Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps.

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多重 PCR 与 DNA 条形码识别容器繁殖蚊种的比较

蚊子的识别很大程度上依赖于形态特征。由于某些物种无法通过形态学方法可靠地区分，因此将分子技术纳入诊断流程非常重要。使用桑格测序的 DNA 条形码目前广泛用于蚊子种类的识别。然而，这种方法不允许在一个样品中检测多个物种，这在分析蚊子卵时非常重要。容器繁殖伊蚊的检测通常是通过使用产卵器收集卵来进行的。这些陷阱由一个装满水的黑色容器和插入的用于产卵支持的木铲组成。不同种类的伊蚊可能会在抹刀上产下一个或多个卵。与特定聚合酶链式反应 (PCR) 产品的桑格测序相比，针对特定目标物种的多重 PCR 方案有利于检测同一样品中的多个物种。为此，我们采用了之前发布的 PCR 方案，用于同时检测与奥地利监测计划相关的四种不同的伊蚊物种，因为它们可以在产卵器中找到：白纹伊蚊、日本伊蚊、朝鲜伊蚊和膝状伊蚊。为了评估多重 PCR 方案，我们分析了 2021 年和 2022 年的 2271 个产卵器蚊子样本，这些样本是在奥地利全国监测计划范围内收集的。我们将多重 PCR 的结果与 DNA 条形码的结果进行了比较。在 2271 个样本中，多重 PCR 可以识别出 1990 个样本，而使用线粒体细胞色素 C 氧化酶亚基 I 基因的 DNA 条形码可以在 1722 个样本中进行物种确定。多重PCR显示47个样本中存在不同物种的混合物，而DNA条形码无法检测到这种混合物。总之，使用多重 PCR 方案比 DNA 条形码方案更成功地鉴定产卵器样品中的伊蚊种类。此外，多重 PCR 使我们能够检测同一样本中的多个物种，而仅使用 DNA 条形码和桑格测序时可能会漏掉这些物种。因此，我们认为多重 PCR 方案在分析产卵器中的蚊卵时非常适合且具有很大优势。