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

摘要 传染病是全球发病率和死亡率的主要原因，占全世界的29%。 死亡。下一代测序(NGS)是病原体检测、菌株鉴定和药物治疗的有用工具 敏感度测试(以及其他应用)。NGS用于病原体基因组快速分析的首要问题 未加工的病人样本细菌含量通常很低，需要培养数周至数月 在产生足够的微生物负荷之前。然而，养殖在经济和后勤上都是不可持续的。 并提出了其他可能会混淆结果的生物学问题。此外，病原体的浓缩- 特定基因在很大程度上依赖于样本提取效率。使用核酸检测(NAT)和NGS 方法：有效的DNA提取是成功和准确鉴定微生物的关键。 或微生物种群。分析临床和环境样本时DNA提取不佳，包括 对具有弹性的微生物的研究会导致不确定或不准确的诊断结果。对高效率的需求 直接从患者样本中提取难以裂解的微生物的核酸以便于临床可靠 诊断工作流。三角生物科技(Triangle Bio)正在开发一种新颖而专有的 高效、高通量、可重复和无偏见的微生物裂解技术，基于 用于低成本超声设备的空化增强纳米液滴试剂。建议数 纳米液滴优先靶向具有弹性细胞壁的微生物，并提供聚焦的机械剪切 力量。在第一阶段，三角生物公司在DNA提取方面表现出6-100倍和2-5倍的改进 污垢分枝杆菌(结核分枝杆菌[Mtb]的模型)和粪肠球菌(一种革兰氏 阳性细菌)，分别与常用的商业试剂盒进行比较。在第二阶段，该公司将 建立适用于多种感染性病原体的纳米液滴制剂平台 对临床有重大影响。三角生物公司将通过以下三个目标完成这项研究：1)识别 与12个代表性微生物物种和3个临床样本相容的靶向配体候选 矩阵(Y1)，2)验证候选配方的粘合和空化性能并优化工作流程 临床样本基质添加四种目标微生物(Y2-Y3)的条件，以及3)评价 通过展示靶向NGS在诊断耐药结核分枝杆菌(Y3)方面的改进性能，实现了工作流程。 这项技术的成功实施可能会对广泛的应用产生重大影响 需要可靠的微生物裂解技术，包括但不限于用于传染病检测和 传染病病原体的诊断、基于NGS的食品安全检测、临床和环境微生物组 在元基因组分析中弹性微生物可能被低估的研究。

Abstract Infectious diseases are a leading cause of global morbidity and mortality, accounting for 29% of worldwide deaths. Next-generation sequencing (NGS) is a useful tool in pathogen detection, strain identification, and drug susceptibility testing (among other applications). A primary issue for NGS for rapid pathogen genomic analysis is that raw patient samples typically have a low bacterial load, requiring culturing that can take weeks to months before a sufficient microbial load is generated. However, culturing is economically and logistically unsustainable and presents with other biological issues that may confound results. Additionally, enrichment of the pathogen- specific genes is highly dependent on sample extraction efficiency. Using nucleic acid testing (NAT) and NGS methods, efficient DNA extraction is essential for the successful and accurate identification of microorganisms or populations of microbes. Poor DNA extraction when analyzing clinical and environmental samples consisting of resilient microbes leads to inconclusive or inaccurate diagnostic results. There is a need for high-efficiency extraction of nucleic acids from hard-to-lyse microorganisms in direct patient samples to facilitate reliable clinical diagnostic workflows. Triangle Biotechnology (Triangle Bio) is developing a novel and proprietary technology for efficient, high-throughput, reproducible, and unbiased microbial lysis, based on a cavitation-enhancing nanodroplet reagent for use with low-cost sonication devices. The proposed nanodroplets preferentially target to microbes with resilient cell walls and deliver focused mechanical shear forces. In Phase I, Triangle Bio demonstrated a 6-100x and 2-5x improvement in DNA extraction from Mycobacterium smegmatis (a model for Mycobacterium tuberculosis [Mtb]) and Enterococcus faecalis (a Gram- positive bacteria), respectively, compared to commonly used commercial kits. In Phase II, the company will establish a platform of nanodroplet formulations applicable to a wide range of infectious pathogens with significant clinical impact. Triangle Bio will accomplish this research through the following three aims: 1) Identify targeting ligand candidates compatible with 12 representative microbial species and three clinical sample matrices (Y1), 2) Validate binding and cavitation performance of candidate formulations and optimize workflow conditions for clinical sample matrices spiked with four target microbial species (Y2-Y3), and 3) Evaluate workflows by demonstrating improved performance of targeted NGS for diagnosis of drug-resistant Mtb (Y3). Successful implementation of this technology could have significant impacts on a wide range of applications requiring reliable microbial lysis techniques, including but not limited to NGS for infectious disease detection and diagnosis, NGS based food safety testing for infectious pathogens, and clinical and environmental microbiome studies where resilient microbes can be underrepresented in metagenomic analysis.