Screening and preliminary application of DNA aptamers against IHNV G protein

Infectious hematopoietic necrosis virus (IHNV) is a major threat to salmonid aquaculture. Aptamers, with their high affinity and specificity, offer an alternative strategy for rapid and sensitive IHNV detection. In this study, recombinant IHNV G protein was expressed in a prokaryotic system and used as the target for aptamer selection via magnetic bead-based systematic evolution of ligands by exponential enrichment (SELEX). After ten rounds, five candidate aptamers (IHg01, IHg02, IHg04, IHg13, and IHg23) were obtained and characterized. Binding affinity analysis showed that all five aptamers had nanomolar-level dissociation constants, with IHg23 exhibiting the highest specificity and curve fitting. Molecular docking analysis revealed that the primary binding sites between IHg23 and the IHNV G protein were located within the single-stranded loop region of the IHg23 hairpin structure. Functional assays demonstrated that IHg23 significantly inhibited IHNV replication in epithelioma papulosum cyprini cells, with an inhibition rate up to 80.27 %. On the basis of IHg23, a sandwich enzyme-linked aptamer assay (ELAA) was developed using anti-G protein polyclonal antibody as the capture element. The method showed good repeatability (CV <10 %), a detection limit of 10^5.5-10^6.4 TCID₅₀/mL, and no cross-reactivity with other aquatic viruses. In conclusion, IHNV-specific aptamers were successfully screened and validated for diagnostic application. The established ELAA provides a promising tool for the rapid, sensitive, and specific detection of IHNV, contributing to epidemiological surveillance and IHN control in aquaculture.