Portable, Rapid, Multiplexed Flow strip Test for Bacteria

便携式、快速、多重流式细菌测试

• 批准号: 9912174
• 负责人: Sylvia Daunert
• 金额: $ 30.32万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9912174
• 关键词: Address; Affinity; American; Bacteria; Base Pairing; Binding; Binding Proteins; Biological Assay; Cells; Cellular Phone; Centers for Disease Control and Prevention (U.S.); Cessation of life; Cytolysis; DNA; DNA Sequence; DNA copy number; DNA-Binding Proteins; Data; Detection; Development; Devices; Diagnosis; Disease Outbreaks; Economic Burden; Environment; Escherichia coli; Escherichia coli O157; Evaluation; Eye; Food; Goals; Health; Hospitalization; Human; Image; Immobilization; Improve Access; Infrastructure; Laboratories; Lateral; Methods; Monitor; Nucleic Acids; Pathogen detection; Pathogenicity; Polymerase; Polymerase Chain Reaction; Preparation; Protein Binding Domain; Proteins; RNA; RNA amplification; RNA-Directed DNA Polymerase; Reagent; Reporter; Research; Resources; Salmonella; Sampling; Screening procedure; Signal Transduction; Site; Surface; Techniques; Technology; Testing; Time; Tube; Validation; Visualization; Water; Work; Zinc Fingers; amplification detection; assay development; base; cost; cost effective; design; detector; ds-DNA; foodborne; foodborne illness; foodborne pathogen; improved; innovation; interest; multiplex detection; nucleic acid detection; pathogen; point of care; portability; recombinase; success; waterborne; waterborne illness; waterborne infection

PROJECT SUMMARY Cost-effective on-site assay technologies for pathogen detection are sorely lacking, even though they are essential in solving many global challenges such as food and water borne infections and disease outbreaks. For example, each year foodborne diseases from E. coli and salmonella contamination alone cost $6.4 billion in the US. One of the most common strategies for foodborne pathogen detection is laboratory-based polymerase chain reaction (PCR) of a single pathogen type that takes about 24 h followed by confirmation using culture-based methods that take a minimum of 1 week. Additionally, a majority of techniques reli only on DNA whereas RNA is a better indicator of viable bacteria. Ideal technology should target detection of multiple pathogens through the detection of specific nucleic acid in a portable on-site detection platform. However, the detection of multiple nucleic acid targets in a single assay is still problematic, and transitioning a robust, multiplexed nucleic acid detection assay into a low-cost, rapid, on-site detection device is a significant challenge. To that end, our overall goal is to develop a flow-strip multiplex pathogen detection platform that will fulfill the following criteria: simple sample preparation, rapid multiplexed DNA/RNA target amplification and incorporation of reporter in a single pot, portable platform with colorimetric detection for on-site and inexpensive monitoring. Here, we propose to use recombinase polymerase amplification (RPA) and reverse transcriptase-RPA to incorporate unique zinc-finger protein (ZFP) binding motifs (referred to as “Ztags”) and reporter moieties into the amplification product from nucleic acid targets of interest in order to detect the presence of viable pathogen. The amplified product will be captured using immobilized ZFPs via specific ZFP- Ztag interaction for real-time reporter-based detection on a flow-strip platform. Our preliminary data demonstrated that we can incorporate both Ztag and reporter into the target nucleic acid using the RPA method and detect as low as 10 copies of target. Additionally, the proposed method does not need any extraction step as our preliminary data indicates that the RPA method is compatible with the cell lysis reagent. We plan to achieve our goal by pursuing the following specific aims, namely, (1)(a) Design and optimization of the amplification as well as Ztag and reporter incorporation using single-step, one-pot RPA for the detection of the pathogenic strains E. coli O157, E. coli O26, and E. coli O121. (b) Evaluation of the binding affinity between ZFP and target-incorporated Ztag; (2) Design and development of a flow-strip-based, multiplex DNA/RNA detection platform for the different E. coli strains; (3)(a) Characterization and validation of the complete assay using water and food samples. (b) Evaluation of the long-term stability of the flow strip platform. The proposed research is significant because it will provide a simple and inexpensive multiplex pathogen detection platform that could achieve a global impact due to reducing the economic burden of food and water borne diseases in the developed world and improving access to detection technologies in low- resource environments.

项目概要 非常缺乏具有成本效益的病原体检测现场检测技术，尽管它们是 对于解决食品和水源感染以及疾病爆发等许多全球挑战至关重要。 例如，每年仅由大肠杆菌和沙门氏菌污染引起的食源性疾病就造成 64 亿美元的损失 在美国，最常见的食源性病原体检测策略之一是基于实验室。 单一病原体类型的聚合酶链反应 (PCR) 大约需要 24 小时，然后进行确认 使用基于培养的方法至少需要 1 周的时间，大多数技术仅依赖于。 DNA 而 RNA 是活细菌的更好指示剂，理想的技术应该针对多种细菌的检测。 通过在便携式现场检测平台中检测特定核酸来检测病原体。 在一次检测中检测多个核酸靶标仍然存在问题，并且需要转变一种稳健的、 将多重核酸检测分析转化为低成本、快速的现场检测装置是一项重要的研究 为此，我们的总体目标是开发一种流式多重病原体检测平台。 满足以下标准：简单的样品制备、快速多重 DNA/RNA 靶标扩增以及 将报告器整合到一个单罐便携式平台中，并具有用于现场和现场比色检测的功能 在这里，我们建议使用重组酶聚合酶扩增（RPA）和反向检测。 转录酶-RPA 整合独特的锌指蛋白 (ZFP) 结合基序（称为“Ztag”）和 将报告基团插入感兴趣的核酸靶标的扩增产物中，以检测 存在活病原体时，将使用固定化 ZFP 通过特定的 ZFP- 捕获扩增产物。 Ztag 交互用于在 Flow-Strip 平台上进行基于报告的实时检测。 证明我们可以使用 RPA 将 Ztag 和报告基因整合到目标核酸中 方法并检测低至 10 个目标副本此外，所提出的方法不需要任何目标。 提取步骤，因为我们的初步数据表明 RPA 方法与细胞裂解试剂兼容。 我们计划通过追求以下具体目标来实现我们的目标，即（1）（a）设计和优化 使用单步、一锅 RPA 进行扩增以及 Ztag 和报告基因掺入，以检测 致病菌株大肠杆菌 O157、大肠杆菌 O26 和大肠杆菌 O121 (b) 结合亲和力评估。 ZFP 和 Target-incorporated Ztag 之间的研究；（2）基于 Flow-Strip 的多重设计和开发 不同大肠杆菌菌株的 DNA/RNA 检测平台；(3)(a) 表征和验证 使用水和食品样品进行完整测定 (b) 流动条的长期稳定性评估。 所提出的研究意义重大，因为它将提供一个简单且廉价的多路复用。 病原体检测平台可因减轻食品经济负担而产生全球影响 和发达国家的水传播疾病，并改善低收入国家获得检测技术的机会 资源环境。