Research Project
6882981
[unreadable] 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. [unreadable] [unreadable]
