Portable, Rapid, Multiplexed Flow strip Test for Bacteria

项目摘要 用于病原体检测的具有成本效益的现场测定技术非常缺乏，尽管它们是 这对解决许多全球性挑战至关重要，例如食物和水传播的感染以及疾病爆发。 例如，每年来自E.仅大肠杆菌和沙门氏菌污染就花费了64亿美元 在美国.食源性病原体检测最常见的策略之一是以实验室为基础 单一病原体类型的聚合酶链反应（PCR），需要约24小时，然后进行确认 使用基于培养的方法，至少需要1周。此外，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检测多种 通过在便携式现场检测平台上检测特异性核酸来检测病原体。但 在单个测定中检测多个核酸靶仍然是有问题的，并且将稳健的， 将多重核酸检测分析转化为低成本、快速、现场检测装置是一个重要的 挑战.为此，我们的总体目标是开发一种流动条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靶扩增， 将报告基因并入具有比色检测的单罐便携式平台中， 廉价的监测。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以掺入独特的锌指蛋白（ZFP）结合基序（称为“Ztags”），和 将报告部分引入来自感兴趣的核酸靶标的扩增产物中，以检测所述扩增产物。 存在可行的病原体。扩增产物将使用固定化的ZFP通过特异性ZFP捕获。 在流条平台上进行基于实时检测器的Ztag交互。我们的初步数据 证明了我们可以使用RPA将Ztag和报告基因掺入靶核酸中 方法和检测低至10个拷贝的目标。此外，所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相容。 我们计划通过以下具体目标来实现我们的目标，即（1）（a）设计和优化 使用单步一锅法RPA进行扩增以及Ztag和报告基因掺入，用于检测 致病菌株E. coliO157、E. coli O26和E. coli O121。(b)结合亲和力评价 ZFP和靶向掺入Ztag之间的关系;（2）基于流式条带的多重 DNA/RNA检测平台用于不同的E.（3）（a）鉴定和验证 使用水和食物样品进行完整的分析。(b)流动测试条的长期稳定性评价 平台这项研究是有意义的，因为它将提供一个简单和廉价的多路复用 病原体检测平台，可以实现全球影响，由于减少粮食的经济负担 和水传播疾病，并改善低收入国家获得检测技术的机会， 资源环境。

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.