Aptamer-linked immobilized sorbent assay for detecting foot and mouth disease (FMD) virus VP1 serotype O

Authors: Quintela, Irwin; Lopez-Magana Ii, Raymondo; HWANG, ANYA - Hispanic Association Of Colleges & Universities (HACU); VASSE, TYLER - Hispanic Association Of Colleges & Universities (HACU); Wu, Vivian

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/31/2025
Publication Date: 2/3/2026
Citation: Quintela, I.A., Lopez-Magana Ii, R., Hwang, A., Vasse, T., Wu, V.C. 2026. Aptamer-linked immobilized sorbent assay for detecting foot and mouth disease (FMD) virus VP1 serotype O. Scientific Reports. 16. Article 5453. https://doi.org/10.1038/s41598-025-34793-8.
DOI: https://doi.org/10.1038/s41598-025-34793-8

Interpretive Summary: Foot and mouth disease (FMD) is considered as one of the most feared livestock diseases due to its high infectivity rate and severely damaging effects on animals. Both wild and domestic cloven-hoofed animals such as sheep, goats, cattle, pigs, etc. are prone to FMD virus (FMDV) infection which can eventually lead to extensive meat and dairy production losses. Vesicular lesions of infected animals are unreliable clinical symptoms for diagnosis purposes due to shared similarities with other lesions i.e., vesicular diseases (SVD) in swine caused by Seneca virus A (SVA) and vesicular stomatitis (VS) in cattle caused by VS virus. Laboratory-based methods are employed to differentiate FMDV and SVA infections, but many of these currently available commercial diagnostic devices such as those that are antibody and molecular-based are difficult to implement in farms and other on-site settings. Aptamers are strong candidates for the detection of targets due to their unique recognition and binding properties. These high-affinity probes can be utilized to recognize, bind, and capture their targets in various types of detection technologies. They are short single-stranded (ss) nucleotides composed of DNA or RNA sequences that are selected based on their affinity and specificity for the target(s). By using combinatorial oligonucleotide libraries and exponential enrichment, this study has designed ssDNA aptamers that specifically bind and detect Foot and Mouth Disease (FMD) Virus VP1 Serotype O in a microplate setup. Due to its flexibility, the aptamers that were identified and developed in this study possess a great potential to be integrated into other detection platforms, such as the lateral flow assay system, to rapidly screen for FMDV and facilitate efficient monitoring of potential biohazard risks in farms, specifically in a pen-side setting.

Technical Abstract: Foot and Mouth Disease (FMD) is one of the most devastating animal diseases that can cause severe diseases, death and/or depopulation of infected livestock. Infected swine forms lesions like other major vesicular diseases. However, FMD can lead to myocardial necrosis and death. Current FMD diagnosis relies on immune and molecular-based methods (i.e ELISA) which often encounter cross-reactivity and cost issues. Therefore, a simple and portable diagnostic tool would greatly aid its detection efforts. This study aims to generate highly-specific aptamers that can bind to Viral Protein 1 (VP1) Serotype O capsid protein of FMD virus (FMDV) through Sequential Evolution of Ligands by Exponential Enrichment (SELEX) and incorporate the aptamers onto a 96-wells microplate to rapidly detect FMDV through Aptamer-linked Immobilized Sorbent Assay (ALISA). A random DNA pool library (100 µM, 81 -nt) with a 45 random nucleotide region flanked by fixed 3’ and 5’ regions was incubated (1 hr) with FMDV VP1 (0.1 nmol) immobilized on magnetic beads. Following incubation, non-binding sequences were discarded. The remaining bound ssDNA pool was amplified using conventional PCR. Amplified single-stranded sequences were recovered by denaturation (95 °C, 5 mins) and magnetic separation. The recovered pool was re-incubated with the target and non-target recombinant proteins for succeeding rounds resulting in the enrichment of target-specific aptamers. The final sequences were used for sandwich ALISA by immobilizing one set of non-biotinylated aptamers prior to adding increasing concentrations (25 ng/mL – 2 µg/mL) of VP1 and non-target proteins in the microplate wells. Finally, biotinylated aptamers, streptavidin horseradish peroxidase, and chromagen substrate were added and washed prior to the absorbance reading (450 nm). Results showed that the generated signals from VP1 samples were significantly higher than non-target samples (Senecavirus A). These aptamer sequences exhibited specificity in their ability to bind only to VP1 but not to non-target protein, thus suggesting that these novel aptamers may find application as specific detection tools for the rapid detection of FMDV. Future efforts will be focused on improving the sensitivity of the assay and verifying specificity by testing additional non-target agents that cause similar clinical symptoms with FMDV infection and related environmental samples.