High-Resolution In Situ Genotyping of Legionella pneumophila Populations in Drinking Water by Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Environmental DNA
High-Resolution In Situ Genotyping of Legionella pneumophila Populations in Drinking Water by Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Environmental DNA
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DOI：
10.1128/aem.00416-10
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发表时间：
2010-09-01
影响因子：
4.4
通讯作者：
Hoefle, Manfred G.
中科院分区：
文献类型：
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作者：
Kahlisch, Leila;Henne, Karsten;Hoefle, Manfred G.
了解水传播病原体嗜肺军团菌感染的核心是克隆水平的检测。目前，嗜肺军团菌分离株的多位点可变数量串联重复（VNTR）分析（MLVA）可用作高分辨率基因分型的工具。由于嗜肺军团菌很难分离，因此由于各自环境种群处于可存活但不可培养（VBNC）状态，爆发菌株的分离常常失败。因此，我们开发了一种独立于培养的方法来检测饮用水中的单克隆。该方法基于从饮用水中提取 DNA，然后使用嗜肺军团菌 MLVA 基因分型所需的一组八个 VNTR 引物对进行 PCR。通过单链构象多态性 (SSCP) 和毛细管电泳分析 PCR 扩增子以获得各自的 MLVA 图谱。与高分辨率分析并行，我们使用相同的环境 DNA，通过使用 16S rRNA 基因靶向引物的实时 PCR 来量化饮用水中嗜肺军团菌细胞的数量。我们使用来自小型饮用水网络的一组饮用水样本来测试我们的方法。通过这些样本，我们证明了所开发的方法可直接适用于从饮用水中获取的 DNA。我们能够在饮用水中检测到比通过分离检测到的更多的嗜肺军团菌 MLVA 基因型。我们的方法可能是一种有价值的工具，即使在疫情爆发后也能识别疫情菌株，并且有可能直接应用于临床材料。
Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.