The ultimate goal of this proposed project is to develop a clinical diagnostic in three years that targets 15 agents that cause infections in the respiratory tract that may be misdiagnosed for the SARS virus. Our preliminary targets have genomes constructed from RNA or DNA and therefore the test will be required to analyze both, possibly in a single tube. The MULTI-CODE platform will be used to for the diagnostic proposed. This platform is extremely simple to perform, is complete in less than three hours, does not require washing, filtration of expensive equipment and is uniquely suited for the clinical lab. Specifically: i. The platform has been shown in a mock experiment using synthetic targets to simultaneously detect RNA and DNA even when the targets differ by a single nucleotide. ii. The platform is fast and robust. Detecting low copy number in less than three hours with minimal "hands-on" requirements. iii. Previously, PCR based tests in both singleplex and low number multiplexes have been able to diagnose these targets in throat swabs as suggested in this application. iv. EraGen's collective expertise in diagnostic platform development (Chemistry, molecular biology, manufacturing) and market implementation makes it uniquely qualified to explore capabilities of this highly innovative technology. The Aims of Phase I are to: 1. Design multiple MULTI-CODE PCR primers and ERA-CODE primer extenders for each target. 2. Design multiple MULTI-CODE PCR primers and ERA-CODE primer extenders for at least two internal positive control RNA and DNA targets. 3. Synthesize all primers and targets from Aim 1. 4. Determine the correct mixture of primers and extenders that include one of the DNA and RNA internal positive control targets that allow for the highest multiplexing possible for both sputum and stool samples that continues to allow 1-100 copy sensitivity. 5. Using a number of sample preparation systems, validate sample preparation methods in conjunction with multiplexed assays using blinded controlled samples spiked with synthetic targets in multiple combinations.
Thesaurus Terms: communicable disease diagnosis, diagnosis design /evaluation, polymerase chain reaction, rapid diagnosis, respiratory infection feces, nucleic acid chemical synthesis, sputum biotechnology, clinical research, human tissue
