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

摘要 传染病是全球发病和死亡的主要原因，占全球发病和死亡的29 死亡下一代测序（NGS）是病原体检测、菌株鉴定和药物治疗中的有用工具。 敏感性测试（以及其他应用）。NGS用于快速病原体基因组分析的主要问题 原始患者样本通常具有较低的细菌载量，需要花费数周至数月的培养 在产生足够的微生物负载之前。然而，养殖在经济上和后勤上都是不可持续的 并存在可能混淆结果的其他生物学问题。另外，病原体的富集- 特异性基因高度依赖于样品提取效率。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 或微生物的种群。分析临床和环境样本时DNA提取不佳， 导致不确定或不准确的诊断结果。需要高效率 从直接患者样品中的难以裂解的微生物中提取核酸， 诊断工作流程。三角生物技术（三角生物）正在开发一种新的和专有的 一种高效、高通量、可重复和无偏见的微生物裂解技术， 用于低成本声处理装置的空化增强纳米液滴试剂。拟议 纳米液滴优先靶向具有弹性细胞壁的微生物， 力.在第一阶段，Triangle Bio在DNA提取方面表现出6- 100倍和2- 5倍的改进， 耻垢分枝杆菌（结核分枝杆菌[Mtb]的模型）和粪肠球菌（革兰氏阴性杆菌） 阳性细菌）与常用的商业试剂盒相比。在第二阶段，公司将 建立适用于各种传染性病原体的纳米液滴制剂平台， 重大临床影响。三角生物将通过以下三个目标来完成这项研究：1）识别 与12种代表性微生物物种和3种临床样品相容的靶向配体候选物 基质（Y1），2）考察候选制剂的结合和空化性能并优化工作流程 加标四种目标微生物菌种（Y2-Y3）的临床样品基质的条件，以及3）评价 通过证明靶向NGS用于诊断耐药Mtb（Y3）的改进性能， 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的NGS， 诊断，基于NGS的传染性病原体食品安全检测，以及临床和环境微生物组 在宏基因组分析中，弹性微生物可能代表性不足的研究。

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.