Swansea University, Department of Biosciences, Singleton Park, Swansea, Wales, United Kingdom.
PLoS One. 2019 Jul 17;14(7):e0219218. doi: 10.1371/journal.pone.0219218. eCollection 2019.
Environmental DNA is increasingly being used for assessing the presence and relative abundance of fish in freshwater, but existing protocols typically rely on filtering large volumes of water which is not always practical. We compared the effects of water volume, filtration type and eDNA extraction procedures in the detection of fish in three freshwater bodies (pond, lake and river) using a short fragment of the 12s rRNA mtDNA gene. Quantification of eDNA capture efficiency after DNA extraction, as well as amplification efficiency, were evaluated by conventional PCR and quantitative PCR. No significant differences on eDNA capture yield were found among freshwater bodies, but increasing water volume had a positive effect on eDNA capture and amplification efficiency. Although highest eDNA capture rates were obtained using 2 L of filtered water, 100 mL syringe filtration in combination with ethanol- sodium acetate precipitation proved to be more practical and increased quantitative PCR amplification efficiency by 6.4%. Our results indicate that such method may be optimal to detect fish species effectively across both lotic and lentic freshwater environments.
环境 DNA 正越来越多地被用于评估淡水鱼类的存在和相对丰度，但现有的方法通常依赖于过滤大量的水，而这在实际操作中并不总是可行的。本研究通过对短的 12s rRNA mtDNA 基因片段进行检测，比较了在三个淡水水体（池塘、湖泊和河流）中使用不同体积的水、过滤类型和 eDNA 提取程序对鱼类的检测效果。通过常规 PCR 和定量 PCR 评估了 DNA 提取后 eDNA 捕获效率的定量以及扩增效率。在淡水体中，eDNA 捕获产量之间没有发现显著差异，但增加水体积对 eDNA 捕获和扩增效率有积极影响。尽管使用 2L 过滤水可获得最高的 eDNA 捕获率，但使用 100mL 注射器过滤与乙醇-醋酸钠沉淀相结合的方法更为实际，可将定量 PCR 扩增效率提高 6.4%。我们的研究结果表明，该方法可能是有效检测溪流和静水淡水环境中鱼类物种的最佳方法。
