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

摘要 传染病夺走了世界29%人口的生命，1500多万人死于 每年都会因细菌和真菌疾病而死亡。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测试(NAT)是一种有效的识别方法 微生物可以在几个小时内(或更快)得出结果。然而，基于NAT的诊断具有 一次次未能取代需要几天(或更长时间)才能得出明确诊断的传统测试。 诊断分析需要满足敏感度、特异度和重复性的严格标准；对于NAT- 在测试中，高效提取高质量核酸的第一步是至关重要的。不幸的是，有生命危险 血液感染和结核性脑膜炎等疾病通常会产生临床样本 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 引起结核病的有机体是出了名的难以分解。事实上，许多革兰氏阳性细菌、病毒和真菌 很难用传统方法打开，在大约一半的有血流的患者中 感染，致病微生物属于这些类别之一。使用物理和机械方法 提高微生物裂解效率的方法包括打珠、高压均质、直接或间接 超声化和冻融/煮沸。这些技术通常只取得一定的成功，尽管 他们需要专门而昂贵的设备或材料，使他们不适合常规的接入点 护理诊断。间接超声化--它通过启动微观结构的形成和崩塌而起作用 气泡(一种称为空化的过程)-与其他裂解方法相比，提供了多种好处，包括 降低了成本和污染，易于使用，并且可扩展以实现可移植性。尽管如此，就像其他 方法，间接超声努力从有弹性的核酸中获得持续的高产量 在不牺牲质量的情况下培养微生物。三角生物科技正在开发新的和专有的超声波 通过降低声能大幅提高核酸提取效率的试剂 空化所需的。这一阶段的SBIR将为我们的试剂辅助超声建立概念验证 通过确定最大限度地提高微生物裂解效率的条件(目标1)并展示改进的技术 与传统方法(目标2)相比，使用我们的方法提取的DNA的NAT结果。非-- 致病目标微生物，污垢分枝杆菌，粪肠球菌和枯草杆菌是常用的模型。 危害公众健康的致病分枝杆菌、革兰氏阳性细菌和芽胞形成细菌 安全。本项目收集的数据将为验证我们的平台的第二阶段研究提供基础 通过使用NAT建立对多种弹性微生物的高灵敏度(低检测下限)的技术 方法：研究方法。三角科技的长期目标是以成套设备(试剂、缓冲剂)的形式生产这项技术。 并将该平台与小型和便携式声学设备的新兴应用程序集成在一起，从 生成优化的协议，以便与现有的低成本声化器一起使用该套件。

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.