ABSTRACT: Monitoring the occurrence and density of parasites and pathogens can identify high infection-risk areas and facilitates disease control and eradication measures. Environmental DNA (eDNA) techniques are increasingly used for pathogen detection due to their relative ease of application. Since many factors affect the reliability and efficacy of eDNA-based detection, rigorous validation and assessment of method limitations is a crucial first step. We evaluated an eDNA detection method using in situ filtration of large-volume water samples, developed to detect and quantify aquatic wildlife parasites by quantitative PCR (qPCR). We assessed method reliability using Batrachochytrium dendrobatidis, a pathogenic fungus of amphibians and the myxozoan Tetracapsuloides bryosalmonae, causative agent of salmonid proliferative kidney disease, in a controlled experimental setup. Different amounts of parasite spores were added to tanks containing either clean tap water or water from a semi-natural mesocosm community. Overall detection rates were higher than 80%, but detection was not consistent among replicate samples. Within-tank variation in detection emphasises the need for increased site-level replication when dealing with parasites and pathogens. Estimated parasite DNA concentrations in water samples were highly variable, and a significant increase with higher spore concentrations was observed only for B. dendrobatidis. Despite evidence for PCR inhibition in DNA extractions from mesocosm water samples, the type of water did not affect detection rates significantly. Direct spiking controls revealed that the filtration step reduced detection sensitivity. Our study identifies sensitive quantification and sufficient replication as major remaining challenges for the eDNA-based methods for detection of parasites in water.

中文翻译：

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验证用于检测水中野生动物病原体的基于 eDNA 的方法

摘要：监测寄生虫和病原体的发生和密度可以识别高感染风险区域，并有助于疾病控制和根除措施。环境 DNA (eDNA) 技术因其相对易于应用而越来越多地用于病原体检测。由于许多因素会影响基于 eDNA 的检测的可靠性和有效性，因此严格验证和评估方法限制是至关重要的第一步。我们评估了一种使用原位过滤大量水样的 eDNA 检测方法，该方法旨在通过定量 PCR (qPCR) 检测和量化水生野生动物寄生虫。我们使用Batrachochytrium dendrobatidis（一种两栖动物和粘虫的致病真菌）评估了方法的可靠性Tetracapsuloides bryosalmonae，鲑鱼增殖性肾病的病原体，在受控的实验装置中。将不同数量的寄生虫孢子添加到装有干净自来水或来自半自然中宇宙群落的水的水箱中。总体检出率高于 80%，但重复样本之间的检出率并不一致。检测中的罐内变异强调了在处理寄生虫和病原体时需要增加站点级复制。水样中估计的寄生虫 DNA 浓度变化很大，仅观察到B. dendrobatidis的孢子浓度显着增加. 尽管有证据表明从中宇宙水样中提取 DNA 有 PCR 抑制作用，但水的类型并没有显着影响检测率。直接加标控制显示过滤步骤降低了检测灵敏度。我们的研究将敏感的量化和充分的复制确定为基于 eDNA 的水中寄生虫检测方法的主要挑战。