Introduction Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus 3 (CyHV-3), is a significant disease that poses a threat to the healthy cultivation of common carp and koi both domestically and internationally. Establishing a sensitive, rapid, and simple detection method suitable for on-site rapid screening is currently the most effective approach to prevent the occurrence of KHVD by enabling the timely detection of pathogens and the interruption of viral transmission.

Objective The purpose of this study is to establish a detection method suitable for on-site rapid screening of CyHV-3, enabling the timely detection of pathogens, the interruption of viral transmission routes, and providing technical support for the prevention and control of KHVD.

Methods In this study, different primer and crRNA combinations were designed based on the specific conserved genes of CyHV-3. Through experimental screening, the optimal "primer-crRNA combination" and reaction conditions were identified, leading to the establishment of an on-site rapid detection method for CyHV-3 using RAA-CRISPR/Cas12a. The specificity, sensitivity, and accuracy of clinical sample detection of this method were evaluated.

Results The method exhibits high sensitivity, with a minimum detection limit of 1.24×10² copies/μL, surpassing the sensitivity of detection methods recommended by both the aquaculture industry standards and the World Organisation for Animal Health (WOAH). It also demonstrates strong specificity, showing no cross-reactivity with common pathogens in cyprinid fish such as CyHV-2 and Grass Carp Reovirus (GCRV). The detection results are reliable, as evidenced by the consistency between the results obtained from laboratory-collected clinical samples and those obtained using the method recommended by aquaculture industry standards. Additionally, the method offers advantages such as intuitive result reading (visual determination with the naked eye), low requirements for equipment and personnel, and rapid detection (reaction time of only 45 minutes).

Conclusion The on-site rapid detection method for CyHV-3 using RAA-CRISPR/Cas12a established in this study provides an effective technical means for the rapid screening and real-time monitoring of pathogen presence in the circulation of common carp and koi seedlings as well as in aquaculture water bodies.