SBIR-STTR Award

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.

Keywords:
Qrt-Pcr; Analyte Specific Reagents; Diseases Of Military Importance; Field Tests; Rickettsia; Typhus; Spotted Fever; Q Fever

During Phase I, probes and primers were designed for six Rickettsial diseases: Rickettsia typhi (murine typhus); Spotted Fever Group (SFG); Orientia tsutsugamushi (scrub typhus); Coxiella burnetii (Q fever); Anaplasma phagocytophilum; and Ehrlichiae. Signature sets (probes and primers) for four of the pathogens were synthesized and tested, as were positive and negative controls. Phase II will extend the work, establishing limits of detection, linearity, sensitivity, and specificity for the assays. In collaboration with the Army, further testing will include clinical samples from infected personnel and comparison to "gold standards". At least three lots of the successful assays will be manufactured under cGMP guidelines according to the FDA, and provided to the military for testing as Analyte Specific Reagents (ASRs). Feedback from military medical personnel will be used to format assays which can be used in existing JBAIDS qPCR instruments.

Keywords:
Rickettsial Detection; Rickettsia Typhi; Spotted Fever Group (Sfg); Orientia Tsutsugamushi; Coxiella Burnetii; Anaplasma Phagocytophilum; Ehrlichiae; Quantitative Polymerase C