range, as determined by SPR. In both colorimetric and ELASA platforms, it enabled the detection of as few as 10 viral particles per milliliter, compared to the 1000 viral particles per milliliter required by conventional PCR. ELASA results demonstrated excellent diagnostic performance, yielding an area under the curve of 0.96. Importantly, this aptamer-based method eliminates the need for DNA extraction, primers, or gel electrophoresis. These findings underscore the aptamer's strong potential as a cost-effective, rapid, and user-friendly point-of-care diagnostic tool for EHV-1, especially in low-resource or field settings. © 2025 Elsevier B.V.
