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

军团病是由嗜肺军团菌(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。由这种系统产生的气雾剂使LP能够传播给人类。这种疾病每年都有多次暴发的报道。例如，魁北克市在2012年遭遇了一次重大疫情。对水系统的监测对于限制感染风险和暴发至关重要。目前，水系统中LP的检测是通过培养或qPCR进行的。这些方法昂贵而繁琐，在培养的情况下，需要长达两周的时间才能完成。与Magnus化学有限公司合作，我们建议开发一种新的检测方法，该方法基于适体对LP的识别，适体是一种折叠成与靶标特异结合的结构的短DNA序列。该适配子将使用“通过指数富集法系统进化配体”(SELEX)技术来开发。这些适配子的特异性和通用性将与从水系统中经常分离的各种LP分离物和其他细菌种类进行测试。最好的适配子将被固定在表面等离子体共振生物传感器(SPR)棱镜的表面，并将评估它们检测人造水样中LP的能力。然后，将通过修改表面化学和使用第二个适配子与金纳米颗粒偶联来优化该系统，以放大信号并提高检测下限(LOD)。为了进一步降低低密度，将开发一种芯片实验室战略，用于在水样中进行低密度浓缩。目标是达到10-100 CFU Lp/ml(维持水平)的LOC。接下来，将用实际水样对新的检测方法进行测试，并与现有的检测方法进行比较。该项目应该能够开发一种新的检测战略，该战略可以小型化，并可用来连续监测水系统中是否存在低密度脂蛋白。在短期内，该项目将改善及时的反应，启动水系统的处理，将退伍军人病的风险降至最低。

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.