Page 27 - 《渔业研究》2026年第3期

320 渔 业 研 究 第 48 卷

Establishment of an RAA-CRISPR/Cas12a detection
method for cyprinid herpesvirus 3

2
1
ZHANG Aoqing ，WANG Qing ，ZOU Jianyang ，WANG Hao ，SHI Wen ，XU Wei ，LI Xiaoyang ，
2,3
2,3
1,2
3
4
1*
5
ZHANG Shu ，YU Meiling ，YIN Jiyuan 2*
(1. College of Animal Science and Technology, Guangxi University, Nanning 530004, China;
2. Key Laboratory of Fishery Drug Development, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of
Aquatic Animal Immunity and Green Culture, Pearl River Fisheries Research Institute, Chinese
Academy of Fishery Sciences, Guangzhou 510380, China;
3. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
4. College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China;
5. School of Computer Science and Technology, Ocean University of China, Qingdao 266100, China)
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
2
1.24×10 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.
Key words: cyprinid herpesvirus 3 (CyHV-3); koi herpesvirus disease (KHVD); RAA-CRISPR/Cas12a; rapid
detection