Development and evaluation of a highly specific lateral flow immunoassay (LFIA) strip for the rapid detection of Tilapia lake virus in fish samples

Tilapia lake virus (TiLV) has emerged as a critical threat to global tilapia aquaculture, necessitating rapid and accessible diagnostic tools to mitigate economic losses. In this study, we developed and evaluated a highly specific lateral flow immunoassay (LFIA) strip designed for the rapid on-field detection of TiLV. The assay uses a pair of TiLV-specific monoclonal antibodies (TV5-A10 as the colloidal gold–labelled detector and TV14-D9 as the test-line capture antibody), which target the TiLV segment 8 structural protein. Performance evaluation demonstrated that the LFIA strip possesses high analytical specificity, exhibiting no cross-reactivity with common non-target tilapia pathogens. Analytical sensitivity, assessed using recombinant TiLV-segment 8 protein, yielded a limit of detection of 0.09 μg/ml, while testing of TiLV-positive gill homogenate remained positive at a dilution of 1:640. Diagnostic performance was evaluated using 61 field-collected clinical samples and compared to a molecular reference method. The LFIA strip achieved a diagnostic accuracy of 98.36%, with a relative sensitivity of 97.92% and a relative specificity of 100.00%. Agreement between the LFIA strip and the molecular reference method was almost perfect (Cohen's kappa = 0.95). Further, accelerated ageing tests indicated that the strips remain stable for approximately two years at ambient temperature. These findings indicate that the developed LFIA strip is a robust, user-friendly tool suitable for routine surveillance and early presumptive diagnosis of TiLV in fish farms.