Real-Time PCR Assays for Direct Detection of Sepsis and CAP Pathogens
Principal Investigator: Virginia Coryell
Affiliation: Northern Arizona University
Abstract: Advanced diagnostic assays for pathogens causing sepsis and community acquired pneumonia (CAP) will be developed, tested in a clinical setting and moved into a manufacturing prototype. The assays will be based upon real-time PCR and TaqMan(r) probe-primer combinations that detect the pathogen genomes. Real-time PCR is considered the gold standard of low level nucleic acid detection and generates data in a few hours, versus more than two days for culturing methods. This technology is capable of single molecule detection and single-nucleotide discrimination, while providing quantitative data across six-orders of magnitude. This high sensitivity, discrimination and quantitation can be accomplished even when there is an overwhelming background of highly related nucleic acids. Advances in signature discovery and assay design from our biodefense efforts will be applied to more common clinical pathogens. A three-way partnership is proposed among the Keim Lab (TGen-NAU), Applied Biosystems (instruments and assay manufacturing), and the clinical expertise of the Arizona Banner Health System (Laboratory Sciences of Arizona). The industrial partners have committed significant resources in the form of reagents, informatics support, labor and instruments towards the success of this project. Using the latest bioinformatics approaches and the rapidly expanding genomic databases, we will identify a large number of potential diagnostic genomic signatures (e.g., single nucleotide polymorphisms-SNPs). Applied Biosystems will use the Assays-on-Demand(r) pipeline to convert these into real-time PCR assays for verification and validation at TGen, NAD and in the Banner Health clinical labs. Validation against large panels of strains and stream of clinical specimens will be used to insure assay specificity. Multiple validated assays will be advanced to the Applied Biosystems manufacturing facility and will utilize the low density array micro-card technology. Low density micro card arrays have great flexibility for up to 768 assays in multiple combinations of pathogens, virulence and antibiotic resistance genes. This manufacturing flexibility will allow us to mass produce customized assay systems targeted at sepsis, CAP or even particular clinical disease presentations of each. In addition, an informatics system will be developed for handling assay data during the validation and eventually for handling associated clinical data in a HIPAA compliant environment. Clinical specimens including blood, sputum, etc. will be analyzed using current standard lab culture and other protocols in parallel to the real-time PCR assays for clinical validation studies. Quantitative data will be generated with every real-time PCR assay and will analyzed together, to better understand bacterial loads in contrast to pathogens.
Funding Period: 2005-07-01 - 2010-06-03
more information: NIH RePORT