Improving diagnostic sensitivity for difficult-to-lyse microbial samples with nanodroplet technology

利用纳米液滴技术提高难以裂解的微生物样品的诊断灵敏度

• 批准号: 10081308
• 负责人: Sandeep Kasoji
• 金额: $ 25.21万
• 依托单位: TRIANGLE BIOTECHNOLOGY, INC.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-03 至 2022-05-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10081308
• 关键词: Acoustics; Address; Adoption; Affect; Bacillus subtilis; Bacteria; Bacterial DNA; Base Pairing; Biological; Biological Sciences; Biotechnology; Buffers; Capillary Electrophoresis; Categories; Cell Wall; Cells; Chemicals; Clinical; Clinical Research; Colony-forming units; Communicable Diseases; Cytolysis; DNA; DNA Fragmentation; Data; Detection; Devices; Diagnosis; Diagnostic; Diagnostic Sensitivity; Diagnostics Research; Disease; Effectiveness; Enterococcus faecalis; Equipment; Fluorometry; Formulation; Foundations; Freezing; Generations; Genes; Genus Mycobacterium; Gram-Positive Bacteria; Hour; Human; Lead; Life; Measures; Mechanics; Medical; Meningeal Tuberculosis; Methodology; Methods; Microbe; Microscopic; Modeling; Molecular Target; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleic Acid Amplification Tests; Nucleic Acids; Organism; Pathogenicity; Patients; Persons; Phase; Population; Predisposition; Process; Protocols documentation; Public Health; Reagent; Recombinant DNA; Reproducibility; Reproduction spores; Resistance; Safety; Sampling; Sensitivity and Specificity; Sepsis; Small Business Innovation Research Grant; Sonication; Techniques; Technology; Test Result; Testing; Thick; Time; Vagina; Virus; Work; Yeasts; accurate diagnosis; bacterial community; base; cost; diagnostic accuracy; diagnostic assay; falls; fungus; improved; innovation; microbial; microbial community; microbiome; microbiota; microorganism; milliliter; nanoDroplet; next generation sequencing; novel; pathogen; phase 2 study; point-of-care diagnostics; portability; preservation; pressure; success; therapeutic development

Abstract Communicable diseases claim the lives of 29% of world's population and more than 15 million persons die as a result of bacterial and fungal diseases every year. Timely and accurate diagnosis can reduce debilitation and save lives, and nucleic acid testing (NAT) of biomedical samples is a powerful method for identifying microorganisms that can return results in just a few hours (or faster). However, NAT-based diagnostics have repeatedly failed to displace traditional tests that can take several days (or more) to deliver a definitive diagnosis. Diagnostic assays need to meet exacting standards of sensitivity, specificity, and repeatability; and for NAT- tests, the initial step of efficiently extracting high-quality nucleic acids is critical. Unfortunately, life-threatening diseases such as bloodstream infections and tuberculous meningitis often produce clinical samples with pathogen concentrations as low as 1 colony-forming unit per milliliter. Notably, Mycobacterium tuberculosis, the organism that causes TB, is notoriously difficult to lyse. In fact, many gram-positive bacteria, viruses and fungi are hard to break open using conventional approaches and, in roughly half of patients with bloodstream infections, the causative microbe falls into one of these categories. Physical and mechanical approaches used to improve microbial lysis efficiencies include bead beating, high-pressure homogenization, direct or indirect sonication, and freeze-thawing/boiling. These techniques commonly achieve only moderate success despite their need for specialized and expensive equipment or materials, making them unsuitable for routine point-of- care diagnostics. Indirect sonication – which works by initiating the formation and collapse of microscopic bubbles (a process called cavitation) – offers multiple benefits compared to alternative lysis methods including reduced cost and contamination, ease of use, and being scalable for portability. Nonetheless, like other approaches, indirect sonication struggles to achieve consistently high yields of nucleic acids from resilient microbes without sacrificing quality. Triangle Biotechnology is developing novel and proprietary sonication reagents that substantially improve the efficiency of nucleic acid extraction by reducing the acoustic energy required for cavitation. This Phase I SBIR will establish a proof-of-concept for our reagent-assisted sonication technology by identifying conditions that maximize microbial lysis efficiency (Aim 1) and demonstrating improved NAT results from DNA extracted using our approach compared to traditional methods (Aim 2). The non- pathogenic target microorganisms, M. smegmatis, E. faecalis, and B. subtilis are commonly used models for pathogenic mycobacteria, Gram-positive bacteria, and spore-forming bacteria that threaten public health and safety. The data collected in this project will provide the foundation for Phase II studies to validate our platform technology by establishing high sensitivity (low limits of detection) for multiple resilient microbes using NAT methods. Triangle's long-term objective is to productize the technology in the form of a kit (reagents, buffers) and integrate the platform with emerging applications for small and portable sonication devices, beginning with the generation of optimized protocols for use of the kit with existing, low-cost sonicators.

抽象的 传染病夺去了世界 29% 人口的生命，超过 1500 万人因传染病而死亡 每年都有细菌和真菌疾病的结果。及时准确的诊断可以减少虚弱和 拯救生命，生物医学样本的核酸检测 (NAT) 是一种强有力的识别方法 微生物可以在短短几个小时（或更快）内返回结果。然而，基于 NAT 的诊断 多次未能取代传统测试，传统测试可能需要几天（或更长时间）才能得出明确的诊断。 诊断分析需要满足严格的灵敏度、特异性和可重复性标准；对于 NAT- 测试中，高效提取高质量核酸的第一步至关重要。不幸的是，有生命危险 血流感染和结核性脑膜炎等疾病通常会产生具有以下特征的临床样本： 病原体浓度低至每毫升 1 个菌落形成单位。值得注意的是，结核分枝杆菌 众所周知，引起结核病的微生物很难裂解。事实上，许多革兰氏阳性细菌、病毒和真菌 使用传统方法很难打开，并且大约一半的患者有血流 感染时，致病微生物属于其中一类。使用的物理和机械方法 提高微生物裂解效率的方法包括珠打浆、高压均质化、直接或间接 超声处理和冻融/煮沸。尽管这些技术通常只取得了一定的成功 他们需要专门且昂贵的设备或材料，使他们不适合常规点 护理诊断。间接超声处理——通过启动微观结构的形成和崩溃来起作用 气泡（称为空化的过程）——与其他裂解方法相比具有多种优势，包括 降低成本和污染，易于使用，并且可扩展以实现便携性。尽管如此，和其他人一样 方法中，间接超声处理很难从弹性体中获得持续高的核酸产量。 微生物而不牺牲质量。 Triangle Biotechnology 正在开发新颖且专有的超声处理 通过降低声能大幅提高核酸提取效率的试剂 空化所需的。第一阶段 SBIR 将为我们的试剂辅助超声处理建立概念验证 通过确定最大化微生物裂解效率的条件（目标 1）并证明改进的技术 使用我们的方法提取的 DNA 与传统方法相比的 NAT 结果（目标 2）。非 致病性目标微生物，耻垢分枝杆菌、粪肠球菌和枯草芽孢杆菌是常用的模型 威胁公众健康的致病性分枝杆菌、革兰氏阳性菌和孢子形成菌 安全。该项目收集的数据将为第二阶段研究提供基础，以验证我们的平台 通过使用 NAT 为多种弹性微生物建立高灵敏度（低检测限）的技术 方法。 Triangle 的长期目标是以套件（试剂、缓冲液）的形式将该技术产品化 并将该平台与小型和便携式超声设备的新兴应用程序集成，首先 生成优化的协议，以便将该套件与现有的低成本超声仪一起使用。