SBIR-STTR Award

Phase I will develop multiple combinations of qRT-PCR primers and probes for some of six diseases of military importance: Dengue, Rift Valley Fever, Chikungunya, Crimean-Congo Hemorrhagic Fever, Sandfly Fever, and Tick-Borne Encephalitis. 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 diseases by the end of Phase I Option.

Keywords:
Qrt-Pcr; Analyte Specific Reagents; Diseases Of Military Importance; Field Tests; Dengue; Rift Valley Fever; Chikungunya; Tick-Borne Encephalitis

During Phase I, probes and primers were designed and tested for six arbovirus diseases: Dengue Fever, Rift Valley Fever, Chikungunya, Crimean-Congo Hemorrhagic Fever, Sand Fly Fever, and Tick-Borne Encephalitis. Positive and negative controls were designed, produced, and tested as well. Phase II will extend the work, establishing limits of detection, linearity, sensitivity, and specificity for the assays. 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:
Arbovirus Detection; Dengue Fever; Rift Valley Fever; Chikungunya Virus; Crimean-Congo Hemorrhagic Fever; Sand Fly Fever; Tick-Borne Encephalitis; Quantitative Polymerase Chai