摘要
病毒性出血性败血症病毒(Viral hemorrhagic septicemia virus,VHSV)是一种对冷水鱼类具有高致死率的弹状病毒,在世界范围内广泛流行,严重危害水产养殖业安全和水生生物多样性。为了实现该病的便捷、快速、灵敏诊断,本文基于RPA和CRISPR/Cas技术,通过RPA引物和crRNA筛选及检测体系优化,结合侧向流层析技术与荧光显色法,构建了VHSV的快速检测体系。研究表明,RT-RPA在39℃下反应30 min可获得最佳扩增效果,通过CRISPR/Cas12a与侧向流层析技术的结合使用可在50 min内完成病毒样本的检测,检测结果可通过肉眼直接判断。该检测方法的检测限为9 copies/μL,与同属的传染性造血器官坏死病毒(IHNV)和牙鲆弹状病毒(HIRRV)之间无交叉反应,表现出较高的灵敏度与特异性。临床样本检测结果显示,本方法与RT-PCR检测结果一致,均表现出较高的准确性与稳定性。本研究构建了一种VHSV的RPA-CRISPR/Cas12a检测方法,为VHSV的便捷、快速诊断提供了重要的技术支撑。
Viral hemorrhagic septicemia virus(VHSV)is an important fish Novirhabdovirus virus that can cause high lethality in cold-water fish and is widely prevalent worldwide,seriously threatening global food security and aquatic biodiversity.To achieve the convenient,rapid,and sensitive diagnosis of VHSV,a rapid detection system was developed through the screening of RPA primers and crRNA basing on RPA and CRISPR/Cas technologies and the optimization of the detection system by combining with the application of lateral flow chromatography and fluorescence colorimetry in the present study.The experimental results showed that RT-RPA reaction at 39℃for 30 min can obtain the best amplification effect,and the detection of the virus samples can be completed in 50 min by combining CRISPR/Cas12a with lateral flow chromatography.The results can be judged by the naked eyes.The system was highly sensitive and specific;the sensitivity of the assay was 9 copies/μL,and there was no cross-reactivity with infectious hematopoietic necrosis virus(IHNV)and hirame rhabdovirus(HIRRV).The clinical sample test showed that the results obtained with RPA-CRISPR/Cas12a method was in consistent with those obtained with RT-PCR method,demonstrating that it is highly accurate and stable.In this study,we have developed an RPA-CRISPR/Cas12a detection method for VHSV,and provided an important technical support for the convenient and rapid diagnosis for VHSV.
作者
王洪升
唐小千
绳秀珍
邢婧
迟恒
战文斌
Wang Hongsheng;Tang Xiaoqian;Sheng Xiuzhen;Xing Jing;Chi Heng;Zhan Wenbin(Key Laboratory of Mariculture(Ocean University of China),Ministry of Education,Qingdao 266003,China;Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao 266237,China)
基金
国家重点研究发展计划项目(2023YFD2400704)资助。
作者简介
王洪升(1999-),男,硕士生。E-mail:1966353205@qq.com;通信作者:唐小千,男,博士,教授。E-mail:tangxq@ouc.edu.cn。
