Automated POC Extraction of Sputum-Borne Bacterial Nucleic Acids In the Clinic

临床上自动 POC 提取痰传播细菌核酸

• 批准号: 8758757
• 负责人: Amy Droitcour
• 金额: $ 20.72万
• 依托单位: WAVE 80 BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8758757
• 关键词: Antibiotics; Area; Bacteria; Bacterial DNA; Bacterial Infections; Bacterial RNA; Biochemical Process; Biological Assay; Blood capillaries; Buffers; Cell Wall; Cell membrane; Cells; Chemicals; Clinic; Clinical Research; Collection; Consumption; Cytolysis; DNA; Development; Devices; Diagnosis; Disease; Drug Resistant Tuberculosis; Electronics; Engineering; Filtration; Genetic Materials; Geometry; Glycogen; HIV; Health; Heterogeneity; Hour; Hydration status; Hydroxide Ion; Individual; Infection; Intervention; Lead; Life; Liquid substance; Lung; Lung diseases; Malignant neoplasm of lung; Mechanics; Methods; Microfluidics; Mitochondrial DNA; Mitochondrial RNA; Molecular Analysis; Moraxella (Branhamella) catarrhalis; Mucins; Mucous body substance; Mycobacterium tuberculosis; Nucleic Acid Amplification Tests; Nucleic Acids; Outcome; Output; Patient Care; Patient Monitoring; Patients; Phase; Pneumonia; Precipitation; Preparation; Process; Protocols documentation; Pulsatile Flow; RNA; Reagent; Recovery; Research; Research Personnel; Resources; Respiratory System; Respiratory tract structure; Running; Rupture; Sampling; Series; Small Business Innovation Research Grant; Solid; Solutions; Sputum; Staphylococcus aureus; System; Technology; Validation; Viral Load result; Viscosity; Work; base; cancer cell; cancer risk; capillary; cost; crosslink; design; improved; innovation; innovative technologies; instrument; manufacturing process; meter; novel; operation; pathogen; public health relevance; respiratory; response; tool; usability; viscoelasticity

DESCRIPTION (provided by applicant): Sputum is an increasingly promising noninvasive matrix for diagnosing and monitoring patients with lung conditions. Tuberculosis drug resistance is determined by culture or molecular analysis of bacteria in sputum; lung cancer cells can appear in sputum as an early indicator of disease; absence of bacteria in sputum is a rule-out criterion for broad-spectrum antibiotics in pneumonia. Yet the enormous challenge of extracting nucleic acids from sputum-with its exceptional viscoelasticity, heterogeneity of mucin amounts and cross-linking, and hydration levels-is a barrier to advances in lung disease clinical research and patient care. This Phase I SBIR encompasses early work on a novel tool for sputum processing: a cartridge that automatically carries out a sequence of chemical, mechanical, and biochemical processes on a sputum sample, culminating in the output of isolated DNA and RNA from the cells contained in the starting sample. The sputum sample processing method to be developed is itself wholly novel, while leveraging two innovative technologies pioneered by the Applicant Organization: slit capillary array fluidic actuators (SCAFAs) and solid- phase extraction. A key step in the sputum sample processing is the disruption of cell membranes by electrolytic effects induced through the passage of electrical current through a specially formulated buffer; in this newly prominent area of research, the Applicant Organization is one of a small number of groups leading the effort to characterize and understand the relevant effects. The starting point for the project is preliminary work which has led to the development of a baseline protocol for homogenizing sputum to degrade the elastic component of its mechanical response and reduce its viscosity to a level where it can be readily manipulated by SCAFA chips. Project work encompasses a parameter space study of geometry and other conditions for initial sputum homogenization; development of a filtration/elution process for concentrating cells; and detailed experimentation around electrical lysis chamber configuration, buffer conditions, and electrical input. The subsequent process steps, including glycogen-facilitated precipitation, washing, and elution, will be developed in Phase II, based on a framework developed by the Applicant Organization for diverse pathogen and host DNA/RNA.

描述（由申请人提供）：痰液是一种越来越有希望的无创矩阵，用于诊断和监测患有肺部疾病的患者。结核病耐药性是通过细菌中细菌的培养或分子分析来确定的。肺癌细胞可以作为疾病的早期指标出现。痰中缺乏细菌是肺炎广谱抗生素的规则标准。然而，从痰中提取核酸的巨大挑战，其特殊的粘弹性，粘蛋白量的异质性和交联的异质性以及水分水平 - 是肺部疾病临床研究和患者护理进展的障碍。 该阶段I SBIR涵盖了用于痰液加工的新工具上的早期工作：一种自动在痰液样品上自动执行化学，机械和生化过程的墨盒，最终导致了从开始样品中包含的细胞的分离DNA和RNA的输出。要开发的痰液样品处理方法本身是完全新颖的，同时利用申请人组织开创的两种创新技术：缝隙毛细管阵列流体液体执行器（SCAFA）和固相提取。痰样品处理的关键步骤是通过通过特殊配方的缓冲液通过电流通过电流引起的电解效应破坏细胞膜。在这个新的研究领域，申请人组织是少数小组之一，努力表征和理解相关效果。 该项目的起点是初步工作，这导致开发了基线方案，用于使痰液均质降解其机械响应的弹性成分，并将其粘度降低到可以通过SCAFA芯片轻松操纵的水平。项目工作涵盖了几何学和其他条件的参数空间研究，用于初始痰液均质化；开发用于浓缩细胞的过滤/洗脱过程； 以及围绕电裂解室配置，缓冲条件和电输入的详细实验。随后的工艺步骤，包括糖原相关的降水，洗涤和洗脱，将基于申请人组织针对多种病原体和宿主DNA/RNA开发的框架，在第二阶段中开发。