Improving efficiency and reliability of environmental DNA analysis for silver carp
Improving efficiency and reliability of environmental DNA analysis for silver carp
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DOI：
10.1016/j.jglr.2015.02.009
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发表时间：
2015-06-01
影响因子：
2.2
通讯作者：
Gaikowski, Mark P.
中科院分区：
文献类型：
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作者：
Amberg, Jon J.;McCalla, S. Grace;Gaikowski, Mark P.
自然资源机构已制定监测计划，利用环境 DNA (eDNA) 在鳙鱼和鲢鱼在五大湖内建立种群之前及早发现它们。这种分子监测技术必须高度准确和精确，以便可靠地解释，并且在检测阈值和成本方面也必须高效。因此，我们比较了两种 DNA 提取技术，并将新的定量 PCR (qPCR) 测定法与监测程序使用的传统 PCR (cPCR) 测定法进行了比较。 qPCR 和 cPCR 检测均能够扩增从鳙鱼混合种群存在地点采集的环境样本中存在的鲢鱼 DNA。然而，与 cPCR 检测相比，qPCR 检测的 PCR 阳性样本要少得多，随后被确定不含鳙鱼 DNA。此外，即使存在阻碍 cPCR 扩增的抑制剂，qPCR 检测也能够扩增鳙鱼的 DNA。此外，选择适当的 DNA 提取方法可以通过降低检测限和降低与样品处理相关的成本来显着改变 eDNA 监测程序的效率。本文报告的结果目前正被纳入 eDNA 监测计划中，以降低成本、增加 DNA 产量并增加检测扩增靶 DNA 的可信度。这些结果对于提高我们准确、自信地解释使用 eDNA 早期检测入侵物种的监测项目报告的结果的能力至关重要。由 Elsevier B.V. 代表国际五大湖研究协会出版。
Natural resource agencies have established surveillance programs which use environmental DNA (eDNA) for the early detection of bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix before they establish populations within the Great Lakes. This molecular monitoring technique must be highly accurate and precise for confident interpretation and also efficient, both in detection threshold and cost. Therefore, we compared two DNA extraction techniques and compared a new quantitative PCR (qPCR) assay with the conventional PCR (cPCR) assay used by monitoring programs. Both the qPCR and cPCR assays were able to amplify the DNA of silver carp present in environmental samples taken from locations where mixed populations of bigheaded carps existed. However, the qPCR assay had substantially fewer PCR positive samples which were subsequently determined not to contain DNA of bigheaded carps than the cPCR assay. Additionally, the qPCR assay was able to amplify the DNA of bigheaded carps even in the presence of inhibitors that blocked amplification with cPCR. Also, the selection of an appropriate DNA extraction method can significantly alter the efficiency of eDNA surveillance programs by lowering detection limits and by decreasing costs associated with sample processing. The results reported herein are presently being incorporated into eDNA surveillance programs to decrease the costs, increase DNA yield and increase the confidence that assays are amplifying the target DNA. These results are critical to enhancing our ability to accurately and confidently interpret the results reported from monitoring programs using eDNA for early detection of invasive species. Published by Elsevier B.V. on behalf of International Association for Great Lakes Research.