检测和定量鱼传传染性造血器官坏死病病毒（鲑鱼疱疹病毒 3 型）的环介导等温扩增检测方法的建立。
Development of a loop-mediated isothermal amplification assay for the detection and quantification of epizootic epitheliotropic disease virus (salmonid herpesvirus-3).
机构信息
Function Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao, 266071, China; Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, MI, 48824, USA.
Comparative Medicine and Integrative Biology, College of Veterinary Medicine, Michigan State University, East Lansing, MI 48824, USA.
出版信息
J Virol Methods. 2019 Feb;264:44-50. doi: 10.1016/j.jviromet.2018.11.006. Epub 2018 Nov 13.
Epizootic Epitheliotropic Disease Virus (EEDV; Salmonid Herpesvirus-3) causes a serious disease hatchery-reared lake trout (Salvelinus namaycush), threatening restoration efforts of this species in North America. The current inability to replicate EEDV in vitro necessitates the search for a reproducible, sensitive, and specific assay that allows for its detection and quantitation in a time- and cost-effective manner. Herein, we describe a loop-mediated isothermal amplification (LAMP) assay that was developed for the quantitative detection of EEDV in infected fish tissues. The newly developed LAMP reaction was optimized in the presence of calcein, and the best results were produced using 2 mM MgCl, 1.8 mM dNTPs and at an incubation temperature of 67.1 °C. This method was highly specific to EEDV, as it showed no cross-reactivity with several fish viruses, including Salmonid Herpesvirus, -2, -4, and -5, Infectious Pancreatic Necrosis Virus, Spring Viremia of Carp Virus, Infectious Hematopoietic Necrosis Virus, Golden Shiner Reovirus, Fathead Minnow Nidovirus, and Viral Hemorrhagic Septicemia Virus. The analytical sensitivity of the EEDV-LAMP method was estimated to be as low as 16 copies of plasmid per reaction. When infected fish tissue was used, a positive reaction could be obtained when an infected gill tissue sample that contained 430 viral copies/μg was diluted up to five orders of magnitude. The sensitivity and specificity of the newly developed LAMP assay compared to the SYBR Green qPCR assay were 84.3% and 93.3%, respectively. The quantitative LAMP for EEDV had a correlation coefficient (R = 0.980), and did not differ significantly from the SYBR Green quantitative PCR assay (p > 0.05). Given its cost- and time-effectiveness, this quantitative LAMP assay is suitable for screening lake trout populations and for the initial diagnosis of clinical cases.
传染性上皮病病毒（EEDV；鲑鱼疱疹病毒-3）引起严重的疾病，孵化场养殖的湖鳟鱼（Salvelinus namaycush），威胁到北美该物种的恢复工作。目前无法在体外复制 EEDV，因此需要寻找一种可重复、敏感和特异的检测方法，以便以经济有效的方式对其进行检测和定量。在此，我们描述了一种用于检测感染鱼组织中 EEDV 的环介导等温扩增（LAMP）检测方法。在钙黄绿素存在的情况下优化了新开发的 LAMP 反应，在 2 mM MgCl2、1.8 mM dNTP 和 67.1°C 的孵育温度下获得最佳结果。该方法对 EEDV 具有高度特异性，因为它与几种鱼类病毒（包括鲑鱼疱疹病毒、-2、-4 和-5、传染性胰腺坏死病毒、鲤鱼春病毒血症病毒、传染性造血坏死病毒、金鳞鱼虹彩病毒、胖头鱼诺达病毒和病毒性出血性败血症病毒）没有交叉反应。EEDV-LAMP 方法的分析灵敏度估计低至每个反应 16 个质粒拷贝。当使用感染的鱼组织时，当包含 430 个病毒拷贝/μg 的感染的鳃组织样本稀释五个数量级时，可以获得阳性反应。与 SYBR Green qPCR 检测相比，新开发的 LAMP 检测的灵敏度和特异性分别为 84.3%和 93.3%。EEDV 的定量 LAMP 与 SYBR Green 定量 PCR 检测具有相关性（R=0.980），且与 SYBR Green 定量 PCR 检测无显著差异（p>0.05）。鉴于其成本效益和时间效益，这种定量 LAMP 检测方法适用于湖鳟鱼群体的筛查和临床病例的初步诊断。
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