Development of novel toxin detection methodologies applicable tomarine and fresh

描述（由申请人提供） 有害藻华（HABs）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有害藻华毒素的情况。此外，研究显示，在过去几十年，赤潮事件的频率和强度有所增加，并将在本世纪继续增加。 在这些事件的生态驱动因素方面仍然存在知识差距，这阻碍了制定成功的缓解战略。大多数有害藻华由几个属组成，每一种都含有有毒和无毒菌株。这使得使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PCR）的进步促进了对这些复杂事件的深入理解的第一步，虽然以前的研究一般集中在一个单一的生物体。 研究人员的目标是开发和采用一种“灵活的”实时多重定量聚合酶链反应（MqPCR）检测方法，用于同时定量共存的有毒HAB属。这将有助我们进一步了解这些复杂的事件，并将大大改善香港政务司的工作与现有的 州和联邦监测和观测方案。 研究人员有四个主要目标：1）开发MqPCR特异性TaqMan引物和探针，用于检测产生类毒素的蓝藻以及有毒的裸藻，以纳入现有的MqPCR方法。 2)设计和现场验证一个“灵活的”MqPCR，可以定制特定的赤潮事件。 通过设计TaqMan引物和探针以在相同的热循环程序中复制，他们将能够选择特定的引物组以匹配不同的水华情况。3)研究人员将使用MqPCR来调查微囊藻毒素/节球藻毒素，柱孢菌素，石房蛤毒素和类毒素a生产蓝藻以及潜在有毒的裸藻对环境变量的反应，以及在水华过程中相互之间的关系。 他们每年将从全国各地的四个地点取样，这些地点通常会出现混合属的密集开花;西部的伊利湖、格兰德湖、俄亥俄州的圣玛丽湖、佛罗里达州的圣约翰河和德克萨斯州的纽塞斯河。 在水华事件之前、期间和之后每周采集一次样本（至少150份田间样本）。 调查人员还将收集营养数据、理化参数和藻类毒素数据。 4)确定对美国淡水中产生毒素的藻类知之甚少的作用。 三名研究人员（2名教师，一名博士后研究员）和一名合作研究员在多个学科（微生物学，遗传学和化学）的丰富专业知识将允许对这项研究采取多学科方法。所开发的MqPCR技术将成为快速监测有害藻华属的宝贵工具，可应用于整个海洋-淡水连续体。这项研究将提供重要的数据建模和预测未来的赤潮事件，以及早期预警网络的发展，以保护人类免受赤潮毒素的暴露。 公共卫生相关性：藻类大量繁殖导致严重的水质和人类健康问题;然而，对其生态仍然知之甚少。使用尖端的遗传方法，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类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.