FilmArray-A Closed System for Multi-Pathogen Screening

FilmArray-多病原体筛查的封闭系统

• 批准号: 6882981
• 负责人: Steven F Dobrowolski
• 金额: $ 13.4万
• 依托单位: BIOFIRE DEFENSE, LLC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2005-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882981
• 关键词: Bacillus; bacterial genetics; biohazard detection; biomedical equipment development; biotechnology; bioterrorism /chemical warfare; clinical biomedical equipment; communicable disease diagnosis; diagnosis design /evaluation; fungal genetics; heat; microarray technology; nucleic acid denaturation; polymerase chain reaction; rapid diagnosis; silicates; yeasts

DESCRIPTION (provided by applicant): Polymerase chain reaction (PCR) has become the method of choice for rapid diagnosis of infectious agents. To identify an infectious agent by molecular means requires a panel of PCR assays. So called "multiplex PCR" concurrently assays for multiple targets in a single reaction. Multiplex PCR has proved valuable in some systems, but shortcomings exist concerning robustness of high level multiplex reactions and difficulties in clear analysis of multiple products. The proposed FilmArray platform solves many problems associated with multiplex PCR and discriminating individual reaction products. FilmArray is homogeneous and performed in a thin-film plastic disposable pouch. A sample is injected into the FilmArray pouch wherein fluidic channels traffic the sample to locations where it undergoes: nucleic acid preparation, primary large volume multiplex PCR, dilution of primary reaction product, and nested secondary PCR, culminating with real-time detection and melting-curve analysis of secondary product. All reagents required for isolation of nucleic acids and PCR are freeze dried in the FilmArray pouch. The long-term goal is a contained system assaying a single sample for a large panel of infectious agents. Feasibility is demonstrated using yeast and DNase treated Bacillus globigii spores as model systems. Specific Aim 1 demonstrates preparation of nucleic acid in the FilmArray pouch using bead milling, followed by binding to silica-based magnetic beads and subsequent elution. Specific Aim 2 demonstrates the 2-stage PCR process contained in the FilmArray pouch. Stage 1 consists of a large volume multiplex reaction for 6 single copy yeast genes. The primary reaction uses a short cycling protocol that does not proceed through the log phase. Primary reaction product is diluted in the FilmArray pouch with a fluidic system and is transferred to sites of the secondary, single-plex, nested PCR reaction. Specific Aim 3 seeks to perform analysis of 2nd stage reactions by real-time PCR and dye-binding/high-resolution thermal denaturation. Specific Aim 4 constructs a prototype instrument with drivers to facilitate chemistries described in Specific Aims 1, 2, and 3. Phase I focuses upon feasibility but long term objectives are using FilmArray to screen a single specimen (blood, sputum, cerebrospinal fluid) for broad panels of infectious agents that are potentially responsible for symptoms observed in patients. Pouches are designed to assay for candidate infectious agents consistent with clinical presentation.

描述（由申请人提供）：聚合酶链式反应（PCR）已成为快速诊断传染原的首选方法。要通过分子手段识别传染源，需要进行一组 PCR 检测。所谓的“多重 PCR”可在一次反应中同时检测多个靶标。多重 PCR 已被证明在某些系统中很有价值，但存在高水平多重反应的稳健性以及多种产物的清晰分析困难等缺点。所提出的 FilmArray 平台解决了与多重 PCR 和区分单个反应产物相关的许多问题。 FilmArray 是均质的，并在薄膜塑料一次性袋中进行。将样品注入 FilmArray 袋中，其中流体通道将样品输送至进行以下操作的位置：核酸制备、初级大容量多重 PCR、初级反应产物的稀释和嵌套次级 PCR，最终实现实时检测和熔解。二次产品曲线分析。核酸分离和 PCR 所需的所有试剂均在 FilmArray 袋中冷冻干燥。长期目标是建立一个封闭的系统，可以分析单个样本中的大量传染原。使用酵母和 DNase 处理的球芽孢杆菌孢子作为模型系统证明了可行性。具体目标 1 演示了使用珠磨在 FilmArray 袋中制备核酸，然后与二氧化硅基磁珠结合并随后进行洗脱。具体目标 2 演示了 FilmArray 袋中包含的 2 阶段 PCR 过程。第一阶段包括 6 个单拷贝酵母基因的大体积多重反应。主要反应使用不进行对数阶段的短循环方案。初级反应产物在 FilmArray 袋中通过流体系统进行稀释，并转移至次级、单重、嵌套 PCR 反应的位点。具体目标 3 旨在通过实时 PCR 和染料结合/高分辨率热变性对第二阶段反应进行分析。具体目标 4 构建了一个带有驱动程序的原型仪器，以促进具体目标 1、2 和 3 中描述的化学反应。第一阶段的重点是可行性，但长期目标是使用 FilmArray 筛选单个样本（血液、痰液、脑脊液）以进行广泛的检测。一组可能导致患者出现症状的传染源。袋子设计用于检测与临床表现一致的候选传染原。