Molecular Monitoring and Rapid-Response Characterization

分子监测和快速响应表征

• 批准号: 7161663
• 负责人: CHRISTOPHER D SINIGALLIANO
• 金额: $ 7.55万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7161663
• 关键词: Dinoflagellates; algae; cell sorting; environmental toxicology; flow cytometry; fluorescence microscopy; fluorescent in situ hybridization; light microscopy; monitoring device; polymerase chain reaction; technology /technique development; water sampling /testing

The annual occurrence of Karenia brevis in the Gulk of Mexico is one of the most significant harmful algal blooms (HABs) in the coastal waters of the United States. This HAB species has a large economic, health, and environmental impact on Gulf coast communities, especially in Florida, through massive fish kills, deaths of marine mammals and birds, human consumption of toxin-contaminated seafood, and respiratory effects from aerosolized toxins. Over the past few decades the incidence of K. brevis blooms appears to be increasing in both frequency and duration. Current government-mandated programs for monitoring this HAB species must rely on microscopic cell counts. These counts are time consuming, have low sample through-put, and require highly-skilled microscopists who are experts at identifying and enumerating Karenia brevis cells of varying morphologies in complex natural phytoplankton assemblages. There is consensus among HAB researchers and public health professionals that monitoring efforts for HABs must evolve towards the use of rapid, accurate, sensitive, and objective molecular-based detection and quantitation methods. The proposed project will first utilize a combination of molecular techniques, such as Fluorescent In Situ Hybridization (FISH) and real-time quantitative polymerase chain reaction (qPCR), with traditional methods (light and fluorescent microscopy) to enumerate Karenia brevis cells in laboratory cultures. The objective of this initial phase is to test the efficacy of, and where feasible optimize, molecular methodologies with a short (3-5 hour) processing time for use in natural populations. Second, we will apply a combination of metabolic measurements (cell division, photosynthesis and respiration rates) and flow cytometry methodologies in cultures, and then natural popualtiosn, to improve our understanding of how environmental factors influence the metabolic state of K. brevis. Third, we will attempt to relate these indices to environmental parameters. The ultimate objective of this research is to facilitate the idenitfication and enumeration of this HAB species, and develop near real-time indices to assess the metabolic state of Karenia brevis in various stages of bloom development. Significance: It is anticipated that the results of this project will enhance our understanding of what factors contribute to Karenia bloom development. It will also synergistically enhance other collaborative HAB research programs through a rapid-response sampling program and sharing of samples and data.

墨西哥峡谷中的Karenia Brevis的年度发生是最重要的有害之一 在美国沿海水域中的藻类开花（HAB）。这种HAB物种具有庞大的经济 健康和环境对墨西哥湾沿岸社区的影响，特别是在佛罗里达州，通过大量鱼类 杀人，海洋哺乳动物和鸟类的死亡，人类对毒素污染的海鲜的消费以及 毒素毒素的呼吸作用。在过去的几十年中，K。BrevisBlooms的发生率 频率和持续时间似乎都在增加。现任政府规定的计划 监测该HAB物种必须依靠微观细胞计数。这些计数很耗时，有 低样本贯穿put，需要高技能的显微镜家，他们是识别和识别和 在复杂的天然浮游植物组合中列举了不同形态的karenia brevis细胞。 HAB研究人员和公共卫生专业人员之间达成共识，以监测 HAB必须发展为使用快速，准确，敏感和客观的基于分子的检测 和定量方法。 拟议的项目将首先利用分子技术的组合，例如原位荧光 杂交（FISH）和实时定量聚合酶链反应（QPCR），传统方法 （光和荧光显微镜）在实验室培养物中枚举karenia brevis细胞。目的 初始阶段是测试用A 短（3-5小时）的处理时间用于自然种群。其次，我们将结合 代谢测量（细胞分裂，光合作用和呼吸速率）和流式细胞术 文化中的方法论，然后是自然的popualtiosn，以提高我们对如何的理解 环境因素影响K. Brevis的代谢状态。第三，我们将尝试将这些 环境参数的指标。这项研究的最终目的是促进同性恋 并列举了这种HAB物种，并开发了接近实时指数以评估代谢状态 Karenia Brevis处于Bloom Development的各个阶段。 意义：预计该项目的结果将增强我们对什么的理解 因素有助于Karenia Bloom开发。它也将协同增强其他协作 HAB研究计划通过快速响应抽样程序以及样本和数据共享。