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 传染病夺走了29％的世界人口的生命，超过1500万人死于 每年细菌和真菌疾病的结果。及时，准确的诊断可以减少衰弱和 挽救生命，生物医学样品的核酸测试（NAT）是一种识别的有力方法 可以返回的微生物仅几个小时（或更快）。但是，基于NAT的诊断有 一再无法取代传统测试，可能需要几天（或更多）才能提供确定的诊断。 诊断测定需要符合敏感性，特异性和可重复性的严格标准；对于nat- 测试，有效提取高质量核酸的第一步至关重要。不幸的是，威胁生命 血液感染和结核性脑膜炎等疾病通常会产生临床样本 每毫升的病原体浓度低至1个菌落形成单位。值得注意的是，结核分枝杆菌， 引起结核病的有机体很难裂解。实际上，许多革兰氏阳性细菌，病毒和真菌 很难使用常规方法打破敞开，大约一半的血液患者 感染，因果微生物属于其中一种类别之一。使用的物理和机械方法 为提高微生物裂解效率包括珠子拍，高压均质化，直接或间接 超声处理和冻融/沸腾。这些技术通常仅实现中等的成功目的地 他们对专业且昂贵的设备或材料的需求，使其不适合常规点 护理诊断。间接社会 - 通过启动显微镜的形成和崩溃来起作用 气泡（一种称为空化的过程） - 与替代性裂解方法相比，有多种好处 降低成本和污染，易用性以及可延伸性的可移植性。尽管如此，还是其他 方法，间接超声处理努力从弹性中获得一致的高产量的核酸产量 微生物而不牺牲质量。三角生物技术正在开发新颖和专有超声处理 通过降低声能来大大提高核酸提取效率的试剂 空化所必需的。 I阶段I SBIR将为我们的试剂辅助超声处理建立概念概念 通过确定最大化微生物裂解效率的条件（AIM 1）并证明改善 与传统方法相比，使用我们的方法提取的DNA结果（AIM 2）。非 - 致病靶标微生物，史氏菌，粪肠球菌和枯草芽孢杆菌是常用的模型 致病性分枝杆菌，革兰氏阳性细菌和变质细菌，威胁到公共卫生和 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建立高灵敏度（检测低限制）来建立高灵敏度（检测低极限） 方法。 Triangle的长期目标是以套件的形式生产技术（试剂，缓冲区） 并将平台与小型和便携式社会设备的新兴应用程序集成 使用该套件与现有低成本超声器的优化协议的生成。