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: 10484601
• 负责人: Sandeep Kasoji
• 金额: $ 100万
• 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 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10484601
• 关键词: Accounting; Acinetobacter baumannii; Acoustics; Address; Antibodies; Bacteria; Binding; Biological; Biological Assay; Biotechnology; Blood; California; Candida albicans; Cell Nucleus; Cell Wall; Cell surface; Cells; Cessation of life; Chemicals; Clinical; Communicable Diseases; Communities; Cytolysis; DNA; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostic Sensitivity; Drug resistance; Drug resistant Mycobacteria Tuberculosis; Enterococcus faecalis; Fluorometry; Food Safety; Formulation; Foundations; Genes; Genus Mycobacterium; Gram-Positive Bacteria; Health; Human; Lead; Lectin; Ligand Binding; Ligands; Logistics; Measures; Mechanics; Metagenomics; Methodology; Methods; Microbe; Modeling; Molecular Analysis; Molecular Diagnosis; Morbidity - disease rate; Mycobacterium smegmatis; Mycobacterium tuberculosis; Nucleic Acid Amplification Tests; Nucleic Acids; Pathogen detection; Patients; Peptides; Performance; Pharmaceutical Preparations; Phase; Polymerase Chain Reaction; Population; Predisposition; Protocols documentation; Public Health; Reagent; Reproducibility; Research; Resistance; Sampling; Sonication; Sputum; Techniques; Technology; Testing; Thick; Tuberculosis diagnosis; Universities; Urine; base; clinical diagnostics; clinically relevant; cost; design; diagnostic platform; direct application; drug testing; improved; in-vitro diagnostics; innovation; mechanical force; microbial; microbiome; microbiome research; microorganism; mortality; nanoDroplet; next generation sequencing; novel; novel diagnostics; pathogen; pathogen genomics; preservation; safety testing; screening; statistics; technology validation; tool

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.

抽象的 传染病是全球发病率和死亡率的主要原因，占全球发病率和死亡率的 29% 死亡人数。下一代测序 (NGS) 是病原体检测、菌株鉴定和药物检测的有用工具 敏感性测试（以及其他应用）。 NGS 快速病原体基因组分析的主要问题 原始患者样本的细菌含量通常较低，需要进行数周至数月的培养 在产生足够的微生物负荷之前。然而，养殖在经济和后勤上是不可持续的 并提出了可能混淆结果的其他生物学问题。此外，病原体的富集- 特定基因高度依赖于样品提取效率。使用核酸检测 (NAT) 和 NGS 方法，高效的 DNA 提取对于成功、准确地鉴定微生物至关重要 或微生物种群。分析临床和环境样本时 DNA 提取效果不佳 具有弹性的微生物会导致不确定或不准确的诊断结果。需要高效率 从直接患者样本中难以裂解的微生物中提取核酸，以促进可靠的临床 诊断工作流程。三角生物技术（Triangle Bio）正在开发一种新颖且专有的 高效、高通量、可重复且公正的微生物裂解技术，基于 空化增强纳米液滴试剂，用于低成本超声处理设备。拟议的 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 力量。在第一阶段，Triangle Bio 展示了从 DNA 中提取 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（革兰氏杆菌） 阳性细菌），分别与常用的商业试剂盒进行比较。在第二阶段，公司将 建立适用于多种传染性病原体的纳米液滴制剂平台 显着的临床影响。 Triangle Bio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 种临床样本兼容的靶向候选配体 矩阵 (Y1), 2) 验证候选配方的结合和空化性能并优化工作流程 添加四种目标微生物物种 (Y2-Y3) 的临床样品基质的条件，以及 3) 评估 通过展示用于诊断耐药 Mtb (Y3) 的靶向 NGS 的改进性能，改进了工作流程。 该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 和 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 研究表明，在宏基因组分析中，弹性微生物的代表性可能不足。