Mas-Carrió Eduard, Schneider Judith, Othenin-Girard Victor, Pigeault Romain, Taberlet Pierre, Christe Philippe, Glaizot Olivier, Fumagalli Luca
Laboratory for Conservation Biology, Department of Ecology and Evolution, Biophore, University of Lausanne, Lausanne, Switzerland.
Department of Ecology and Evolution, Biophore, University of Lausanne, Lausanne, Switzerland.
PeerJ. 2025 Mar 18;13:e19107. doi: 10.7717/peerj.19107. eCollection 2025.
Accurate detection and identification of vector-host-parasite systems are key to understanding their evolutionary dynamics and to design effective disease prevention strategies. Traditionally, microscopical and serological techniques were employed to analyse arthropod blood meals for host/parasite detection, but these were limited in taxonomic resolution and only to pre-selected taxa. In recent years, molecular techniques have emerged as a promising alternative, offering enhanced resolution and taxonomic range. While singleplex polymerase chain reaction (PCR) assays were used at first to identify host, vector and parasite components in separate reactions, today multiple primer pairs can be combined in a single reaction, , multiplex, offering substantial time and cost savings. Nonetheless, despite the potential benefits of multiplex PCR, studies quantifying its efficacy compared to singleplex reactions are scarce. In this study, we used partially digested mosquito blood meals within an avian malaria framework to jointly identify the host, vector and parasite using multiplex DNA metabarcoding, and to compare it with separate singleplex PCRs. We aimed to compare the detection probabilities and taxonomic assignments between both approaches. We found both to have similar performances in terms of detection for the host and the vector, but singleplex clearly outperformed multiplex for the parasite component. We suggest adjusting the relative concentrations of the PCR primers used in the multiplex assay could increase the efficiency of multiplex in detecting all the components of the studied multi-species system. Overall, the results show that multiplex DNA metabarcoding can be an effective approach that could be applied to any vector-borne interaction involving blood-feeding arthropods. Our insights from this proof-of-concept study will help improve laboratory procedures for accurate and cost-efficient medical diagnosis of vector-borne diseases, the spread of which is globally exacerbated by current climate change.
准确检测和识别病媒-宿主-寄生虫系统是理解其进化动态以及设计有效疾病预防策略的关键。传统上，人们采用显微镜和血清学技术来分析节肢动物的血餐以检测宿主/寄生虫，但这些技术在分类分辨率上有限，且仅适用于预先选定的分类群。近年来，分子技术已成为一种有前景的替代方法，具有更高的分辨率和分类范围。虽然最初使用单重聚合酶链反应（PCR）分析在单独反应中鉴定宿主、病媒和寄生虫成分，但如今多个引物对可在单个反应中组合，即多重PCR，可大幅节省时间和成本。尽管如此，尽管多重PCR有潜在优势，但与单重反应相比量化其功效的研究却很少。在本研究中，我们在禽疟框架内使用部分消化的蚊虫血餐，通过多重DNA元条形码技术联合鉴定宿主、病媒和寄生虫，并将其与单独的单重PCR进行比较。我们旨在比较两种方法在检测概率和分类归属方面的差异。我们发现两者在宿主和病媒检测方面表现相似，但在寄生虫成分检测上，单重PCR明显优于多重PCR。我们建议调整多重分析中使用的PCR引物的相对浓度，可能会提高多重分析检测所研究的多物种系统所有成分的效率。总体而言，结果表明多重DNA元条形码技术可以是一种有效的方法，可应用于任何涉及吸血节肢动物的病媒传播相互作用。我们从这项概念验证研究中获得的见解将有助于改进实验室程序，以准确且经济高效地诊断病媒传播疾病，当前气候变化在全球范围内加剧了此类疾病的传播。
