Techniques for the practical collection of environmental DNA: filter selection, preservation, and extraction
Techniques for the practical collection of environmental DNA: filter selection, preservation, and extraction
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DOI：
10.1007/s10201-015-0457-4
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发表时间：
2016-01-01
期刊：
影响因子：
1.6
通讯作者：
Maruyama, Atsushi
中科院分区：
文献类型：
--
作者：
Minamoto, Toshifumi;Naka, Takafumi;Maruyama, Atsushi
环境DNA （Environmental DNA, eDNA）分析最近被用于水生大型生物的检测；然而，在以前的研究中使用的分析方法并没有优化实际应用。在此，我们比较了几种从水样中富集和提取DNA的方法，以建立广泛适用的以鲤鱼为模型物种的eDNA分析技术。首先，对几种类型的滤波器进行了比较，以确定最优滤波器类型。其次，比较了苯酚提取、乙醇沉淀（苯酚处理）和超滤等不同提取和分离步骤后的eDNA得率。第三，用乙醇固定DNA，测试eDNA在过滤器上的保存情况。乙醇沉淀法得到的eDNA拷贝数最多，其次是用0.2 μ m聚碳酸酯过滤器和0.7 μ m玻璃纤维过滤器过滤。苯酚处理比超滤处理收集到更多的eDNA拷贝数。用15ml乙醇固定DNA，使eDNA在室温下在过滤器上保存至少6天。最后，使用现场水样比较了不同过滤类型和DNA富集程序的组合。根据这些结果，我们建议根据上下文选择合适的eDNA分析选择方法。例如，当期望有高浓度的目标DNA时，例如在水族馆实验中，乙醇沉淀是有利的。然而，当目标DNA是罕见的，这是在大多数实地研究的情况下，过滤，然后冷冻或DNA固定乙醇和苯酚处理建议。过滤器类型应在调查前根据感兴趣的水的特性决定。因此，利用这些技术的自适应组合，eDNA分析可以应用于各种情况。
Environmental DNA (eDNA) analysis has recently been used for detection of aquatic macro-organisms; however, the analytical procedures used in previous studies have not been optimized for practical use. Here, we compared several methods for DNA enrichment and extraction from water samples to establish widely applicable techniques for eDNA analysis using common carp as the model species. First, several types of filters were compared to identify the optimal filter type. Second, the eDNA yield was compared after a variety of extraction and isolation steps, including a combination of phenol extraction, ethanol precipitation (phenol treatment), and ultrafiltration. Third, DNA fixation with ethanol was tested for the preservation of eDNA on filters. Ethanol precipitation yielded the largest number of eDNA copies, followed by filtering using a 0.2-mu m polycarbonate filter and a 0.7-mu m glass fiber filter. Phenol treatment resulted in collection of a higher number of eDNA copies than that collected using ultrafiltration. DNA fixation with 15 ml ethanol enabled eDNA preservation on the filters at ambient temperatures for at least 6 days. Finally, combinations of different filter types and DNA enrichment procedures were compared using field water samples. From these results, we propose that the appropriate selection method for eDNA analysis should be chosen based on context. For example, when a high concentration of the target DNA is expected, such as in an aquarium experiment, ethanol precipitation is advantageous. However, when the target DNA is rare, which is the case in most field studies, filtration followed by freezing or DNA fixation by ethanol and phenol treatment are recommended. The filter type should be decided prior to the survey based on the characteristics of the water of interest. Thus, eDNA analysis could be applied to various situations using adaptive combinations of these techniques.