Screening and Application of Novel DNA Aptamers for Targeting Tilapia Lake Virus‐Infected Cells

Abstract

Tilapia Lake Virus (TiLV) is an emerging pathogen that poses a significant threat to global tilapia aquaculture, leading to substantial economic losses. The development of rapid, sensitive, and specific diagnostic methods is crucial for disease control and prevention. In this study, TiLV‐infected tilapia brain cell line (TiB cells) was used as the target for screening specific aptamers via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. The affinity, specificity, stability, cytotoxicity, and antiviral activity of the obtained aptamers were systematically analysed through flow cytometry, confocal laser scanning microscopy, real‐time quantitative PCR, and cell viability assays. This study successfully screened and obtained two aptamers, LH‐1 and LH‐2. Both aptamers specifically recognize and bind to TiLV‐infected TiB cells while showing no cross‐reactivity with normal cells or cells infected with other viruses. Their secondary structures exhibit typical stem‐loop configurations, with dissociation constants (K d ) reaching nanomolar levels at 506.9 nM and 326.4 nM, respectively, and they demonstrate no cytotoxicity toward host cells. Aptamer LH‐2 is capable of detecting as few as 1 × 10 3 cells/mL within 5 min at temperatures ranging from 4°C to 28°C. The detection capability of LH‐2 for TiLV infection was consistent with that of RT‐qPCR, as validated in vivo. These results demonstrate that LH‐2 possesses significant potential to be developed as a core recognition component for on‐site rapid detection kits. This study provides critical molecular tools and a theoretical foundation for the rapid diagnosis of TiLV and the development of novel targeted prevention and control strategies. It strongly advances the practical application of aptamer technology in the precise prevention and control of aquatic diseases.

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