Development of novel toxin detection methodologies applicable tomarine and fresh

描述(由申请人提供) 有害藻华(HAB)会对生态、经济和人类健康造成严重影响。至少有32个州报告了人类或哺乳动物接触HAB毒素的情况。此外，研究表明，过去几十年来，赤潮发生的频率和强度有所增加，本世纪还将继续增加。关于这些事件的生态驱动因素的知识差距仍然存在，这阻碍了成功的缓解战略的制定。大多数赤潮由几个属组成，每个物种都含有有毒和无毒的菌株。这使得用传统方法理解这些事件的生态变得极其困难。分子技术(如定量聚合酶链式反应)的进步促进了深入了解这些复杂事件的第一步，尽管以前的研究一般集中在单个生物体上。研究人员的目标是开发和使用一种“灵活的”实时多重定量聚合酶链式反应(MqPCR)分析方法，用于同时定量共存的有毒HAB属。这将有助于下一步进一步加深我们对这些复杂事件的了解，并将显著改善民政事务局活动与现有活动的整合 州和联邦的监测和观测计划。 研究人员有四个主要目标：1)开发MqPCR特异的TaqMan引物和探针，用于检测产生类毒素的蓝藻以及有毒的真细菌，以整合到现有的MqPCR方法中。2)设计和现场验证了可针对特定HAB事件量身定做的灵活的MqPCR。通过设计TaqMan引物和探针在同一热循环程序中复制，他们将能够选择特定的引物集来匹配不同的开花情况。3)研究人员将使用MqPCR来研究在水华发生过程中，产生微囊藻毒素/根瘤素、圆柱状精视蛋白、岩藻毒素和类毒素a的蓝藻以及潜在有毒的真球藻对环境变量的反应，以及它们之间的相互关系。他们每年将从全国四个经常出现密集混合属水华的地点进行采样：西部的伊利湖、俄亥俄州的格兰德湖圣玛丽、佛罗里达州的圣约翰斯河和德克萨斯州的努切斯河。每周将在水华活动之前、期间和之后收集样本(至少150个现场样本)。调查人员还将收集营养数据、理化参数和藻毒素数据。4)确定鲜为人知的产毒藻类在美国淡水中的作用。三名研究人员(两名教员，一名博士后研究人员)和一名合作研究人员在多个学科(微生物学、遗传学和化学)方面的丰富专业知识将允许对这项研究采取多学科方法。开发的MqPCR技术将是快速监测可应用于海洋-淡水连续体的HAB属的宝贵工具。这项研究将为未来赤潮事件的建模和预测以及开发早期预警网络以保护人类免受赤潮毒素的影响提供重要数据。 公共卫生相关性：藻类水华导致严重的水质和人类健康问题；然而，对其生态仍知之甚少。使用尖端的遗传学方法，研究人员将进一步了解促进这些有毒藻类的环境因素。这将有助于进一步开发成功的预测模型和藻类补救战略，帮助保护人类免受藻毒素暴露对人类健康的负面影响。

DESCRIPTION (provided by applicant) Harmful algal blooms (HABs) can have severe ecological, economical, and human health impacts. At least 32 states have reported human or mammal exposures to HAB toxins. Furthermore, studies suggest that the frequency and intensity of HAB events have increased over the past few decades and will continue to increase this century. Knowledge gaps remain regarding the ecological drivers of these events which have hindered the development of successful mitigation strategies. Most HABs are comprised of several genera with each species containing toxic and non-toxic strains. This has made understanding the ecology of these events using traditional methods extremely difficult. Advances in molecular techniques (e.g. quantitative PCR) have facilitated the first steps towards a deeper understanding of these complex events although previous studies have generally focused on a single organism. The investigators' goal is to develop and employ a "flexible" real-time multiplex quantitative polymerase chain reaction (MqPCR) assay for the simultaneous quantification of co-occurring toxic HAB genera. This will facilitate the next important step in furthering our understanding of these complex events and will significantly improve the integration of HAB activities into existing state and federal monitoring and observational programs. The investigators have four primary objectives: 1) Develop MqPCR specific TaqMan primers and probes for the detection of anatoxin-a producing cyanobacteria as well as toxic eugleniods to incorporate into the existing MqPCR method. 2) Design and field validate a "flexible" MqPCR that can be tailored to specific HAB events. By designing both TaqMan primers and probes to be replicated in the same thermal cycling program, they will be able to select specific primer sets to match different bloom situations. 3) The investigators will use the MqPCR to investigate the response of microcystin/nodularin-, cylindrospermopsin-, saxitoxin-, and anatoxin-a-producing cyanobacteria as well as potentially toxic euglenoids to environmental variables individually, as well as in relation to each other, over the course of a bloom. They will sample from four sites across the nation each year that routinely develop dense blooms of mixed genera; western Lake Erie, Grand Lake St. Marys, OH, St. Johns River, FL, and Nueces River, TX. Samples will be collected weekly before, during and after bloom events (150 field samples minimum). The investigators will also collect nutrient data, physiochemical parameters and algal toxin data. 4) Identify the role of poorly understood toxin-producing algae with U.S. freshwaters. The three investigators (2 faculty members, one post-doctoral researcher) and a co-investigator's vast expertise in a number of disciplines (microbiology, genetics, and chemistry) will allow for a multi-disciplinary approach toward this research. The MqPCR technique developed will be a valuable tool for the rapid monitoring of HAB genera that can be applied across the marine- freshwater continuum. This study will provide important data for the modeling and prediction of future HAB events as well as the development of early warning networks to protect humans from exposure to HAB toxins. Public Health Relevance: Algal blooms cause serious water quality and human health issues; however, their ecology remains poorly understood. Using a cutting-edge genetic method the investigators will further their understanding of the environmental factors promoting these toxic algae. This will allow for the further development of successful predictive models and algal remediation strategies that will help protect humans from the negative human health effects of algal toxin exposure.