Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter
Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter
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DOI：
10.1111/2041-210x.12683
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发表时间：
2017-05-01
影响因子：
6.6
通讯作者：
Hellstrom, Micaela
中科院分区：
文献类型：
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作者：
Spens, Johan;Evans, Alice R.;Hellstrom, Micaela
水环境DNA （eDNA）是一种新兴的、高效的、无创的物种清查工具。为了最大限度地提高下游定量PCR （qPCR）和下一代测序（NGS）应用的性能，起始材料的质量和数量至关重要，这需要优化eDNA的捕获、储存和提取技术。之前对eDNA捕获/存储的比较研究已经测试了沉淀和开放式过滤器。然而，实际封闭的过滤器，减少不必要的处理没有包括在内。在这里，我们通过比较过滤胶囊（Sterivex-GP聚醚砜，孔径为022m，以下简称SX）与常用方法来填补这一空白。我们的实验设置，总共涵盖了41种处理方法，结合沉淀或过滤捕获，不同的保存技术和储存时间，在一个湖泊（和一个无鱼的对照池）取样。我们选择了记录在案的捕获方法，这些方法已经成功地瞄准了广泛的动物群。eDNA的提取采用DNeasy((R)) Blood & Tissue kit （Qiagen）改进后的优化方案。我们测量了两种当地关键鱼类的总eDNA浓度和cq值（用于qPCR DNA定量的循环），以针对特定的mtDNA细胞色素b （cyt b）序列。与聚碳酸酯轨迹蚀刻过滤器（PCTE）、玻璃纤维过滤器（GF）或乙醇沉淀过滤器（EP）相比，SX产生了更高数量的总eDNA和更低的cq值。对于其中一个目标物种，SX产生的cq值也低于硝酸纤维素过滤器（CN）。与GF和PCTE相比，SX样品的DNA完整性在储存2周后没有明显降低。在储存前添加防腐剂可改善SX结果。总之，我们推荐SX过滤器（最初设计用于过滤微生物）作为一种有效的捕获方法来取样微生物eDNA。建议过滤后立即用乙醇或朗迈尔缓冲液保存SX。保存的SX胶囊可以在室温下保存至少2周而不会有明显的降解。与其他过滤器相比，减少处理和较少暴露于外界压力可能有助于更好的eDNA结果。SX胶囊易于运输，并且可以在偏远和恶劣的现场条件下进行eDNA采样，因为样品可以在现场过滤/保存。
Aqueous environmental DNA (eDNA) is an emerging efficient non-invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next-generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and open' filters. However, practical enclosed' filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex-GP polyethersulfone, pore size 022m, hereafter called SX) with commonly used methods. Our experimental set-up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish-free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy((R)) Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq-values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq-values than polycarbonate track-etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq-values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro-organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.