Trap Array Chips Enabling Rapid, Automated, and Portable Antibiotic Resistance Screening

项目目标简述：将开发一种微型系统，能够在15分钟内快速、多路、便携式地在护理点识别耐药细菌。摘要：抗生素耐药性的快速增长被认为是美国和世界范围内最严重的公共卫生威胁之一，耐药细菌数量的增加导致临床结果不佳和死亡率升高，同时消耗了更多的卫生保健资源。在护理点或护理点附近快速识别多种细菌并评估其抗生素敏感性的能力将大大提高临床医生的能力，通过根据特定感染处方适当的药物或通过隔离高风险患者以防止进一步传播来优化治疗。在这个项目中，我们将开发一种使用一次性芯片实验室设备的新技术，该设备能够将患者样本分离到1000多个单独的反应室中，在这些反应室中可以快速分析与抗生素耐药性相关的不同微生物基因片段，从而在15分钟内高效筛选多个潜在目标。由此产生的技术被设想为一种简单而廉价的诊断平台，用于护理点。在这项工作中开发的基础芯片实验室技术也将有助于推进其他诊断平台，这些诊断平台需要简单的操作和最少的设备，用于缺乏中心实验室资源的临床环境。技术摘要：抗生素耐药性是对公共卫生的一个主要且日益严重的威胁，耐药病原体显著增加了感染患者的发病率和死亡率。与抗菌素耐药性增加有关的一个主要挑战是，在治疗的最初阶段，缺乏快速检测方法来确定致病病原体及其耐药概况。在这个项目中，我们将开发一种低成本的一次性热塑性微流控平台，采用一种新的陷阱阵列技术，有望为在护理点进行抗生素耐药性筛查的聚合酶链反应（PCR）的常规临床应用打开大门。陷阱阵列平台将支持超过1000个同时PCR反应，使用引物用于多个抗生素抗性基因目标，而不需要外部泵送，阀门，底物制备或试剂引入。由此产生的平台将专门用于开发多药耐药碳青霉烯酶产生细菌的快速多重检测，目前在多种临床环境中提出了重大挑战。为了实现这些目标，这些努力将导致新技术的发展，从而实现(1)将连续样本量的单步离散化到单个样品陷阱阵列中，以实现高度并行分析；(2)在芯片制造过程中，将所有所需的PCR试剂（包括用于多路分析的定义引物集）直接集成到样品陷阱阵列中。(3)在一次性热塑性芯片上对扩增子进行快速PCR和高分辨率熔融分析（HRMA），只需一个手动移液步骤即可操作，大大简化了在接近患者的环境中使用的分析实施，而无需进入中央生化实验室。

Proposal Title: Trap Array Chips Enabling Rapid, Automated, and Portable Antibiotic Resistance Screening Brief description of project Goals: A miniature system enabling rapid, multiplexed, and portable point-of-care identification of antibiotic-resistant bacteria in under 15 minutes will be developed.Nontechnical Abstract:The rapid growth of antibiotic resistance is recognized as one of the most serious threats to public health in the U.S. and worldwide, with increasing numbers of drug-resistant bacteria leading to poor clinical outcomes and higher death rates, while consuming greater health-care resources. The ability to rapidly identify multiple bacteria and evaluate their antibiotic susceptibility at or near the point of care would greatly enhance the ability of clinicians to optimize treatment through the prescription of appropriate drugs based on the specific infections, or through the isolation of high risk patients to prevent further transmission. In this project we will develop a new technology using disposable lab-on-a-chip devices capable of segregating a patient sample into over 1000 individual reaction chambers where different microbial gene fragments associated with antibiotic resistance may be rapidly analyzed, allowing highly effective screening of multiple potential targets in under 15 min. The resulting technology is envisioned as a simple and inexpensive diagnostic platform for use at the point of care. The underlying lab-on-a-chip technologies developed in this work will also serve to advance other diagnostic platforms requiring simple operation and minimal equipment for use in clinical settings lacking central laboratory resources. Technical Abstract:Antibiotic resistance represents a major and growing threat to public health, with drug-resistant pathogens significantly increasing rates of morbidity and mortality for infected patients. A major challenge associated with the increase in antimicrobial drug resistance is the lack of rapid assays for identifying causative pathogens and their drug resistance profiles during the earliest stages of treatment. In this project we will develop a low-cost and disposable thermoplastic microfluidic platform employing a novel trap array technology expected to open the door to routine clinical application of polymerase chain reaction (PCR) for antibiotic resistance screening at the point of care. The trap array platform will support over 1000 simultaneous PCR reactions using primers for multiple antibiotic-resistance gene targets, without the need for external pumping, valving, substrate preparation, or reagent introduction. The resulting platform will specifically be applied to the development of a rapid multiplexed assay for multi-drug resistant carbapenemase-producing bacteria, which currently presents a significant challenge across multiple clinical environments. To achieve these goals, the effort will result in the development of new technologies enabling (1) single-step discretization of a continuous sample volume into an array of individual sample traps to enable highly parallel analysis, (2) integration of all required PCR reagents, including defined primer sets for multiplexed analysis, directly into the array of sample traps during chip manufacture, and (3) rapid PCR and high resolution melt analysis (HRMA) of the resulting amplicons in a disposable thermoplastic chip requiring only a single manual pipetting step for operation, vastly simplifying assay implementation for use in near-patient settings without access to a central biochemical laboratory.