鲤疱疹病毒3型RAA-CRISPR/Cas12a检测方法的建立OA

[Introduction]Koi herpesvirus disease(KHVD),caused by cyprinid herpesvirus 3(CyHV-3),is a significant disease that posing 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 the 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 timely pathogen detection,interrup-tion 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 con-served genes of CyHV-3.Through experimental screening,the optimal"primer-crRNA combination"and reac-tion 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 using 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 in-dustry standards and the World Organisation for Animal Health(WOAH).It also demonstrates strong spe-cificity,showing no cross-reactivity with common pathogens in cyprinid fish such as CyHV-2 and grass carp re-ovirus(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 the aquacul-ture industry standards.Additionally,the method offers advantages such as intuitive result reading(visual de-termination 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 aquacul-ture water bodies.