Institute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES), Vienna, Austria.
Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
Parasit Vectors. 2024 Apr 2;17(1):171. doi: 10.1186/s13071-024-06255-z.
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.
蚊子的鉴定主要依赖于形态学特征。由于一些物种无法通过形态学方法可靠地鉴别，因此将分子技术纳入诊断管道非常重要。使用 Sanger 测序的 DNA 条形码目前广泛用于鉴定蚊子种类。然而，这种方法不允许在一个样本中检测多种物种，这在分析蚊子卵时非常重要。容器滋生的埃及伊蚊的检测通常通过收集卵来进行，这些卵是用诱卵器收集的。这些诱卵器由一个装满水的黑色容器和一个木制的刮刀组成，用于产卵支持。不同物种的埃及伊蚊可能会在刮刀上产下单个或多个卵。与特定聚合酶链反应 (PCR) 产物的 Sanger 测序相比，针对特定感兴趣物种的多重 PCR 方案对于在同一样本中检测多种物种具有优势。
为此，我们改编了先前发表的一种 PCR 方案，用于同时检测奥地利监测计划中与监测相关的四种不同的埃及伊蚊物种，因为它们可以在诱卵器中发现：白纹伊蚊、日本伊蚊、朝鲜伊蚊和埃及伊蚊。为了评估多重 PCR 方案，我们分析了 2021 年和 2022 年在奥地利全国监测计划范围内收集的 2271 个诱卵器蚊子样本。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 可以鉴定 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 条形码对 1722 个样本进行物种鉴定是可行的。多重 PCR 显示 47 个样本中存在不同物种的混合物，而这些物种无法通过 DNA 条形码检测到。
总之，与 DNA 条形码相比，使用多重 PCR 方案鉴定诱卵器样本中的埃及伊蚊种类更为成功。此外，多重 PCR 允许我们在同一样本中检测多种物种，而单独使用 Sanger 测序的 DNA 条形码可能会遗漏这些物种。因此，我们建议在分析诱卵器中的蚊子卵时，使用多重 PCR 方案非常合适且具有很大的优势。
