Institute for Ecology, Evolution and Diversity, Faculty of Biological Sciences, Goethe University Frankfurt, Frankfurt am Main, Hessen, Germany.
Conservation Genetics Group, Senckenberg Nature Research Society, Gelnhausen, Hessen, Germany.
PeerJ. 2024 Sep 30;12:e17782. doi: 10.7717/peerj.17782. eCollection 2024.
The monitoring of mosquitoes is of great importance due to their vector competence for a variety of pathogens, which have the potential to imperil human and animal health. Until now mosquito occurrence data is mainly obtained with conventional monitoring methods including active and passive approaches, which can be time- and cost-consuming. New monitoring methods based on environmental DNA (eDNA) could serve as a fast and robust complementary detection system for mosquitoes. In this pilot study already existing marker systems targeting the three invasive mosquito species , and were used to detect these species from water samples via microfluidic array technology. We compared the performance of the high-throughput real-time PCR (HT-qPCR) system Biomark HD with real-time PCR (qPCR) and also tested the effect of different filter media (Sterivex 0.45 µm, Nylon 0.22 µm, PES 1.2 µm) on eDNA detectability. By using a universal qPCR protocol and only 6-FAM-MGB probes we successfully transferred these marker systems on the HT-qPCR platform. All tested marker systems detected the target species at most sites, where their presence was previously confirmed. Filter media properties, the final filtration volume and observed qPCR inhibition did not affect measured Ct values via qPCR or HT-qPCR. The Ct values obtained from HT-qPCR were significantly lower as Ct values measured by qPCR due to the previous preamplification step, still these values were highly correlated. Observed incongruities in eDNA detection probability, as manifested by non-reproducible results and false positive detections, could be the result of methodological aspects, such as sensitivity and specificity issues of the used assays, or ecological factors such as varying eDNA release patterns. In this study, we show the suitability of eDNA-based detection of mosquito species from water samples using a microfluidic HT-qPCR platform. HT-qPCR platforms such as Biomark HD allow for massive upscaling of tested species-specific assays and sampling sites with low time- and cost-effort, thus this methodology could serve as basis for large-scale mosquito monitoring attempts. The main goal in the future is to develop a robust (semi)-quantitative microfluidic-based eDNA mosquito chip targeting all haematophagous culicid species occurring in Western Europe. This chip would enable large-scale eDNA-based screenings to assess mosquito diversity, to monitor species with confirmed or suspected vector competence, to assess the invasion progress of invasive mosquito species and could be used in pathogen surveillance, when disease agents are incorporated.
由于蚊子具有携带多种病原体的能力，这些病原体有可能危及人类和动物的健康，因此对蚊子进行监测非常重要。到目前为止，蚊子的发生数据主要是通过传统的监测方法获得的，包括主动和被动方法，这些方法既耗时又费钱。基于环境 DNA（eDNA）的新监测方法可以作为一种快速而强大的蚊子补充检测系统。在这项初步研究中，已经使用针对三种入侵蚊子物种（ 和 ）的现有标记系统，通过微流控阵列技术从水样中检测这些物种。我们比较了高通量实时 PCR（HT-qPCR）系统 Biomark HD 的性能与实时 PCR（qPCR）的性能，还测试了不同过滤介质（Sterivex 0.45 µm、Nylon 0.22 µm、PES 1.2 µm）对 eDNA 可检测性的影响。通过使用通用的 qPCR 方案和仅 6-FAM-MGB 探针，我们成功地将这些标记系统转移到 HT-qPCR 平台上。在大多数先前确认存在目标物种的地点，所有测试的标记系统都检测到了目标物种。过滤介质特性、最终过滤体积和观察到的 qPCR 抑制作用并没有通过 qPCR 或 HT-qPCR 影响测量的 Ct 值。由于之前的预扩增步骤，HT-qPCR 获得的 Ct 值显著低于 qPCR 测量的 Ct 值，但这些值高度相关。在 eDNA 检测概率方面观察到的不一致性，表现为不可重复的结果和假阳性检测，可能是由于方法学方面的原因，例如所用测定法的敏感性和特异性问题，或生态因素，例如不同的 eDNA 释放模式。在这项研究中，我们展示了使用微流控 HT-qPCR 平台从水样中检测蚊子物种的 eDNA 的适用性。HT-qPCR 平台（如 Biomark HD）允许大规模扩展测试的物种特异性测定法和采样点，同时减少时间和成本投入，因此该方法可以作为大规模蚊子监测尝试的基础。未来的主要目标是开发一种针对西欧所有吸血库蚊物种的基于微流控的强大（半）定量 eDNA 蚊子芯片。该芯片将能够进行大规模的基于 eDNA 的筛查，以评估蚊子的多样性，监测具有确认或疑似媒介能力的物种，评估入侵蚊子物种的入侵进度，并可用于病原体监测，当纳入疾病因子时。
