Research Institute for Nature and Forest, Geraardsbergen, Belgium.
Aquatic Research Facility, Environmental Sustainability Research Centre, University of Derby, Derby, UK.
J Fish Biol. 2021 Feb;98(2):399-414. doi: 10.1111/jfb.14315. Epub 2020 Mar 30.
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 environmental DNA (eDNA)-based approaches offer clear advantages over conventional field methods for monitoring rare and endangered species, accurate detection and quantification remain difficult and quality assessment is often poorly incorporated. In this study, we developed and validated a novel digital droplet PCR (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 the definition of the limit of detection (LOD) and the limit of quantification (LOQ), which were set at concentrations of 0.07 and 0.14 copies μl , respectively. A series of aquarium experiments revealed a significant and positive relationship between the number of individuals and the eDNA concentration measured. During a 3 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 litre of water, only 12 sites appeared to harbour relatively dense populations. The other 31 sites gave a relatively weak positive signal that was typically situated below the LOQ. Combining sample-specific estimates of effective DNA quantity (Q ) and conventional field sampling, we concluded that each of these weak positive sites still likely harboured the species and therefore they 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 being accommodated and higher sensitivity obtained.
欧洲泥鳅（Misgurnus fossilis）是一种具有高保护价值的神秘且知之甚少的鱼类。该物种在其原生范围内的数量急剧减少，甚至在某些地区已经灭绝。尽管基于环境 DNA（eDNA）的方法在监测稀有和濒危物种方面具有明显的优势，但仍然难以进行准确的检测和定量，而且质量评估通常也不完善。在这项研究中，我们开发并验证了一种新颖的基于数字液滴 PCR（ddPCR）的 eDNA 检测方法，该方法可用于可靠检测和定量，从而能够准确监测多种生境类型下的 M. fossilis。实验室条件下的稀释实验允许定义检测限（LOD）和定量限（LOQ），分别设定在 0.07 和 0.14 拷贝 μl 浓度下。一系列水族馆实验表明，个体数量与测量的 eDNA 浓度之间存在显著的正相关关系。在 2017 年至 2019 年的 3 年调查中，我们在佛兰德斯（比利时）评估了 96 个地点是否存在 M. fossilis。对这些样本的 eDNA 分析显示，该物种的阳性检出率为 45%。根据每升水中的 eDNA 浓度，只有 12 个地点显示出相对密集的种群。其余 31 个地点的阳性信号较弱，通常低于 LOQ。结合样本特异性有效 DNA 量（Q）的估计值和常规野外采样，我们得出结论，这些弱阳性位点中的每一个仍可能存在该物种，因此它们并非假阳性。此外，只有 7 个分类为阴性的样本需要进一步采样，因为我们的分析表明这些地点存在假阴性检测（即第二类错误）的风险很大。最后，我们表明 ddPCR 优于常规 qPCR 分析，特别是在目标 DNA 浓度非常低的情况下，这可能归因于 ddPCR 对抑制效应的敏感性降低、能够容纳更高的样本浓度以及获得更高的灵敏度。
