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

这个PFI：空气技术翻译项目的重点是翻译以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，在发展中国家令人担忧。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发现的，但当它们被生贝类食用时，或者在罕见的情况下，与海滩水域接触时，也会对人类健康构成风险。该项目将把从先前的生态学研究中获得的知识转化为允许设计用户友好的、快速的分子检测试剂盒的技术。现有的检测水和贝类样本中弧菌物种的方法已经有几十年的历史，需要24-96小时才能得出结果。即使是检测创伤弧菌和副溶血性弧菌致病类型的新的分子方法也受到缺乏特异性和/或需要通过浓缩步骤来提高灵敏度的影响，这使得它们几乎需要一天的时间才能完成。该项目将使用一种新的方法为试剂盒确定有用的靶标，从而产生新的、快速的创伤弧菌和副溶血性弧菌毒力类型的分子诊断方法。据设想，这些试剂盒可用于1)证明生食用贝类是安全的，2)保护接触水的高危人群的健康，3)确定与危险形式的细菌有关的模式，以改进预警系统。资助的项目将导致设计出易于使用的商业化分子检测试剂盒，并将在2-3小时内提供结果。这些功能将允许准确的公共通知和公共健康保护。该项目解决了从研究向商业应用过渡过程中的一个具体知识缺口。弧菌属是一种复杂的细菌体，自然存在于河口和沿海水域，但只有一小部分能够致病。组建的团队在新一代序列和序列数据方法弧菌方面拥有宝贵的专业知识。生态学和弧菌属(Vibrio sp.)致病性。这些知识的结合使人们能够分析已知的毒力(致病)弧菌样本，以确定与细菌致病能力有关的特定“DNA基序”。通过分析已知的毒力和无毒细胞的重复过程，项目团队可以识别DNA基序或签名，这些DNA基序或签名可用于设计DNA检测试剂盒。随后，通过该项目的支持，分子测试试剂盒将被开发、优化、验证和商业化。除了技术活动，该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。该中心将参与广泛的研究和技术转让活动，包括市场评估、现场采样和研究、实验室样品准备、样品培养和基于分子的分析、下一代序列数据注释和分析，以及试剂盒设计和优化。该项目将发挥协同作用，并直接为Pi Noble运营的分子培训设施做出贡献，在那里，水质专业人员将学习如何正确使用定量聚合酶链式反应方法，并解释用于水质管理的定量聚合酶链式反应数据。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，猎户座集成生物科学公司是项目合作伙伴。猎户座集成生物科学公司是一家致力于发展独特和颠覆性计算方法的公司，以调查序列和分子应用的“大数据”。该项目将直接影响海洋元基因组学的进展，因为项目团队正在利用一种整体的方法来瞄准复杂的生物体。该项目的新颖之处在于，该方法摒弃了“单基因靶标”的定量聚合酶链式反应设计方法，允许产生快速、成本效益高的诊断试剂盒，供贝类捕捞和水质管理人员用来管理宝贵的资源和保护公众健康。

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.