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）已成为传染病病原体快速诊断的首选方法。通过分子方法鉴定传染因子需要一组聚合酶链反应测定。所谓的“多重PCR”在一个反应中同时检测多个目标。多重聚合酶链反应在一些系统中已被证明是有价值的，但存在高水平多重反应的鲁棒性和难以清晰分析多种产物的缺点。提出的FilmArray平台解决了多重PCR和区分单个反应产物相关的许多问题。filarray是均匀的，并在薄膜塑料一次性袋中执行。将样品注入FilmArray袋中，其中流体通道将样品运送到其经历的位置：核酸制备，一次大体积多重PCR，一次反应产物稀释，嵌套二次PCR，最终进行实时检测和二次产物熔化曲线分析。分离核酸和PCR所需的所有试剂在FilmArray袋中冷冻干燥。长期目标是建立一个封闭的系统，分析单个样本中大量的感染因子。以酵母和dna酶处理的球状芽孢杆菌孢子为模型系统，论证了其可行性。特异性目标1演示了在FilmArray袋中使用珠磨制备核酸，随后与硅基磁珠结合并随后洗脱。特异性目标2演示了包含在FilmArray袋中的两阶段PCR过程。阶段1包括6个单拷贝酵母基因的大体积多重反应。主反应使用不经过日志阶段的短循环协议。一级反应产物在FilmArray液囊中用流体系统稀释，然后转移到二级、单plex、巢式PCR反应的位点。特异性目标3寻求通过实时PCR和染料结合/高分辨率热变性进行第二阶段反应分析。具体目标4构建了一个原型仪器的驱动器，以促进具体目标1,2和3中描述的化学。第一阶段侧重于可行性，但长期目标是使用FilmArray筛选单个标本（血液、痰液、脑脊液），以寻找可能导致患者观察到的症状的广泛感染因子。药袋设计用于检测符合临床表现的候选感染因子。