https://phaidra.vetmeduni.ac.at/o:5395 CRISPR‐Enhanced RAA‐SHERLOCK Assay for Point‐of‐Care Detection of Cyprinid Herpesvirus‐3: Development, Validation and Clinical Application Soliman, Hatem Hatem Soliman 0000-0003-1641-6759 University of Veterinary Medicine Vienna Akram, Naveed Naveed Akram University of Veterinary Medicine Vienna Saleh, Mona Mona Saleh 0000-0001-9273-1502 University of Veterinary Medicine Vienna Wiley journal article open access eng Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance. CRISPR-Cas13a CyHV-3 Lateral Flow Detection Point-of-care Diagnostics Recombinase-aided Amplification SHERLOCK http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:5395/download 10.1111/jfd.70079 https://phaidra.vetmeduni.ac.at/o:605 application/pdf Journal of Fish Diseases issn:0140-7775 VoR Journal of Fish Diseases 49 4 585.39 kB 2026