摘要
赤点石斑鱼神经坏死病毒(Red spotted grouper nervous necrosis virus,RGNNV)是危害中国海水养殖鱼类的主要病原体之一,尽早检出RGNNV是防控该病毒感染和减少养殖损失的关键措施。为建立快速检测RGNNV的方法,将重组酶聚合酶扩增(Recombinase polymerase amplification,RPA)技术与CRISPR/Cas12a(CRISPR,成簇规律间隔短回文重复序列;Cas12a,CRISPR相关蛋白)检测系统相结合,通过设计和筛选RPA引物与crRNA(CRISPR RNA),优化RPA反应条件与CRISPR/Cas12a检测体系,评估该方法的灵敏度与特异性。同时,对临床石斑鱼样本进行检测,并与行业标准《SC/T 7216—2022鱼类病毒性神经坏死病诊断方法》中规定的qPCR法进行一致性比较。结果显示,该检测方法最短可在45 min内得到结果,灵敏度为1.5×10^(1)拷贝·μL^(−1),检测特异性良好,实际样本检测与行标qPCR法对比符合率为100%。研究建立的RPA-CRISPR/Cas12a-RGNNV检测方法具有快速、便捷、高灵敏度、强特异性和可视化检测结果等特点,为RGNNV的现场快速检测提供了新的技术手段。
Red spotted grouper nervous necrosis virus(RGNNV)is one of the most significant pathogens threatening marine fish farming in China,so early detection of RGNNV is a critical measure for preventing viral infection and reducing aquaculture losses.To develop a rapid RGNNV detection method,we developed a rapid visual detection method by integrating recombinase polymerase amplification(RPA)with the CRISPR/Cas12a system.Besides,we designed specific RPA primers and crRNA,then optimized both RPA reaction conditions and CRISPR/Cas12a detection parameters.The method's sensitivity and specificity were systematically evaluated through clinical testing of 20 suspected-infected grouper samples,with comparison to the standardized qPCR method(SC/T 7216—2022).Results show a detection limit of 1.5×10^(1)copies·μL^(−1)and complete concordance with qPCR findings.The established RPA-CRISPR/Cas12a method demonstrates rapid detection,high sensitivity,excellent specificity,and visual interpretability,providing an efficient field-deployable solution for RGNNV surveillance in aquaculture operation.
作者
闫光达
马红玲
徐力文
邓益琴
郭志勋
冯娟
YAN Guangda;MA Hongling;XU Liwen;DENG Yiqin;GUO Zhixun;FENG Juan(Tianjin Agricultural University,Tianjin 300384,China;South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences/Guangdong Provincial Key Laboratory of Fishery Ecology and Environment,Guangzhou 510300,China)
出处
《南方水产科学》
北大核心
2025年第4期11-21,共11页
South China Fisheries Science
基金
中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助(2024XT0604,2023TD44)
国家重点研发计划项目(2024YFD2401403)
广东省科技计划项目(2022B0202110001)
广东省现代农业产业技术体系创新团队建设项目(2024CXTD27)。
