Aptamer-linked Immobilized Sorbent Assay for detecting Foot and Mouth Disease (FMD) Virus VP1 Serotype O

Authors: Quintela, Irwin; HWANG, ANYA - Volunteer; VASSE, TYLER - Volunteer; Wu, Vivian

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 2/28/2024
Publication Date: N/A
Citation: N/A

Interpretive Summary:

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 screening 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 it onto a 96-wells microplate and 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 in a microplate by immobilizing one set of non-biotinylated aptamers before adding increasing concentrations of VP1 and non-target proteins. Finally, biotinylated aptamers, streptavidin horseradish peroxidase (S-HRP) and chromagen substrate were added prior to the absorbance reading (450 nm). Results showed that the aptamer (FMDV Apt) had an estimated dissociation constant (Kd) of 14 ± 8.6 nM. A concentration-dependent relationship within a concentration range (0.05 ng/mL – 100 ng/mL) of FMDV VP1 protein was observed. Moreover, the FMDV ALISA did not experience cross-reactivity when tested with non-targets with a detection limit of 500 ng/mL, suggesting its robustness and potential use in other detection platforms. Future efforts will focus on improving the assay's sensitivity and verifying specificity by testing additional non-target agents that cause similar clinical symptoms with FMDV infection and other related environmental samples.