Abstract
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.