BACKGROUND Accurate Anopheles species identification is key for effective malaria vector control. Identification primarily depends on morphological analysis of field samples as well as molecular species-specific identifications. During an intra-laboratory assessment (proficiency testing) of the Anopheles funestus group multiplex PCR assay, it was noted that Anopheles arabiensis can be misidentified as Anopheles leesoni, a zoophilic member of the An. funestus group. The aim of this project was, therefore, to ascertain whether other members of the Anopheles gambiae complex can also be misidentified as An. leesoni when using the standard An. funestus multiplex PCR. METHODS The An. funestus multiplex PCR was used to amplify DNA from An. gambiae complex specimens. These included specimens from the laboratory colonies and field samples from the Democratic Republic of Congo. Amplified DNA from these specimens, using the universal (UV) and An. leesoni species-specific primers (LEES), were sequence analysed. Additionally, An. leesoni DNA was processed through the diagnostic An. gambiae multiplex PCR to determine if this species can be misidentified as a member of the An. gambiae complex. RESULTS Laboratory-colonized as well as field-collected samples of An. arabiensis, An. gambiae, Anopheles merus, Anopheles quadriannulatus, Anopheles coluzzii as well as Anopheles moucheti produced an amplicon of similar size to that of An. leesoni when using an An. funestus multiplex PCR. Sequence analysis confirmed that the UV and LEES primers amplify a segment of the ITS2 region of members of the An. gambiae complex and An. moucheti. The reverse was not true, i.e. the An. gambiae multiplex PCR does not amplify DNA from An. leesoni. CONCLUSION This investigation shows that An. arabiensis, An. gambiae, An. merus, An. quadriannulatus, An. coluzzii and An. moucheti can be misidentified as An. leesoni when using An. funestus multiplex PCR. This shows the importance of identifying specimens using standard morphological dichotomous keys as far as possible prior to the use of appropriate PCR-based identification methods. Should there be doubt concerning field-collected specimens molecularly identified as An. leesoni, the An. gambiae multiplex PCR and sequencing of the internal transcribed spacer 2 (ITS2) can be used to eliminate false identifications.

中文翻译：

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冈比亚按蚊复合体的成员物种可能被误认为是李氏按蚊。

背景技术准确的按蚊种类识别是有效控制疟疾媒介的关键。鉴定主要取决于野外样品的形态分析以及分子种类特异性鉴定。在实验室内对按蚊氏菌群多重PCR分析的评估（能力验证）期间，应注意的是，阿拉伯按蚊可能被错误地识别为按蚊的嗜食性人按蚊（Anopheles leesoni）。funestus组。因此，该项目的目的是确定冈比亚按蚊复合体的其他成员是否也可以被误认为An。使用标准An。真菌多重PCR。方法 使用funestus多重PCR扩增An。冈比亚复杂标本。其中包括来自实验室殖民地的标本和来自刚果民主共和国的野外样品。使用通用（UV）和An。从这些样本中扩增DNA。对葡萄球菌属物种特异性引物（LEES）进行了序列分析。此外，利索尼DNA通过诊断An。冈比亚多重PCR，以确定是否可以将该物种错误地识别为An。冈比亚情结。结果实验室定殖的和野外采集的An。阿拉伯半岛 冈比亚按蚊，梅氏按蚊，按蚊按蚊，库氏按蚊和莫奇按蚊产生的扩增子的大小与按蚊相似。leesoni当使用An。真菌多重PCR。序列分析证实UV和LEES引物扩增了An成员的ITS2区域的片段。冈比亚情结和安。莫切蒂。反之则不正确，即An。冈比亚多重PCR无法扩增An。leesoni。结论本研究表明An。阿拉伯半岛 冈比亚 梅鲁斯，安。Quadriannulatus，安 coluzzii和An。moucheti可能会误认为An。leesoni使用An时。真菌多重PCR。这显示了在使用适当的基于PCR的识别方法之前，尽可能使用标准形态学二分法密钥识别标本的重要性。是否对分子鉴定为An的野外采集标本有疑问？leesoni，安 冈比亚多重PCR和内部转录间隔区2（ITS2）的测序可用于消除错误的识别。结论本研究表明An。阿拉伯半岛 冈比亚 梅鲁斯，安。Quadriannulatus，安 coluzzii和An。moucheti可能会误认为An。leesoni使用An时。真菌多重PCR。这显示了在使用适当的基于PCR的识别方法之前，尽可能使用标准形态学二分法密钥识别标本的重要性。是否对分子鉴定为An的野外采集标本有疑问？leesoni，安 冈比亚多重PCR和内部转录间隔区2（ITS2）的测序可用于消除错误的识别。结论本研究表明An。阿拉伯半岛 冈比亚 梅鲁斯，安。Quadriannulatus，安 coluzzii和An。moucheti可能会误认为An。leesoni使用An时。真菌多重PCR。这显示了在使用适当的基于PCR的识别方法之前，尽可能使用标准形态学二分法密钥识别标本的重要性。是否对分子鉴定为An的野外采集标本有疑问？leesoni，安 冈比亚多重PCR和内部转录间隔区2（ITS2）的测序可用于消除错误的识别。这显示了在使用适当的基于PCR的识别方法之前，尽可能使用标准形态学二分法密钥识别标本的重要性。是否对分子鉴定为An的野外采集标本有疑问？leesoni，安 冈比亚多重PCR和内部转录间隔区2（ITS2）的测序可用于消除错误的识别。这表明在使用适当的基于PCR的识别方法之前，尽可能使用标准形态学二分法密钥识别标本的重要性。是否对分子鉴定为An的野外采集标本有疑问？leesoni，安 冈比亚多重PCR和内部转录间隔区2（ITS2）的测序可用于消除错误的识别。