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）：聚合酶链反应（PCR）已成为快速诊断传染性病原体的首选方法。为了通过分子手段鉴定感染因子，需要一组PCR测定。所谓的“多重PCR”在单个反应中同时测定多个靶标。多重PCR已被证明在一些系统中是有价值的，但存在关于高水平多重反应的鲁棒性和难以清楚地分析多种产物的缺点。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是同质的，并在薄膜塑料一次性袋中进行。将样本注入FilmArray测试条中，其中流体通道将样本运送到进行以下操作的位置：核酸制备、一级大容量多重PCR、一级反应产物稀释和巢式二级PCR，最终进行二级产物的实时检测和熔解曲线分析。核酸分离和PCR所需的所有试剂均在FilmArray测试条中冻干。长期目标是建立一个封闭的系统，分析一个单一的样本，一个大面板的传染性病原体。使用酵母和DNA酶处理的球形芽孢杆菌孢子作为模型系统证明了可行性。特定目的1证明了使用珠磨在FilmArray测试条中制备核酸，然后与基于二氧化硅的磁珠结合并随后洗脱。特定目的2证明了FilmArray测试条中包含的2阶段PCR过程。阶段1由6个单拷贝酵母基因的大体积多重反应组成。初级反应使用不通过对数期进行的短循环方案。使用流体系统在FilmArray测试条中稀释一级反应产物，并将其转移至二级、单重、巢式PCR反应位点。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。Specific Aim 4构建了一个带有驱动程序的原型仪器，以促进Specific Aim 1、2和3中描述的化学反应。I期重点关注可行性，但长期目标是使用FilmArray筛查单个标本（血液、痰液、脑脊液）中可能导致患者症状的多种传染性病原体。测试条设计用于检测与临床表现一致的候选感染原。