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）：痰是一种越来越有前景的无创基质，用于诊断和监测肺部疾病患者。通过痰中细菌培养或分子分析来确定结核耐药性；肺癌细胞可能出现在痰中，作为疾病的早期指标；痰中没有细菌是肺炎广谱抗生素的排除标准。然而，从痰液中提取核酸所面临的巨大挑战——因其特殊的粘弹性、粘蛋白含量和交联的异质性以及水合水平——成为肺部疾病临床研究和患者护理进展的障碍。 第一阶段 SBIR 包含了一种新型痰液处理工具的早期工作：一种能够对痰液样本自动执行一系列化学、机械和生化过程的盒，最终从起始样本中包含的细胞中分离出 DNA 和 RNA。待开发的痰样本处理方法本身是全新的，同时利用了申请人组织首创的两项创新技术：狭缝毛细管阵列流体致动器（SCAFA）和固相萃取。痰样本处理的一个关键步骤是通过电流通过特殊配制的缓冲液而引起的电解效应破坏细胞膜；在这个新的突出研究领域，申请组织是领导表征和理解相关影响的少数团体之一。 该项目的起点是前期工作，该工作导致开发了一个基线协议，用于均质化痰液，以降低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。项目工作包括几何参数空间研究和初始痰均质化的其他条件；开发浓缩细胞的过滤/洗脱工艺； 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，包括糖原促进沉淀、洗涤和洗脱，将在第二阶段开发，基于申请人组织针对不同病原体和宿主 DNA/RNA 开发的框架。