鲍疱疹病毒原位LAMP检测方法的建立与初步应用  

Establishment and application of in situ LAMP for detection of Haliotid herpesvirus 1(HaHV-1)

作　　者：谷莉 郑玉东 张翔 白昌明[2] 刘金兰 辛鲁生 李晨[2] 王崇明[2] GU Li;ZHENG Yudong;ZHANG Xiang;BAI Changming;LIU Jinlan;XIN Lusheng;LI Chen;WANG Chongming(Tianjin Key Laboratory of Aqua-Ecology and Aquaculture,College of Fishery,Tianjin Agriculture University,Tianjin 300384,China;Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Affairs,Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Laboratory of Pathology and Immuunology of Aquatic Animals,Ocean University of China,Qingdao 266071,China)

机构地区：[1]天津农学院水产学院,天津市水产生态及养殖重点实验室,天津300384 [2]中国水产科学研究院黄海水产研究所,青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室,农业农村部海水养殖病害防治重点实验室,青岛市海水养殖流行病学与生物安保重点实验室,山东青岛266071 [3]中国海洋大学,水产动物病害与免疫学实验室,山东青岛266000

出　　处：《水产学报》2022年第5期885-894,共10页Journal of Fisheries of China

基　　金：青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室开放课题(2019-BHA02);国家自然科学基金(32073014);国家现代农业产业技术体系专项。

摘　　要：为精确定位鲍疱疹病毒(HaHV-1)在宿主不同组织器官中的分布,明确HaHV-1的组织亲嗜性和侵染进程,实验基于环介导等温扩增技术(LAMP)和原位杂交技术建立了HaHV-1的原位LAMP检测方法。利用该方法研究了HaHV-1人工感染实验不同时间节点,病毒在杂色鲍主要器官的分布规律和组织亲嗜性。并对已报道的HaHV-1 LAMP扩增引物进行优化,实现对载玻片上原位固定靶组织内病毒DNA的稳定、特异扩增,筛选最佳显色时间等原位杂交反应条件,最后通过免疫酶标技术分析HaHV-1在组织样本内的分布情况。结果显示,HaHV-1原位LAMP检测方法最适显色时间为60 min。利用该方法对攻毒后24、36、48、60和72 h,HaHV-1在杂色鲍外套膜、鳃、肝胰腺和腹足神经节4种样本的组织分布情况进行检测和分析。病毒阳性信号最早于36 h出现在腹足神经节,48 h在部分外套膜样本中观察到病毒阳性信号,分布局限于外周神经中。在感染实验后期,病毒阳性信号出现在肝胰腺结缔组织中。病毒阳性信号出现的部位常有大量细胞渗出和浸润,渗出的细胞中可见被病毒感染的血淋巴细胞。本研究建立的HaHV-1原位LAMP检测方法,同步实现了高灵敏度和精确定位的功能。该检测方法适用于病毒感染的确诊、不同组织器官的亲嗜性、病毒侵染途径和致病机理等相关研究。To elucidate the tissue tropism and spreading routes of Haliotid herpesvirus 1(HaHV-1) in susceptible Haliotis diversicolor aquatilis after infection, an in situ loop-mediated isothermal amplification(LAMP) method for HaHV-1 was developed in this study based on LAMP and in situ hybridization techniques. The developed in situ LAMP method was then applied on pedal nerve branches, mantle, hepatopancreas and gill of specimens collected across the experimental infection process. For the establishment of the HaHV-1 in situ LAMP detection method, we firstly optimized LAMP primers for specific detection of HaHV-1, improving the specificity and stability of the LAMP reaction on slides. We then optimized the time for color development(about 60 min) on pedal nerve branches, which was identified as the most suitable target for pathological investigation and in situ LAMP detection. Finally, the HaHV-1 in situ LAMP method was established after color development with immunoenzymatic labeling technique. The results showed that the incorporation of the designed loop primers yielded stabile results during LAMP amplification. The developed in situ LAMP detection method was applied on major tissue collected at 24、36、48、60 and 72 h across the experimental infection process. The in situ LAMP signals were firstly detected at 36 h on pedal nerve branches, and at 48 h on mantles after infection. The viral signals detected at mantle were confined to the peripheral nerves scattered throughout the mantle. Finally, in situ LAMP signals were identified occasionally in connective tissue of hepatopancreas of samples collected at the end of the experimental infection. The viral infection signals were always accompanied by tissue lesions and infiltrated haemocytes infected with HaHV-1. The in situ LAMP method developed in this study achieved the goal of rapid detection and definite diagnosis of HaHV-1, and is helpful for investigating the tissue tropism and pathological characteristics in different susceptible species. The detection method

关 键 词：杂色鲍 鲍疱疹病毒 原位LAMP 检测方法 

分 类 号：S941.41[农业科学—水产养殖]