渔业研究2026，Vol.48Issue(3)：311-320,10.DOI:10.14012/j.jfr.2025124
鲤疱疹病毒3型RAA-CRISPR/Cas12a检测方法的建立
Establishment of an RAA-CRISPR/Cas12a detection method for cyprinid herpesvirus 3
摘要
Abstract
[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.关键词
鲤疱疹病毒3型(CyHV-3)/锦鲤疱疹病毒病(KHVD)/RAA-CRISPR/Cas12a/快速检测Key words
cyprinid herpesvirus 3(CyHV-3)/koi herpesvirus disease(KHVD)/RAA-CRISPR/Cas12a/rapid detection分类
农业科技引用本文复制引用
张澳晴,尹纪元,王庆,邹建洋,王浩,施雯,徐伟,李笑阳,张述,于美玲..鲤疱疹病毒3型RAA-CRISPR/Cas12a检测方法的建立[J].渔业研究,2026,48(3):311-320,10.基金项目
中国水产科学研究院院级基本科研业务费(2024XT0603) （2024XT0603）
北海市科技计划项目(北科合2023174005) （北科合2023174005）
2024年省级乡村振兴战略专项资金种业振兴项目资金(2024-SPY-00-017) （2024-SPY-00-017）
