Massively Multiplexed dsDNA Invasion Arrays

项目摘要 由侵入性微生物血流感染（BSI）引起的败血症是一个重大的医疗负担， 仅在美国每年就有超过170万例。以高死亡率（~20%）为特征，预后 脓毒症患者的病情在没有适当治疗的情况下每小时恶化一次。及时准确病原 识别对于使治疗与感染相匹配至关重要。不幸的是，目前的诊断方法 BSI依赖于血液培养，这是缓慢的（1-3天），在存在先前的抗菌剂的情况下是不可靠的 治疗与此同时，医生必须依靠广谱治疗，这往往错过了感染， 导致并发症，并导致耐药性。需要新的诊断方法， 直接从血液中快速检测和识别传染源，无需培养 为了解决这一未满足的需求，HelixBind开发了一个样本回答过程，可以识别 细菌和真菌的BSIs直接从血液中。利用专有的检测方法， 合成的双链体DNA侵入核酸，该测定提供单个CFU/ml的物种水平细节 灵敏度基于dsDNA侵入的检测（而不是ssDNA杂交）提供单bp特异性， 能够可靠地区分密切相关的物种，并减少由于随机 污染。然而，到目前为止，入侵从未以适合大规模使用的形式进行过演示。 并行检测在这项提案中，HelixBind将首次开发一种DNA入侵阵列， 同时检测和识别数百种病原体。该阵列将被纳入一个样本到- 在台式仪器上操作的应答流体盒。由此产生的测试将为临床医生提供 全面覆盖基本上所有与BSI相关的病原体， 在患者就诊后数小时内感染，能够早期应用适当的抗菌剂， 改善护理，拯救无数生命。 HelixBind先前已经建立了基于液体的侵入能力，以从临床上识别BSI。 样品和在这个应用程序中，我们提出了初步的数据，固相阵列为基础的入侵，允许 大规模多重检测在本提案中，我们将讨论与以下方面相关的生物物理挑战： 创建高度多重dsDNA入侵阵列以及与整合该阵列相关的技术障碍， 阵列到样品到答案的流体装置中。具体目标，每个目标都有可量化的可交付成果， 开发过程中的主要风险和渐进步骤。在第二期工程完成后，我们 将拥有功能齐全的检测试剂盒和适用于生产规模扩大和临床试验的产品。我们 还将开发出一种新的，高度多路复用的检测格式，具有单bp分辨率，可以 适用于需要在密切相关的产品之间进行敏感和特异性区分的广泛应用。 目标的

PROJECT SUMMARY Septicemia, induced by an invasive microbial bloodstream infection (BSI), is a significant healthcare burden, accounting for over 1.7M cases annually just in the US. Characterized by high mortality rates (~20%), prognosis for septic patients deteriorates hourly in the absence of appropriate treatment. Timely and accurate pathogen identification is critical to match treatment to the infection. Unfortunately, current diagnostic approaches for BSIs rely on blood culture, which is slow (1-3 days) and unreliable in the presence of prior antimicrobial treatment. In the meantime, physicians must rely on broad-spectrum treatment, which often misses the infection, leads to complications, and drives antimicrobial resistance. New diagnostic approaches are needed which can quickly detect and identify the infectious agent directly from blood, without cultures To address this unmet need, HelixBind has developed a sample to answer process which can identify bacterial and fungal BSIs directly from blood in 3 hours. Utilizing a proprietary detection approach leveraging synthetic, duplex DNA invading, nucleic acids, the assay provides species level detail at single CFUs/ml sensitivity. dsDNA invasion-based detection (rather than ssDNA hybridization) affords single-bp specificity and the ability to reliably differentiate between closely related species and reduce false-positives due to random contaminations. To date, though, invasion has never been demonstrated in a format appropriate for massively parallel detection. In this proposal, HelixBind will develop, for the first time, a DNA invasion array which can detect and identify hundreds of pathogens simultaneously. This array will be incorporated into a sample-to- answer fluidic cassette operated on a benchtop instrument. The resulting test will provide clinicians with comprehensive coverage of essentially all pathogens associated with BSIs and unequivocal identification of an infection within hours of patient presentation, enabling early application of appropriate antimicrobials, improving care and saving countless lives. HelixBind has previously established the capability of fluid-based invasion to identify BSIs from clinical samples and in this application, we present preliminary data for solid-phase array-based invasion, allowing massively multiplexed detection. In this proposal we will address the biophysical challenges associated with creating a highly multiplexed dsDNA invasion array and the technical hurdles associated with integrating this array into a sample-to-answer fluidic device. Specific Aims, each with quantifiable deliverables, serve to address the key risks and progressive steps in the development process. Upon completion of this Phase II project, we will have a fully functional assay and a product appropriate for manufacturing scale up and clinical testing. We will also have developed a new, highly multiplexed assay format, with single-bp resolution, which can be adapted for a wide range of applications requiring sensitive and specific differentiation among closely related targets.