Portable, Rapid, Multiplexed Flow strip Test for Bacteria

便携式、快速、多重流式细菌测试

• 批准号: 9751325
• 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/9751325
• 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; Imagery; 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; 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.

项目摘要 具有成本效益的现场测定技术可用于病原体检测，即使它们是 解决许多全球挑战至关重要的，例如食物和水传播感染和疾病暴发。 例如，仅大肠杆菌和沙门氏菌污染的粮食生存疾病每年耗资64亿美元 在美国。食源性病原体检测最常见的策略之一是基于实验室 单个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 使用至少需要1周的基于培养的方法。此外，大多数技术仅依赖于 DNA，而RNA是可行细菌的更好指标。理想的技术应针对多个检测 通过在便携式现场检测平台中检测特异性核酸检测病原体。但是， 在单个测定中检测多个核酸靶标仍然是有问题的，并且过渡了强大的， 多路复用的核酸检测测定法进入低成本，快速的现场检测装置是重要的 挑战。为此，我们的总体目标是开发一个流动多重病原体检测平台，该平台将 符合以下标准：简单样品制备，快速的多路复用DNA/RNA靶标放大和 将记者纳入单个锅，便携式平台，并带有比色检测的现场检测 廉价的监视。在这里，我们建议使用重组酶聚合酶扩增（RPA）和反向 转录酶-RPA以结合独特的锌指蛋白（ZFP）结合基序（称为“ Ztags”）和 记者部分从感兴趣的核酸靶标进入放大产物，以检测 存在生存的病原体。放大产品将通过特定的ZFP-使用固定的ZFP捕获 基于实时记者的检测在流条纹平台上的ZTAG相互作用。我们的初步数据 证明我们可以使用RPA将ZTAG和记者同时纳入目标核酸 方法并检测到目标低至10份目标。此外，提出的方法不需要任何 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特定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进行的放大和ZTAG和记者合并用于检测 致病菌株大肠杆菌O157，大肠杆菌O26和大肠杆菌O121。 （b）评估结合亲和力 在ZFP和目标ZTAG之间； （2）基于流条的多重设计和开发 不同大肠杆菌菌株的DNA/RNA检测平台； （3）（a）表征和验证 使用水和食物样品完成测定。 （b）评估流条的长期稳定性 平台。拟议的研究很重要，因为它将提供简单且廉价的多路复用 病原体检测平台，由于降低了食物的经济伯恩，可以实现全球影响 以及发达国家中的水传播疾病，并改善了低 - 低 - 资源环境。