生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (3): 114-.doi: 10.3969/j.issn.2095-1736.2024.03.114

• 技术方法 • 上一篇    下一篇

鱼类环境DNA保存条件的比较研究

刘　洋1， 应　方2， 杨　俊3， 王焕英1， 洪文杰1， 王庭璋1   

  1. 1. 浙江天科高新技术发展有限公司 浙江省微生物技术与生物信息研究重点实验室， 杭州 310012；
    2. 浙江省杭州生态环境监测中心， 杭州 310012； 3. 杭州市西湖水域管理处， 杭州 310002
  • 出版日期:2024-06-18 发布日期:2024-06-17
  • 通讯作者: 王庭璋，博士，副研究员，研究方向为环境微生物、生物信息学，E-mail:wtz8615@tkgeneclub.com
  • 作者简介:刘洋，硕士，工程师，研究方向为环境微生物、环境DNA技术应用，E-mail:ly1587@tkgeneclub.com
  • 基金资助:
    国家重点研发计划项目（2022YFE0128600）； 杭州市农业与社会发展科研引导项目（20220919Y182）

Study on the environmental DNA preservation conditions of fishes

LIU Yang1, YING Fang2, YANG Jun3, WANG Huanying1, HONG Wenjie1, WANG Tingzhang1   

  1. 1. Zhejiang Tianke High-tech Development Co., Ltd., Key laboratory of Microbiol technology and Bioinformatics of
    Zhejiang Province, Hangzhou 310012, China; 2. Zhejiang Hangzhou Ecological and Environmental Monitoring Center,
    Hangzhou 310012, China; 3. Hangzhou West Lake Administration, Hangzhou 310002, China
  • Online:2024-06-18 Published:2024-06-17

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摘要： 为探索环境水样中鱼类DNA的最佳保存方法和保存时间，选取饲养鱼的水样为研究对象，在无核酸污染的环境中，采用0.45 μm孔径滤膜对相同体积的水样进行预处理，并对富集环境DNA（eDNA）的滤膜进行不同保存方法和不同保存时间捕获的eDNA质量、鱼类12S rRNA 基因文库浓度、基于ASVs方法的鱼类12S rRNA 扩增子测序结果的比较研究。研究结果显示：－20 ℃冷冻、－80 ℃冷冻、无水乙醇-常温、75%乙醇-常温、Longmire’s保存液-常温以及TK保存液-常温等6种保存方法捕获的eDNA效果最好的是TK保存液-常温，其次为冷冻保存（－20 ℃、－80 ℃）。－20 ℃冷冻、－80 ℃冷冻和TK保存液-常温等3种保存方法在同一保存时间下具有良好的稳定性，且在保存时间2周内的测试条件下，也能稳定保存滤膜，最大程度还原水样中鱼类的遗传信息。研究对eDNA样本最佳保存条件的探索，将为第三方基因检测实验室对鱼类eDNA保存策略提供技术基础，便于eDNA宏条形码技术的开展。

关键词: eDNA, 保存条件, DNA提取, 扩增子测序, ASVs方法

Abstract: The optimal preservation method and preservation time for DNA from the simulated water samples of reared fish was studied. Water samples at the same volume were pretreated with 0.45 μm filter membranes inanucleic acid-free environment. Comparison of the obtained quality of eDNA, PCR concentrations of fish 12S rRNA gene, and the amplicon sequencing results based on the available amplicon sequence variant method by combination of different preservation method and preservation time were studied. The results showed that with six preservation methods (－20 ℃ freezing, －80 ℃ freezing, ethyl alcohol at room temperature, 75% ethanol at room temperature, Longmire buffer at room temperature, and TK buffer at room temperature), the TK buffer at room temperature was the best eDNA captured methods, followed by freezing (－20 ℃, －80 ℃). And the －20 ℃, －80 ℃, and TK buffer at room temperature had good stability under the same preservation time respectively, and could stably preserve the filter membrane within two weeks to maximize the genetic information of fish in water samples. This study provided technical reference for third-party gene detection laboratories for fish eDNA preservation strategies and facilitating the development of eDNA metabarcoding technology.

Key words: environmental DNA, preservation conditions, DNA extraction, amplicon sequencing, available amplicon sequence variant method

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