Development and Commercialization of Analyte Specific Reagents (ASRs) for the Diagnosis of Rickettsial Diseases on FDA-cleared Real-time PCR Platforms

Phase I will develop multiple combinations of qRT-PCR primers and probes for some of the causative agents of six diseases with military importance: rickettsia, murine and scrub typhus, spotted fever, anaplasmosis, ehrlichioses, and Q fever. Based on known sequence information from the literature and contacts at disease labs, probes and primer pairs will be designed and synthesized for testing; expected probe formats include hydrolysis probes, scorpions, and molecular beacons. Positive control plasmids containing target amplicon sequences will be designed, synthesized, and sequence-verified. Assays will be optimized for cycle profile, magnesium concentration, presence of competing nucleic acids, melting temperature, and gels to verify predicted amplicon length. Specificity and sensitivity will be established. Final probes and primer pairs will be provided to a Army Testing Lab for evaluation against disease targets. Acceptable probes and primers will be manufactured under cGMP, and a minimum of 100 vials of at least one analyte assay provided to the military for testing; if successful, the expectation is assays for three of the six agents by the end of Phase I Option.