鲍疱疹病毒原位LAMP检测方法的建立与初步应用

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...

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

In order to understand the tissue tropism and spreading routes of Haliotid herpesvirus 1 (HaHV-1) after infecting Haliotis diversicolor aquatilis, an in situ LAMP method for HaHV-1 was developed in this study. In situ LAMP an alternative in situ nucleic acid amplification technique that relies on LAMP to improve specificity and sensitivity. Then experimental infection of HaHV-1 to H. diversicolor aquatilis was carried out, and 3 samples were collected from infected and control groups respectively at the following time points: 24, 36, 48, 72, 96 and 120 hours post injection (hpi). For the establishment of the HaHV-1 in situ LAMP detection method, we firstly selected a set of LAMP primers designed for specific detection of HaHV-1, and a pair of loop primers was designed by us to improve the spread 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. Our result indicated that the incorporation of the designed loop primers yields stabile results during LAMP amplification. The developed in situ LAMP detection method was applied on pedal nerve branches, mantle, hepatopancreas and gill of specimens collected across the experimental infection process. Our results indicated that the in situ LAMP signals were firstly observed at 36 hpi on pedal nerve branches, gills and mantles, and appeared on hepatopancreas until 72 hpi. The viral infection signals were always accompanied by tissue lesions and infiltrated haemocytes that was infected by HaHV-1. The key feature of in situ LAMP is the mild isothermal condition, an isothermal and low reaction temperature, which causes less damage than PCR-based method. Compared with traditional culture methods and PCR-based methods, the in situ LAMP method needs shorter experiment cycle. Therefore, the in situ LAMP method developed in this study constitutes a potentially valuable tool for rapid detection and definite diagnosis of HaHV-1, for investigating the tissue tropism and pathological characteristics in different susceptible species.