Aptamer-based surface plasmon resonance detection of legionella pneumophila in water systems

军团病是由嗜肺军团菌（Legionella pneumophila，Lp）引起的。许多水系统，如冷却塔和热水分配系统，都可能受到Lp的污染。通过这种系统产生的气溶胶使得Lp能够传播给人类。每年都有许多这种疾病爆发的报道。例如，魁北克市在2012年遭遇了一次大规模疫情。监测水系统对于限制感染风险和疫情至关重要。水系统中Lp的检测目前通过培养或qPCR进行。这些方法昂贵且繁琐，并且在培养的情况下，需要长达两周的时间来执行。与Magnus Chemical Ltd合作，我们建议开发一种新的检测方法，该方法基于通过适体识别Lp，适体是折叠成与靶标特异性结合的结构的短DNA序列。将使用“通过指数富集的配体系统进化”（SELEX）技术开发适体。将针对多种Lp分离株和经常从水系统中分离的其他细菌物种测试适体的特异性和通用性。将最好的适体固定在表面等离子体共振生物传感器（SPR）棱镜的表面上，并评估它们检测人造水样中Lp的能力。然后通过修改表面化学和通过在“夹心测定”中使用与金纳米颗粒偶联的第二适体来放大信号并提高检测限（LOD）来优化系统。为了进一步降低LOD，将开发用于水样中Lp富集的芯片实验室策略。目标是达到10-100 CFU Lp /ml的浓度（维持水平）。接下来，将用真实的水样测试新的检测方法，并与当前的检测方法进行比较。该项目应允许开发一种新的检测策略，该策略可以小型化并用于以连续的方式监测水系统中Lp的存在。在任期内，该项目将改善及时反应，启动水系统的处理，以尽量减少退伍军人病的风险。

Legionnaires' disease is caused by the bacterium Legionella pneumophila (Lp). Many water systems, such as cooling towers and hot water distribution systems, are potentially contaminated by Lp. Production of aerosols by such systems enables the transmission of Lp to humans. Many outbreaks of this disease are reported each year. For example, Quebec City suffered from a major outbreak in 2012. Surveillance of water systems is crucial for limiting infection risks and outbreaks. Detection of Lp in water systems is currently performed by culturing or qPCR. These methods are expensive and tedious, and in the case of culturing, take up to two weeks to perform. In collaboration with Magnus Chemical Ltd, we propose to develop a novel detection method based on the recognition of Lp by aptamers, short DNA sequences that folds into structures binding specifically to a target. The aptamers will be developed using the "Systemic Evolution of Ligands through EXponential enrichment" (SELEX) technique. Specificity and versatility of the aptamers will be tested against a diversity of Lp isolates and other bacterial species frequently isolated form water systems. The best aptamers will be immobilized on the surface of a Surface Plasmon Resonance biosensor (SPR) prism and their ability to detect Lp in man-made water samples will be evaluated. The system will then be optimized by modifying the surface chemistry and by using a second aptamer coupled to gold nanoparticle in a "sandwich assay" to amplify the signal and improve the limit of detection (LOD). To further lower the LOD, a Lab-on-a-Chip strategy will be developed for the Lp enrichment in water samples. The aim is to reach a LOC of 10-100 CFU Lp /ml (maintenance levels). Next, the novel detection method will be tested with real water samples and compared to the current detection methods. This project should allow the development of a novel detection strategy that could be miniaturized and used to monitor the presence of Lp in water systems in a continuous manner. At term, this project will improve timely response to initiate treatment of water systems to minimize risk of Legionnaires' disease.