Abstract:
Legionella are opportunistic intracellular pathogens that are found throughout the environment. The Legionella contamination of water systems represents a serious social problem that can lead to severe diseases, which can manifest as both Pontiac fever and Legionnaires\' disease (LD) infections. Fluorescence in situ hybridization using nucleic acid mimic probes (NAM-FISH) is a powerful and versatile technique for bacterial detection. By optimizing a peptide nucleic acid (PNA) sequence based on fluorescently selective binding to specific bacterial rRNA sequences, we established a new PNA-FISH method that has been successfully designed for the specific detection of the genus Legionella. The LEG22 PNA probe has shown great theoretical performance, presenting 99.9% specificity and 96.9% sensitivity. We also demonstrated that the PNA-FISH approach presents a good signal-to-noise ratio when applied in artificially contaminated water samples directly on filtration membranes or after cells elution. For water samples with higher turbidity (from cooling tower water systems), there is still the need for further method optimization in order to detect cellular contents and to overcome interferents\' autofluorescence, which hinders probe signal visualization. Nevertheless, this work shows that the PNA-FISH approach could be a promising alternative for the rapid (3-4 h) and accurate detection of Legionella.
摘要:
军团菌是在整个环境中发现的机会性细胞内病原体。水系统的军团菌污染是一个严重的社会问题,可能导致严重的疾病,可表现为庞蒂亚克热和军团病(LD)感染。使用核酸模拟探针的荧光原位杂交(NAM-FISH)是用于细菌检测的强大且通用的技术。通过基于与特定细菌rRNA序列的荧光选择性结合来优化肽核酸(PNA)序列,我们建立了一种新的PNA-FISH方法,该方法已成功设计用于军团菌的特异性检测。LEG22PNA探针具有良好的理论性能,呈现99.9%的特异性和96.9%的灵敏度。我们还证明了PNA-FISH方法在直接在滤膜上或在细胞洗脱后应用于人工污染的水样品时具有良好的信噪比。对于浊度较高的水样(来自冷却塔水系统),仍然需要进一步的方法优化,以检测细胞内容物和克服干扰物的自发荧光,这阻碍了探针信号的可视化。然而,这项工作表明,PNA-FISH方法可能是快速(3-4h)和准确检测军团菌的有希望的替代方法。