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珠江河口水体环境DNA提取方法的建立及优化

李红婷 张帅 邹柯姝 陈作志 陈晓雷 蒋佩文 曹漪婷 李敏

李红婷, 张帅, 邹柯姝, 陈作志, 陈晓雷, 蒋佩文, 曹漪婷, 李敏. 珠江河口水体环境DNA提取方法的建立及优化[J]. 南方水产科学. doi: 10.12131/20210304
引用本文: 李红婷, 张帅, 邹柯姝, 陈作志, 陈晓雷, 蒋佩文, 曹漪婷, 李敏. 珠江河口水体环境DNA提取方法的建立及优化[J]. 南方水产科学. doi: 10.12131/20210304
LI Hongting, ZHANG Shuai, ZOU Keshu, CHEN Zuozhi, CHEN Xiaolei, JIANG Peiwen, CAO Yiting, LI Min. Establishment and optimization of environmental DNA extraction method from water of Pearl River Estuary[J]. South China Fisheries Science. doi: 10.12131/20210304
Citation: LI Hongting, ZHANG Shuai, ZOU Keshu, CHEN Zuozhi, CHEN Xiaolei, JIANG Peiwen, CAO Yiting, LI Min. Establishment and optimization of environmental DNA extraction method from water of Pearl River Estuary[J]. South China Fisheries Science. doi: 10.12131/20210304

珠江河口水体环境DNA提取方法的建立及优化

doi: 10.12131/20210304
基金项目: 国家科技基础调查专项 (2019FY101905);广东省科技计划项目(2019B121201001);南方海洋科学与工程广东省实验室 (广州) 人才团队引进重大专项 (GML2019ZD0605);中国水产科学研究院南海水产研究所中央级公益性科研院所基本科研业务费专项资金 (2019TS13, 2021SD18)
详细信息
    作者简介:

    李红婷  (1997— ),女,硕士研究生,研究方向为海洋生物环境DNA。E-mail: 1604790319@qq.com

    通讯作者:

    李 敏 (1984—),男,副研究员,博士,从事海洋生物多样性保护研究。E-mail: limin@scsfri.ac.cn

  • 中图分类号: S 932

Establishment and optimization of environmental DNA extraction method from water of Pearl River Estuary

  • 摘要: 近年来,环境DNA (eDNA) 技术广泛应用于水生生物多样性研究。以珠江河口咸淡水生态系统为研究区域,采用滤膜法富集eDNA,选取直径47 mm、孔径0.45 μm的硝酸纤维膜、醋酸纤维膜、玻璃纤维膜和聚碳酸酯膜共4种材质的滤膜,采用4种滤膜保存方法,结合2种试剂盒提取eDNA,评估不同组合方案对eDNA提取效果的影响。结果显示,滤膜材质和保存方法对eDNA产量具有显著影响;其中,醋酸纤维膜获取的eDNA浓度最高。在DNA提取物质量相当的情况下,海洋动物组织基因组DNA提取试剂盒提取的eDNA浓度高于DNeasy Blood and Tissue kit试剂盒。在不同保存条件下,“液氮”保存法获取的eDNA浓度最高。在不具备冷冻条件时,可添加乙醇,常温保存滤膜。采样后立即过滤水样能够有效防止eDNA降解。文章建立了珠江口咸淡水生态系统水样eDNA的获取、保存和提取方案,可为相似水域的eDNA研究提供参考。
  • 图  1  4种滤膜与2种试剂盒的实验设计方案

    Figure  1.  Experimental design of four kinds of filtration membranes and two kinds of kits

    图  2  滤膜与保存方法的不同组合

    Figure  2.  Different combinations of membrane and preservation methods

    图  3  滤膜与试剂盒组合提取的总eDNA电泳图

    Figure  3.  Electrophoresis diagram of total eDNA extracted by combination of membrane and kit

    图  4  滤膜与试剂盒组合获得的eDNA浓度比较

    Figure  4.  Comparison of eDNA concentration obtained by combination of membrane and extraction kit

    图  5  滤膜与试剂盒组合获得的eDNA纯度比较

    Figure  5.  Purity comparison of eDNA obtained by combination of membrane and extraction kit

    图  6  不同保存方法下提取的eDNA浓度比较

    Figure  6.  Comparison of eDNA concentrations extracted by different preservation methods

    图  7  不同保存方法下提取的eDNA纯度比较

    Figure  7.  Purity comparison of eDNA extracted by different preservation methods

    图  8  不同滤膜过滤1 L水样所需时间比较

    Figure  8.  Comparison of time used for filtration of 1 L water sample by different membranes

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出版历程
  • 收稿日期:  2021-10-21
  • 修回日期:  2021-11-22
  • 录用日期:  2021-11-25
  • 网络出版日期:  2021-12-09

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