The isolation of high-affinity ssDNA aptamer for the detection of ribavirin in chicken

Abstract

The abuse of ribavirin, an antiviral drug, in poultry breeding can cause quality degradation and drug resistance. So it is of great importance to establish a simple and effective method for detecting ribavirin in foods. In this work, aptamers that could especially bind to ribavirin with high affinity were obtained by the Capture-SELEX method. After 15 rounds of enrichment, ssDNA library pool was enriched and then analyzed by high-throughput sequencing. The seven most enriched sequences were selected as candidate aptamers for affinity and specificity characterization. Among the candidate aptamers, APT-1 was proved to be the optimal aptamer. The dissociation constant (K_d) values of APT-1 obtained by the two methods of colorimetric and fluorescence were 34.34 ± 6.038 nmol L⁻¹, 61.19 ± 21.48 nmol L⁻¹, respectively. To study the binding mechanism of the selected aptamer, molecular docking was conducted and results indicated that hydrogen bonds were formed at binding sites located at G37, T38, A40, T53 and A54. Furthermore, to confirm the practicability of the selected aptamer, a fluorescence assay was designed, showing the liner range within 1.0–50 ng mL⁻¹ and the low detection limit of 0.67 ng mL⁻¹. Besides, the aptamer was applied for the detection of ribavirin in chicken samples and the recoveries ranged from 87.26% to 105.57%, which showed great application potential in food safety.