Biosecurity Group, Cawthron Institute, Nelson, New Zealand.
Institute of Marine Science, University of Auckland, Auckland, New Zealand.
PeerJ. 2024 Feb 23;12:e16969. doi: 10.7717/peerj.16969. eCollection 2024.
Molecular biomonitoring programs increasingly use environmental DNA (eDNA) for detecting targeted species such as marine non-indigenous species (NIS) or endangered species. However, the current molecular detection workflow is cumbersome and time-demanding, and thereby can hinder management efforts and restrict the "opportunity window" for rapid management responses. Here, we describe a direct droplet digital PCR (direct-ddPCR) approach to detect species-specific free-floating extra-cellular eDNA (free-eDNA) signals, ., detection of species-specific eDNA without the need for filtration or DNA extraction, with seawater samples. This first proof-of-concept aquarium study was conducted with three distinct marine species: the Mediterranean fanworm , the ascidian clubbed tunicate , and the brown bryozoan to evaluate the detectability of free-eDNA in seawater. The detectability of targeted free-eDNA was assessed by directly analysing aquarium marine water samples using an optimized species-specific ddPCR assay. The results demonstrated the consistent detection of and free-eDNA when these organisms were present in high abundance. Once organisms were removed, the free-eDNA signal exponentially declined, noting that free-eDNA persisted between 24-72 h. Results indicate that organism biomass, specimen characteristics (., stress and viability), and species-specific biological differences may influence free-eDNA detectability. This study represents the first step in assessing the feasibility of direct-ddPCR technology for the detection of marine species. Our results provide information that could aid in the development of new technology, such as a field development of ddPCR systems, which could allow for automated continuous monitoring of targeted marine species, enabling point-of-need detection and rapid management responses.
分子生物监测计划越来越多地使用环境 DNA（eDNA）来检测目标物种，如海洋外来物种（NIS）或濒危物种。然而，目前的分子检测工作流程繁琐且耗时，因此可能会阻碍管理工作，并限制快速管理反应的“机会窗口”。在这里，我们描述了一种直接液滴数字 PCR（direct-ddPCR）方法，用于检测特定物种的游离细胞外 DNA（free-eDNA）信号，即无需过滤或 DNA 提取即可检测特定物种的 eDNA，用于海水样本。这项概念验证水族馆研究使用了三个不同的海洋物种：地中海扇虫、有柄海鞘和棕色苔藓虫，以评估海水中游离 eDNA 的可检测性。通过使用优化的种特异性 ddPCR 测定法直接分析水族馆海水样本，评估了靶向游离 eDNA 的可检测性。结果表明，当这些生物大量存在时，可以一致地检测到 和 的游离 eDNA。一旦去除生物体，游离 eDNA 信号呈指数下降，请注意游离 eDNA 可在 24-72 小时之间保持存在。结果表明，生物量、标本特征（例如，压力和活力）以及特定物种的生物学差异可能会影响游离 eDNA 的可检测性。这项研究代表了评估直接-ddPCR 技术检测海洋物种可行性的第一步。我们的研究结果提供了信息，可帮助开发新技术，例如 ddPCR 系统的现场开发，这可以允许对目标海洋物种进行自动化连续监测，实现现场检测和快速管理反应。
