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

项目摘要 能够快速识别和定量腹腔内细菌的新诊断工具 感染，以帮助临床医生选择适当的抗生素在关键的早期阶段， 治疗目前的分子诊断仍然昂贵，需要进入重要的实验室 基础设施，使其不适合用于医院内的小型卫星实验室 环境在这里，我们寻求通过开发一种简单，廉价， 能够直接评估来自腹腔内脓肿的细菌的自动化微流体平台 液体，以指导感染的初步治疗。该技术将联合收割机芯片上核酸提取， 多重病原体的快速定量PCR（qPCR）和高分辨率熔解分析（HRMA） 该平台将由一次性热塑性塑料制成， 微孔芯片和一个紧凑的USB供电的阅读器包含所有组件的分析操作。 值得注意的是，整个测定将仅需要两次移液器冲程来将样品离散化在密集的样品池中。 含有所有引物和执行测定所需的其他试剂的微升反应威尔斯孔阵列。 在芯片期间将多重PCR引物整合到热塑性微孔阵列中 制造将能够在PCR期间实现自动热控制释放，而被动样品 一次性微孔阵列内的离散化将进一步简化分析操作。测定时间 通过集成薄膜电极和独特的热塑性制造， 支持最佳热传输的技术。一种集成到压电元件中的一次性压电元件， 芯片入口管将能够在样品离散化之前有效释放核酸，并且该测定 将通过支持所有功能的嵌入式混合信号处理器进行操作， 以及通过到膝上型计算机的单个USB连接提供的通信。使用此 系统，用于细菌鉴定的多重qPCR加上用于产品验证的HRMA， 证明，然后使用qPCR-HRMA进行基于病原体鉴定的研究 16 S rRNA基因中的独特特征，理论检测限为50 CFU/mL。的 该技术将在医院环境中通过对50名患者的临床研究进行验证， 同时定量8种常见于腹腔内感染、条件 治疗目前依赖于主观视觉评估和经验性治疗， 快速的近患者细菌鉴定将改变临床实践。

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.