Affiliation: | aCreatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac, MD 20854, United States bU.S. Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Environmental Microbial Safety Laboratory, Bldg 173, BARC-East, 10300 Baltimore Ave, Beltsville, MD 20705, United States cUSDA, ARS, Plant Sciences Institute, Invasive Insect Biocontrol and Behavior Laboratory, Beltsville, MD 20705, United States |
Abstract: | Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 103 spores was achieved by incubating spores simultaneously with capture and detection antibodies (“liquid-phase” assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis. |