PFI:AIR - TT: Rapid, quantitative, molecular diagnostics for virulent Vibrio pathogens in water and shellfish

这个PFI: AIR技术翻译项目的重点是翻译之前对河口和沿海系统中发现的一组细菌病原体弧菌进行的生态研究。霍乱弧菌是一种众所周知的淡水病原体，在发展中国家引起关注。在美国，两种鲜为人知但重要的人类病原体来自同一细菌群：创伤弧菌和副溶血性弧菌。这些细菌是在河口系统中自然存在的，但当食用生贝类或在极少数情况下在海滩水域接触时，它们也会对人类健康构成威胁。该项目将把通过先前的生态研究获得的知识转化为技术，使设计用户友好、快速的分子检测试剂盒成为可能。现有的测试水和贝类样品中弧菌种类的方法已经有几十年的历史了，它们需要24-96小时才能得到结果。即使是用于检测创伤弧菌和副溶血性弧菌致病性形式的新分子方法，也由于缺乏特异性和/或需要富集步骤来提高灵敏度而受到损害，使得它们几乎需要一天才能完成。该项目将允许对创伤弧菌和副溶血性弧菌的毒力形式进行新的、快速的分子诊断，使用一种确定试剂盒有用靶点的新方法。设想这些试剂盒可用于1)证明贝类可以安全食用，2)保护接触水的危险人群的健康，3)确定与危险细菌形式相关的模式，以改进预警系统。该资助项目将设计出易于使用的市售分子检测试剂盒，并在2-3小时内提供结果。这些功能将允许准确的公众通知和保护公众健康。该项目解决了从研究到商业应用过渡中的特定知识差距。弧菌属是复杂的细菌有机体，自然存在于河口和沿海水域，但其中只有一小部分能够引起疾病。该团队在下一代序列和序列数据方法、弧菌生态学和弧菌致病性方面具有宝贵的专业知识。这些知识的结合允许对已知的毒力（致病性）弧菌样本进行分析，以寻找与细菌致病能力相关的特定“DNA基序”。通过重复分析已知的毒性和无毒性细胞，项目团队可以识别DNA基序，或可用于设计DNA测试试剂盒的特征。随后，通过该项目的支持，分子检测试剂盒将被开发、优化、验证和商业化。除技术活动外，本项目还将支持本科生、高级研究生、博士后研究助理以及研究助理教授。他们将参与广泛的研究和技术转移活动，包括市场评估、实地取样和研究、实验室样品制备、培养和基于分子的样品分析、下一代序列数据注释和分析，以及试剂盒设计和优化。该项目将产生协同效应，并直接为PI Noble运营的分子培训设施做出贡献，水质专业人员将在这里学习如何正确使用qPCR方法和解释qPCR数据，以进行水质管理。该项目还与海洋生物技术创新中心合作，这是一个致力于推动海洋科学研究转化为商业应用的组织。此外，Orion Integrated Biosciences也是项目合作伙伴。Orion Integrated Biosciences是一家致力于开发独特的、颠覆性的计算方法来调查序列和分子应用的“大数据”的公司。该项目将直接影响海洋宏基因组学的发展，因为项目团队正在利用整体方法来瞄准复杂的生物。该项目的新颖之处在于，该方法摆脱了“单基因目标”qPCR设计方法，允许生成快速且具有成本效益的诊断试剂盒，贝类捕捞和水质管理人员可以使用该试剂盒来管理宝贵的资源并保护公众健康。

This PFI: AIR Technology Translation project focuses on translating ecological research that has been previously conducted on a set of bacterial pathogens, Vibrios, found in estuaries and coastal systems. Vibrio cholerae is a well-known freshwater pathogen that is of concern in developing countries. Two lesser known, but important human pathogens in the USA are from the same bacterial group: Vibrio vulnificus and Vibrio parahaemolyticus. These are bacteria that are naturally found in estuarine systems, but they can also present a risk to human health when consumed in raw shellfish, or in rare cases, contacted in beach waters. This project will translate knowledge gained through prior ecological research into technology that permits the design of user-friendly, rapid, molecular testing kits. The existing ways to test for Vibrio species in water and shellfish samples are decades old, and they require 24-96 hours for results. Even new molecular approaches for testing of pathogenic forms of V. vulnificus and V. parahaemolyticus are compromised by a lack of specificity and/or a need for an enrichment step to improve sensitivity, making them require almost a day for completion. This project will permit generation of new, rapid molecular diagnostics for virulent forms of V. vulnificus and V. parahaemolyticus using a new approach for determining useful targets for the kits. It is envisioned that these kits could be used to 1) certify that shellfish is safe for raw consumption, 2) protect the health of at risk populations for water contact, 3) determine the patterns associated with dangerous forms of the bacteria for improved warning systems. The funded project will result in the design of commercially available molecular testing kits that will be easy to use, and will provide results in 2-3 hours. These features will permit accurate public notification and protection of public health.This project addresses a specific knowledge gap in the transition from research to commercial application. Vibrio sp. are complex bacterial organisms, that are naturally found in estuarine and coastal waters, but only a small subset of them are capable of causing disease. The assembled team has valuable expertise in next generation sequence and sequence data approaches, Vibrio sp. ecology, and Vibrio sp. pathogenicity. The combination of knowledge permits known virulent (pathogenic) Vibrio samples to be analyzed for specific sets of "DNA motifs" that are related to the capability of the bacteria to cause disease. From a repetitive process of analyzing known virulent and avirulent cells, the project team can identify the DNA motifs, or signatures that can be used to design a DNA test kit. Subsequently, through support from this project the molecular test kits will be developed, optimized, validated, and commercialized.In addition to the technical activities, undergraduates, senior graduate students, and post doctoral research associates as well as research assistant professors will be supported on this project. The will be exposed to a wide range of research and technology transfer activities, including market assessment, field based sampling and study, laboratory sample preparation, culture and molecular based analyses of samples, next generation sequence data annotation and analyses, and kit design and optimization. The project will be synergistic and contribute directly to the Molecular Training Facility run by PI Noble, where water quality professionals come to learn appropriate use of qPCR approaches and interpretation of qPCR data for water quality management. The project also engages the Marine Biotechnologies-Center of Innovation, a group dedicated to advancing the translation of marine science research to commercial application. In addition, Orion Integrated Biosciences is a project partner. Orion Integrated Biosciences is a company dedicated to the advancement of unique and disruptive computational approaches to survey "big data" for sequence and molecular application. This project will directly impact the advancement of marine metagenomics due to the fact that the project team is utilizing a holistic approach to target complex organisms. The project is novel in that the approach moves away from the "single gene target" qPCR design approach, permitting the generation of diagnostic kits that are rapid, and cost-effective that shellfish harvesting, and water quality managers can use to manage precious resources and protect public health.