A rapid, automated system for bacteria profiling of intra-abdominal infections

一种快速、自动化的腹内感染细菌分析系统

• 批准号: 10535472
• 负责人: Don L DeVoe
• 金额: $ 58.13万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-28 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10535472
• 关键词: Abdomen; Abdominal Abscess; Abdominal Infection; Abscess; Address; Antibiotics; Appendicitis; Automation; Bacteria; Bacterial Antibiotic Resistance; Biological Assay; Budgets; Cell Culture Techniques; Child; Clinical; Clinical Research; Communication; Computer software; Computers; Coupled; Cytolysis; Deposition; Detection; Diagnosis; Diagnostic; Disease; Diverticulitis; Electrodes; Elements; Enterocolitis; Environment; Evaluation; Film; Fluorescence; Goals; Hospitals; Hour; Infection; Infrastructure; Injury; Intra-abdominal; Investigation; Laboratories; Lead; Liquid substance; Manuals; Membrane; Methods; Microfabrication; Microfluidics; Mucous Membrane; Nucleic Acids; Oils; Operative Surgical Procedures; Organ; Patient-Focused Outcomes; Patients; Performance; Pharmaceutical Preparations; Phenotype; Polymerase; Porosity; Powder dose form; Preparation; Prevalence; Reaction; Reader; Reagent; Reporting; Resolution; Sampling; Sepsis; Signal Transduction; Silicon; Source; Speed; Stroke; System; Techniques; Technology; Temperature; Testing; Thick; Thinness; Time; Tube; Validation; Visual; Work; antimicrobial; antimicrobial drug; aspirate; chromatin immunoprecipitation; clinical practice; clinically significant; cohort; controlled release; cost; detection limit; diagnostic tool; fabrication; fluorescence imaging; improved; laptop; manufacture; meetings; melting; meter; millimeter; molecular diagnostics; mortality; multiplex assay; novel diagnostics; operation; pathogen; prevent; rRNA Genes; repository; seal; standard of care; surfactant; tool

Project Summary New diagnostic tools capable of rapidly identifying and quantifying bacteria within intra-abdominal infections are needed to help clinicians select appropriate antibiotics during the critical early stages of treatment. Current molecular diagnostics remain costly and require access to significant laboratory infrastructure, making them inappropriate for use in small satellite laboratories within the hospital environment. Here we seek to address this urgent need by developing a simple, inexpensive, and automated microfluidic platform capable of evaluating bacteria directly from intra-abdominal abscess fluids to guide initial treatment of infection. The technology will combine on-chip nucleic acid extraction, rapid quantitative PCR (qPCR), and high resolution melt analysis (HRMA) for multiplexed pathogen identification and quantification in under 10 min. The platform will consist of disposable thermoplastic microwell chips and a compact USB-powered reader containing all components for assay operation. Significantly, the entire assay will require only two pipette strokes to discretize sample within a dense array of microliter reaction wells containing all primers and other reagents needed for assay execution. The integration of multiplexed PCR primers into the thermoplastic microwell array during chip manufacture will enable automated thermally-controlled release during PCR, while passive sample discretization within the disposable microwell arrays will further simplify assay operation. Assay times below 10 min will be enabled by integrated thin film electrodes and a unique thermoplastic fabrication technique supporting optimal thermal transport. A disposable piezoelectric element integrated into the chip inlet tube will enable efficient release of nucleic acids prior to sample discretization, and the assay will be operated through an embedded mixed-signal processor supporting all functions, with power and communication provided through a single USB connection to a laptop computer. Using this system, multiplexed qPCR for bacteria identification plus HRMA for product validation will be demonstrated, followed by an investigation of qPCR-HRMA based pathogen identification using unique signatures in the 16S rRNA gene, with a theoretical detection limit of 50 CFU/mL. The technology will be validated within a hospital environment through a clinical study with 50 patients for the simultaneous quantification of 8 bacteria commonly found in intra-abdominal infections, conditions where treatment currently relies on subjective visual assessment and empiric treatment, and where rapid near-patient bacterial identification would transform clinical practice.

项目摘要 能够快速识别和量化腹内细菌的新诊断工具 感染是必要的，以帮助临床医生在关键的早期阶段选择适当的抗生素。 治疗。目前的分子诊断仍然昂贵，需要重要的实验室 基础设施，使其不适合在医院内的小型卫星实验室使用 环境。在这里，我们试图通过开发一种简单、廉价和 可直接评估腹内脓肿细菌的自动微流控平台 用于指导感染的初始治疗的液体。这项技术将结合芯片上的核酸提取， 多重病原体的快速定量聚合酶链式反应(QPCR)和高分辨熔融分析(HRMA) 在不到10分钟内进行鉴定和定量。站台将由一次性热塑性塑料组成 Microwell芯片和一个紧凑的USB供电的读取器，其中包含用于分析操作的所有组件。 值得注意的是，整个分析只需要两次吸管行程就可以将样品分散在稠密的 微升反应井阵列，包含执行检测所需的所有底物和其他试剂。 芯片过程中多重聚合酶链式反应与热塑性微孔阵列的整合 制造将在聚合酶链式反应期间实现自动热控释放，而被动样本 一次性微孔阵列内的离散化将进一步简化分析操作。化验次数 通过集成薄膜电极和独特的热塑性制造，可以实现10分钟以下的时间 支持最佳热传输的技术。一次性压电元件集成到 芯片入口管将在样品离散化之前实现核酸的有效释放，并且该检测 将通过支持所有功能的嵌入式混合信号处理器进行操作，并带有电源 以及通过到膝上型计算机的单个USB连接提供的通信。使用这个 系统，用于细菌鉴定的多重qPCR和用于产品验证的HRMA将是 基于qPCR-HRMA的病原菌鉴定的研究 在16S rRNA基因中具有独特的特征，理论检测下限为50cfu/mL。这个 这项技术将在医院环境中通过对50名患者进行临床研究来验证 同时定量检测腹内感染常见的8种细菌 目前的治疗依赖于主观视觉评估和经验性治疗，以及 近病人的快速细菌鉴定将改变临床实践。