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

描述(申请人提供)：痰是一种越来越有前途的非侵入性基质，用于诊断和监测肺部疾病患者。肺结核的耐药性是由痰中细菌的培养或分子分析确定的；肺癌细胞可以出现在痰中作为疾病的早期指标；痰中是否有细菌是肺炎中广谱抗生素的排除标准。然而，从痰中提取核酸的巨大挑战--具有特殊的粘弹性、粘蛋白数量和交联度的异质性以及水合水平--是推动肺部疾病临床研究和患者护理的障碍。 这项第一阶段的SBIR包括一种新的痰处理工具的早期工作：一种自动对痰样本执行一系列化学、机械和生化过程的试剂盒，最终从起始样本中包含的细胞中输出分离的DNA和RNA。将开发的痰样本处理方法本身是全新的，同时利用了申请组织开创的两项创新技术：狭缝毛细管阵列流体致动器(SCAFA)和固相萃取。痰样本处理中的一个关键步骤是通过电流通过特殊配方的缓冲液而引起的电解效应对细胞膜的破坏；在这一新的重要研究领域，申请组织是为数不多的领导确定和了解相关影响的小组之一。 该项目的起点是初步工作，这导致制定了一项使痰均质化的基线方案，以降低其机械反应的弹性部分，并将其粘度降低到SCAFA芯片可以轻易操纵的水平。项目工作包括对初始痰均质的几何形状和其他条件的参数空间研究；开发浓缩细胞的过滤/洗脱过程； 并围绕电裂解室的配置、缓冲条件和电输入进行了详细的实验。随后的过程步骤，包括糖原促进的沉淀、洗涤和洗脱，将在第二阶段根据申请组织为不同病原体和宿主DNA/RNA开发的框架而制定。

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.