Eawag (Swiss Federal Institute for Aquatic Science and Technology), Dübendorf, Switzerland.
Cytometry A. 2010 Mar;77(3):264-74. doi: 10.1002/cyto.a.20858.
Legionella is a pathogenic bacterium that establishes and proliferates well in water storage and distribution systems. Worldwide it is responsible for numerous outbreaks of legionellosis, which can be fatal. Despite recent advances in molecular and immunological methods, the official, internationally accepted detection method for Legionella spp. in water samples (ISO 11371) is still based on cultivation. This method has major disadvantages such as a long assay time of 10 days and the detection of cultivable cells only. Therefore, we developed a cultivation-independent, quantitative, and fast detection method for Legionella pneumophila in water samples. It consists of four steps, starting with (1) a concentrating step, in which cells present in one litre of water are concentrated into 5 ml by filtration (pore size 0.45 microm), (2) then cells are resuspended with sterile filtered buffer and double-stained with FITC- and Alexa-conjugated Legionella-specific antibodies, (3) subsequently, the cells are immunomagnetically caught, and (4) finally, fluorescently labeled Legionella cells were flow cytometrically detected and quantified. The efficiency of each step was tested separately. The whole method allows detection of L. pneumophila in 180 min with a detection limit of around 500 cells/l and a recovery of Legionella cells of 52.1 % out of spiked tap water. Fluorescence microscopy and flow cytometric cell-counting correlated well.
军团菌是一种致病细菌，在水储存和分配系统中能够很好地建立和繁殖。在全球范围内，它导致了许多军团病的爆发，这些爆发可能是致命的。尽管最近在分子和免疫学方法方面取得了进展，但军团菌属在水样中的官方、国际公认的检测方法（ISO 11371）仍然基于培养。这种方法有很大的缺点，例如检测时间长（10 天），只能检测可培养的细胞。因此，我们开发了一种非培养、定量和快速的水样中嗜肺军团菌检测方法。它由四个步骤组成，首先是（1）浓缩步骤，通过过滤（孔径 0.45 微米）将一升水中的细胞浓缩到 5 毫升，（2）然后用无菌过滤缓冲液重悬细胞，并与 FITC 和 Alexa 标记的军团菌特异性抗体双重染色，（3）随后，细胞被免疫磁捕获，（4）最后，用流式细胞术检测和定量荧光标记的军团菌细胞。每个步骤的效率都分别进行了测试。整个方法允许在 180 分钟内检测到 L. pneumophila，检测限约为 500 个细胞/升，从加标自来水回收的军团菌细胞回收率为 52.1%。荧光显微镜和流式细胞术细胞计数相关性良好。
