A Droplet-based single cell platform for pathogen identification and AST

用于病原体识别和 AST 的基于 Droplet 的单细胞平台

基本信息

批准号：
9038992
负责人：
JOSEPH C LIAO
金额：
$ 118.49万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-04-01 至 2020-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9038992
关键词：
Address Affect Age Antibiotic susceptibility Antibiotics Arizona Bacterial Infections Biological Assay Bypass Cell Culture Techniques Cells Clinical Culture Media Cytolysis Data Detection Development Plans Device or Instrument Development Devices Diagnosis Diagnostic Diffusion Encapsulated Escherichia coli Future Generations Goals Growth Guidelines Health Healthcare Hour Human Individual Infection Institutes Laboratories Life Measurement Methods Microfluidic Microchips Microfluidics Minimum Inhibitory Concentration measurement Molecular Molecular Diagnosis Molecular Probes Nucleic Acids Optics Peptide Nucleic Acids Predisposition Process Reagent Reporting Research Research Project Grants Research Subjects Ribosomal RNA Sampling Sepsis Source Specificity System Systems Integration Technology Testing Time Translations Universities Urinary tract Urinary tract infection Urine Validation Vertebral column base combinatorial community-acquired UTI design effective therapy evidence base improved industry partner innovation instrument molecular diagnostics mortality multi-drug resistant pathogen pathogen product development prospective sample collection urinary validation studies

项目摘要

DESCRIPTION (provided by applicant): The ability for clinicians to provide effective treatments to bacterial infections with targeted antibiotics hinges on molecular diagnostics capable of identifying the pathogen and determining its susceptibility to antibiotics in a timely manner. Urinary tract infection (UTI) is a particularly representative infection because it one of the most common bacterial infections that affect all ages but is currently only diagnosed in centralized laboratories via bacterial culture, which typically takes 2-3 days for definitive diagnosis. The significant time delay between sample collection and result reporting contributes to widespread empiric use of broad-spectrum antibiotics, which has contributed towards emergence of multidrug-resistant pathogen. Toward addressing this important unmet need, our overall goal is to develop and validate an integrated diagnostic platform for bacterial pathogen identification (ID) and antibiotic susceptibility testing (AST) in a "sample-to-answer" manner in under 3 hours. Such rapid molecular diagnostics will transform the clinicians' ability to provide evidence-based diagnosis of bacterial infections, expedite treatments based on objective data, promote effective utilization of antibiotics. Specifically, we propose to develop an innovative droplet microfluidic network capable of combinatorially generating millions of picoliter (pL)-sized droplets of different compositions, i.e. mixtures of samples and probes or antibiotics at varying concentration levels, as the backbone technology. The microfluidic chip enables a streamlined approach for sample-reagent mixing, compartmentalization of mixtures into a massive number of droplets, and serial dilutions to simultaneously carry out pathogen ID and AST. In the pathogen ID module, single bacterial cells are encapsulated in droplets, achieving an effective concentration equivalent to 108-109 cfu/ml and thereby enabling rapid identification via the hybridization of molecular beacon probes in an amplification-free approach. In the AST module, individual bacterial cells are encapsulated and cultured in droplets that enhance local culture condition for bacterial growth and enable direct measurements of single bacterial doublings, thereby facilitating direct phenotypic AST from urine samples. We have assembled an academic-industry partnership consisted of Johns Hopkins University (droplet microfluidics and diagnostics), Stanford University (UTI, molecular probes, validation studies), University of Arizona (microfluidic AST), and GE Global Research (manufacturing and system integration). We propose the following aims: 1) to achieve single cell, amplification-free pathogen ID in a droplet-based microfluidic chip using a panel of peptide nucleic acid molecular beacons that target bacterial 16S rRNA; 2) to develop a droplet-based single cell AST capable of determining the minimum inhibitory concentration (MIC) for commonly used antibiotics for UTI; 3) to perform system integration and instrument development through partnership with our industry partner; and 4) to perform analytical and clinical validation with the integrated device. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.

描述（由申请人提供）：忠诚者使用靶向抗生素对细菌感染提供有效治疗的能力取决于能够及时识别病原体并确定其对抗生素敏感性的分子诊断，尤其是尿路感染（UTI）。代表性感染，因为它是影响所有年龄段的最常见的细菌感染之一，但目前只能在集中实验室通过细菌培养进行诊断，通常需要 2-3 天才能做出明确诊断，样本采集和结果之间存在显着的时间延迟。报告有助于广谱抗生素的广泛经验使用，从而导致多重耐药病原体的出现。为了解决这一重要的未满足的需求，我们的总体目标是开发和验证用于细菌病原体识别（ID）和诊断的综合诊断平台。在 3 小时内以“从样本到结果”的方式进行抗生素敏感性测试 (AST)，这种快速的分子诊断将改变人群提供细菌感染循证诊断、基于客观数据加快治疗、促进有效治疗的能力。具体来说，我们建议开发一种创新的液滴微流体网络，能够组合产生数百万皮升（pL）大小的液滴。不同成分的液滴，即不同浓度水平的样品和探针或抗生素的混合物，作为微流控芯片的骨干技术，可以实现样品试剂混合、将混合物划分为大量液滴以及连续稀释的简化方法。同时进行病原体 ID 和 AST 在病原体 ID 模块中，单个细菌细胞被封装在液滴中，达到相当于 108-109 cfu/ml 的有效浓度，并能够实现。通过无扩增方法中的分子信标探针杂交进行快速识别在 AST 模块中，单个细菌细胞被封装并培养在液滴中，从而增强细菌生长的局部培养条件，并能够直接测量单个细菌倍增，从而促进直接测量。我们与约翰·霍普金斯大学（液滴微流体和诊断）、斯坦福大学（UTI、分子探针、验证研究）、美国大学建立了学术-工业合作伙伴关系。亚利桑那州（微流控 AST）和 GE 全球研究（制造和系统集成）提出以下目标：1）使用一组肽核酸分子在基于液滴的微流控芯片中实现单细胞、无扩增的病原体 ID。靶向细菌 16S rRNA 的信标；2) 开发基于液滴的单细胞 AST，能够确定尿路感染常用抗生素的最低抑制浓度 (MIC)；通过与我们的行业合作伙伴合作进行系统集成和仪器开发；4) 使用集成设备进行分析和临床验证，为促进技术转化，提出了未来临床部署的产品开发计划。