College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China.
Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Key Laboratory of Maricultural Organism Disease Control, Ministry of Agriculture, Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity, Qingdao 266071, China.
Viruses. 2022 Jul 5;14(7):1475. doi: 10.3390/v14071475.
Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.
潜隐核型多角体病毒（CMNV）是病毒性潜隐死亡病（VCMD）的病原体，在过去十年中给东南亚国家和中国的虾类养殖业造成了严重的经济损失。鉴于快速准确的实验室检测 CMNV 在有效控制 VCMD 的传播中起着重要作用，中国自 2021 年开始进行潜隐核型多角体病毒的国家能力验证（NPT）。在本研究中，根据 CMNV 衣壳蛋白基因保守区设计了特异性引物和探针，建立了一种比以往报道更灵敏的新型 TaqMan 实时 RT-qPCR 检测方法，用于 NPT 中 CMNV 的分子检测。优化的 RT-qPCR 反应程序确定为 54.9°C 反转录 15min，95°C 变性 1min，然后进行 40 个循环，包括 95°C 变性 10s，54.9°C 退火和延伸 25s。新建立的 RT-qPCR 方法的检测限低至每反应 2.15 拷贝 CMNV 质粒模板，相关系数（R）高于 0.99。该新方法与六种常见水生动物病原体无交叉反应，可在一小时内完成，与之前报道的方法相比，可节省 50%的检测时间，代表一种快速检测方法。本研究中建立的基于 CMNV TaqMan 探针的 RT-qPCR 方法为 NPT 活动和养殖甲壳类动物中 CMNV 的快速诊断和定量提供了一种新颖、敏感和特异的工具，将有助于水产养殖行业的从业者有效预防和控制 VCMD。
