The European weather loach (Misgurnus fossilis) is a cryptic and poorly known fish species of high conservation concern. The species is experiencing dramatic population collapses across its native range to the point of regional extinction. Although eDNA-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remains difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel ddPCR eDNA-based method for reliable detection and quantification, which allows accurate monitoring of M. fossilis across a number of habitat types. A dilution experiment under laboratory conditions allowed to define the limit of detection (LOD) and limit of quantification (LOQ), which were set at a concentration of respectively 0.07 copies per μl for the LOD and 0.14 copies per μl for the LOQ. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a three year survey (2017-2019), we assessed 96 locations for the presence of M. fossilis in Flanders (Belgium). eDNA analyses on these samples highlighted 45% positive detections of the species. On the basis of the eDNA concentration per liter water, only twelve sites appeared to harbor relatively dense populations. The others, 31 sites, gave a relatively weak positive signal, that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Qe ) and conventional field sampling, we concluded that each of these weak positive sites still likely harbored the species and therefore do not represent false positives. Further, only seven of the classified negative samples warrant additional sampling as our analyses identified a substantial risk of false-negative detections (i.e., type II errors) at these locations. Finally, we illustrated that ddPCR outcompetes conventional qPCR analyses, especially when target DNA concentrations are critically low, which could be attributed to a reduced sensitivity of ddPCR to inhibition effects, higher sample concentrations that can be accommodated and higher sensitivity that is obtained. This article is protected by copyright. All rights reserved.

中文翻译：

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可靠的 eDNA 检测和量化欧洲天气泥鳅（ Misgurnus boltis ）

欧洲天气泥鳅（Misgurnus boltis）是一种神秘且鲜为人知的鱼类，具有高度的保护意义。该物种正在其原生范围内经历急剧的人口崩溃，直至区域灭绝。尽管基于 eDNA 的方法在监测稀有和濒危物种方面比传统的现场方法具有明显的优势，但准确的检测和量化仍然很困难，而且质量评估往往很少纳入。在这项研究中，我们开发并验证了一种新的基于 ddPCR eDNA 的方法，用于可靠的检测和量化，可以准确监测多种栖息地类型的化石分枝杆菌。实验室条件下的稀释实验允许定义检测限 (LOD) 和定量限 (LOQ)，LOD 的浓度分别设置为每微升 0.07 拷贝和 LOQ 的浓度为每微升 0.14 拷贝。一系列水族馆实验揭示了个体数量与测量的 eDNA 浓度之间存在显着的正相关关系。在为期三年的调查（2017-2019 年）中，我们评估了佛兰德斯（比利时）96 个地点是否存在化石 M.化石。对这些样本的 eDNA 分析突出了该物种的 45% 阳性检测。根据每升水中的 eDNA 浓度，似乎只有 12 个地点拥有相对密集的种群。其他 31 个站点给出了相对较弱的阳性信号，通常位于 LOQ 以下。结合样本特定的有效 DNA 数量 (Qe) 估计和常规现场采样，我们得出的结论是，这些弱阳性位点中的每一个仍然可能包含该物种，因此不代表假阳性。此外，由于我们的分析确定了在这些位置出现假阴性检测（即 II 类错误）的重大风险，因此只有 7 个分类的阴性样本需要额外抽样。最后，我们说明 ddPCR 胜过传统的 qPCR 分析，尤其是当目标 DNA 浓度非常低时，这可能是由于 ddPCR 对抑制效应的敏感性降低、可以容纳的样品浓度更高以及获得的灵敏度更高。本文受版权保护。版权所有。由于我们的分析确定了在这些位置出现假阴性检测（即 II 类错误）的重大风险，因此只有 7 个分类的阴性样本需要额外抽样。最后，我们说明 ddPCR 胜过传统的 qPCR 分析，尤其是当目标 DNA 浓度非常低时，这可能是由于 ddPCR 对抑制效应的敏感性降低、可以容纳的样品浓度更高以及获得的灵敏度更高。本文受版权保护。版权所有。由于我们的分析确定了在这些位置出现假阴性检测（即 II 类错误）的重大风险，因此只有 7 个分类的阴性样本需要额外抽样。最后，我们说明 ddPCR 胜过传统的 qPCR 分析，尤其是当目标 DNA 浓度非常低时，这可能是由于 ddPCR 对抑制效应的敏感性降低、可以容纳的样品浓度更高以及获得的灵敏度更高。本文受版权保护。版权所有。这可能是由于 ddPCR 对抑制作用的敏感性降低，可以容纳更高的样品浓度以及获得更高的敏感性。本文受版权保护。版权所有。这可能是由于 ddPCR 对抑制作用的敏感性降低，可以容纳更高的样品浓度以及获得更高的敏感性。本文受版权保护。版权所有。