Massively Multiplexed dsDNA Invasion Arrays

大规模多重 dsDNA 侵袭阵列

• 批准号: 10392969
• 负责人: Alon Singer
• 金额: $ 100万
• 依托单位: HELIXBIND, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-14 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392969
• 关键词: Accounting; Address; Antimicrobial Resistance; Biological Assay; Biophysics; Blood; Budgets; Caring; Cessation of life; Clinical; Clinical Microbiology; Clinical Research; DNA; Data; Detection; Development; Devices; Diagnostic; Ensure; Feedback; Future; Healthcare; Hour; Infection; Infectious Agent; Intervention; Invaded; Liquid substance; Molecular; Nucleic Acid Amplification Tests; Nucleic Acids; Organism; Patient Care; Patients; Performance; Phase; Physicians; Process; Prognosis; Resolution; Risk; Sampling; Savings; Sepsis; Septicemia; Solid; Specificity; Specimen; Techniques; Technology; Test Result; Testing; Time; TimeLine; Validation; Whole Blood; antimicrobial; assay development; base; cost; diagnostic strategy; ds-DNA; experience; improved; instrument; manufacturing scale-up; member; microbial; microorganism; mortality; multiplex assay; multiplex detection; novel; novel diagnostics; optimal treatments; pathogen; research clinical testing; scale up; septic patients; success

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.

项目概要 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 万例病例。特点是死亡率高（~20%），预后差 如果没有适当的治疗，脓毒症患者的病情每小时都会恶化。及时准确的病原 识别对于将治疗与感染相匹配至关重要。不幸的是，目前的诊断方法 BSI 依赖于血培养，该培养速度较慢（1-3 天），并且在先前使用过抗菌药物的情况下不可靠 治疗。与此同时，医生必须依赖广谱治疗，这往往会错过感染， 导致并发症，并增加抗菌素耐药性。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 开发了一个示例回答流程，可以识别 3 小时内直接从血液中检测细菌和真菌 BSI。利用专有的检测方法 合成、双链 DNA 入侵、核酸，该测定提供单 CFU/ml 的物种水平详细信息 敏感性。基于 dsDNA 入侵的检测（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性造成的误报 污染。然而，迄今为止，入侵从未以适合大规模大规模攻击的形式被证明。 并行检测。在该提案中，HelixBind 将首次开发一种 DNA 入侵阵列，该阵列可以 同时检测和识别数百种病原体。该阵列将被纳入样本到 回答在台式仪器上操作的流体盒。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，并明确识别 患者就诊后数小时内感染，从而能够及早应用适当的抗菌药物， 改善护理并拯救无数生命。 HelixBind 之前已经建立了基于液体的侵入从临床中识别 BSI 的能力 样本，在此应用中，我们提供了基于固相阵列的入侵的初步数据，允许 大规模多重检测。在本提案中，我们将解决与以下相关的生物物理挑战： 创建高度复用的 dsDNA 入侵阵列以及与集成此阵列相关的技术障碍 阵列到样品到答案的流体装置中。每个具体目标都有可量化的可交付成果，旨在解决 开发过程中的主要风险和进展步骤。当这个二期项目完成后，我们 将拥有功能齐全的检测方法和适合生产规模扩大和临床测试的产品。我们 还将开发出一种新的、高度多重化的检测格式，具有单碱基对分辨率，可以 适合需要在密切相关的之间进行敏感和特定区分的广泛应用 目标。