Joint Biological Agent Identification and Diagnostic System (JBAIDS): Stability and Sensitivity of Freeze-Dried Real-Time PCR Assays

The goal of the JBAIDS program was to develop a portable system capable of identifying multiple bio-threat organisms simultaneously. Freeze-dried assays are desirable for use in this system because reagents are generally stable in this form over a wide range of temperatures (4°C45oC). Thirty candidate assays were assessed for performance at multiple time points under various temperature conditions (4°C, 28°C, and 45oC). At each time point freeze-dried reagents were evaluated by running a dilution series, NALOD, and positive controls at varying concentrations. Testing runs were completed on the JBAIDS real-time PCR instrument and evaluated using the JBAIDS software for amplification (positive or negative) calls, as well as crossing threshold and mean maximum fluorescence. NALOD’s for the candidate assays ranged from 5 GE to 350 GE for bacterial assays, and 1.99 to 5000 plaque forming unit equivalents (pfu-e) for viral assays. Freeze-dried bacterial assays were found to be sensitive and stable for at least 8 weeks at 45oC, and at least 12 months for both 4°C and 28oC. RNA Viral assays are stable at 45oC for at least 8 weeks. Room temperature and 4oC testing is ongoing, but data has shown that assays stable at 6 months will also be stable out to at least 12 months.