Comparative Evaluation and Optimization of a CRISPR/Cas-Based Assay for Rapid Detection of Tilapinevirus tilapiae...

Tilapia lake virus (Tilapinevirus tilapiae; TiLV) remains a significant threat to global tilapia aquaculture, underscoring the urgent need for rapid, sensitive, and field-ready diagnostic approaches. In this study, we developed a CRISPR-Cas12a assisted reverse transcriptase-recombinase polymerase amplification assay (Cr-TiLV) targeting a conserved region of segment 6 of the TiLV genome. The integration of RT-RPA with Cas12a significantly enhanced analytical sensitivity, enabling the detection of viral RNA at levels as low as 10 copies. Diagnostic assessment using 40 clinical samples demonstrated that the Cr-TiLV assay achieved sensitivities of 86.67% and 82.29%, and accuracies of 90% and 85%, respectively, relative to semi-nested PCR and RT-qPCR, with specificity approaching 99.9% for both benchmark methods. Although less sensitive than RT-qPCR for samples with low viral loads, the assay consistently detected low to moderate viral concentrations and showed substantial concordance with semi-nested PCR. Operating isothermally at 39 °C and requiring minimal laboratory resources, the Cr-TiLV assay offers a rapid and field-deployable diagnostic solution with strong potential for routine TiLV surveillance in resource-limited aquaculture settings.

The online version contains supplementary material available at https://doi.org/10.1007/s12088-026-01550-y.