The project will develop a decaplex assay to detect varieties of the Category C Priority Pathogen, influenza, listed in Table 1, within 30 minutes of taking a raw patient sample. The subtypes that are detected are all highly pathogenic strains of H5 and H7, which are classed as Select Agents by the USDA. The assay will be developed for the 6-color Bio-Seeqll, a portable, battery operable, thermocycler / fluorometer designed with automated sample preparation for ease of use in the point-of-care clinical market. The requested support will accelerate the development of the human diagnostic version of the influenza assay, and provides a vehicle to gain access to government expertise and resources (e.g., influenza strains). The following activities are planned to advance assay development through pre-clinical validation: 1. Construct a decaplex RT-LATE PCR assay to detect of any of the clinically relevant influenza varieties (including highly pathogenic varieties), in a single tube through iterative interaction modeling and empirical verification. 2. Investigate factors affecting assay performance: Optimize RNA extraction / purification protocols for influenza samples, and lyophilized RT-PCR reagent formulation, for best influenza assay sensitivity, specificity, and robustness in the integrated cartridge (USPD). 3. Perform pre-clinical validation of assay, including comparability testing against available, validated PCR assays. Table 1. Assay targets and subtype identification.Influenza TypeHA SubtypeNA SubtypeHigh vs. Low PathogenicityAH1N1, N2, other H3N1, N2, other H5N1, N2, otherYes H7N1, N2, otherYesBType B HA Relevance: This assay will enable faster, more effective diagnosis of influenza to detect outbreaks (especially of highly pathogenic varieties), reduce unneeded antibiotic use, reduce hospital stays, and enable more effective use of neuraminidase inhibitors, preserving Strategic National Stockpile of drugs for response to pandemic influenza.
