| 摘要: |
| 近年来，环境DNA(Environmental DNA, eDNA)技术作为一种新的水生生物调查方法发展迅速，在水生生态系统的研究领域被广泛应用到物种检测、生物多样性评价、生物量评估等方面。然而，很少有研究专门评价eDNA技术操作流程中不同的eDNA富集方法与提取方法对研究结果的影响，从而针对具体研究对象建立一套最佳的eDNA技术操作流程。此外，由于物种间生活习性的差异，不同物种释放到环境中的DNA量及DNA片段大小不同，因而，针对不同研究对象需采用不同的eDNA富集与提取方法。本研究以中国对虾(Fenneropenaeus chinensis)为研究对象，采用滤膜法富集eDNA，结合血液与组织DNA提取试剂盒提取eDNA。选取直径为47 mm的玻璃纤维膜、硝酸纤维膜、聚碳酸酯膜、尼龙膜共4种材质的滤膜，每种滤膜根据其孔径大小设置0.45、0.8、1.2、5 μm共4个梯度，取样水量设置500 ml、1 L、2 L共3个梯度。结果显示，滤膜材质、滤膜孔径大小及取样水体体积均对中国对虾的定性与定量分析具有一定的影响，其中，0.45 μm的玻璃纤维滤膜过滤2 L水样能够检测到的DNA拷贝数最多，并依据此建立了一套中国对虾eDNA技术的操作流程，提高了中国对虾的检出率，为后续中国对虾的分布监测及生物量评估提供了基础。 |
| 关键词: 中国对虾 eDNA 建立 优化 |
| DOI：10.19663/j.issn2095-9869.20180620001 |
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| Establishment and Optimization of Environmental DNA Detection Techniques for Assessment of Fenneropenaeus chinensis Biomass |
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LI Miao1,2,3, SHAN Xiujuan4,5, WANG Weiji4,5, LÜ Ding5, DAI Fangqun4,5, DING Xiaosong1,6, WU Huanhuan6,7
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1.College of Marine Sciences, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs;3.Shandong Provincial Key Laboratory of Fishery Resources and Eco-Environment, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;4.Laboratory for Marine Fisheries Science and Food Production Processes, Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071;5.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Shandong Provincial Key Laboratory of Fishery Resources and Eco-Environment, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;6.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture and Rural Affairs, Shandong Provincial Key Laboratory of Fishery Resources and Eco-Environment, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;7.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306
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| Abstract: |
| In recent years, environmental DNA (eDNA) technology has developed rapidly as a new method for investigation of aquatic organisms and has been widely used in the field of aquatic ecosystem research for species detection, biodiversity evaluation, and biomass assessment. However, few studies have specifically evaluated the effects of different eDNA enrichment and extraction methods on the results of the eDNA technology operation process to establish a set of optimal eDNA technology operation procedures for specific research subjects. In addition, because of differences in living habits between species, the amount of DNA released from different species and the size of DNA fragments are different. Therefore, different eDNA enrichment and extraction methods should be adopted for different research subjects. In this study, Fenneropenaeus chinensis was used as the research object, and eDNA was enriched by the membrane method, and eDNA was extracted by combining a blood and tissue DNA extraction kit. A filter membrane with a diameter of 47 mm, a nitrocellulose membrane, a polycarbonate membrane, and a nylon membrane were used, and each membrane was set to 0.45 μm, 0.8 μm, 1.2 μm, and 5 μm according to pore size. With 4 gradients, the sampling water volume was set to three gradients of 500 ml, 1 L, and 2 L. The experimental results showed that the membrane material size, pore size of the membrane, and the volume of the sampled water had certain influences on the qualitative and quantitative analysis of F. chinensis. The 0.45 μm glass fiber membrane filter and 2 L water sample detected the greatest DNA copy number. Based on this, a set of operational procedures for F. chinensis prawn eDNA technology was established to improve the detection rate of F. chinensis, which provided the basis for subsequent distribution monitoring and biomass assessment of F. chinensis. |
| Key words: Fenneropenaeus chinensis Environmental DNA Establishment Optimization |
