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Title: Development of a Rapid and Sensitive Immunoassay for Detection and Subsequent Recovery of Bacillus anthracis Spores in Environmental Samples

Author
item HANG, J - CREATV MICROTECH, INC.
item SUNDARAM, A - CREATV MICROTECH, INC.
item ZHU, P - CREATV MICROTECH, INC.
item Shelton, Daniel
item Karns, Jeffrey
item Martin, Phyllis
item LI, S - CREATV MICROTECH, INC.
item AMSTUTZ, P - CREATV MICROTECH, INC.
item TANG, C - CREATV MICROTECH, INC.

Submitted to: Journal of Microbial Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/26/2008
Publication Date: 5/2/2008
Citation: Hang, J., Sundaram, A.K., Zhu, P., Shelton, D.R., Karns, J.S., Martin, P.A., Li, S., Amstutz, P., Tang, C.M. 2008. Development of a Rapid and Sensitive Immunoassay for Detection and Subsequent Recovery of Bacillus anthracis Spores in Environmental Samples. Journal of Microbial Methods. 73:242–246.

Interpretive Summary: Anthrax is caused by the bacterium Bacillus anthracis. This bacterium produces spores that are resistant to normal methods of disinfection and is readily disseminated in the air. Consequently, there is great concern regarding its use as an agent of bioterrorism. Methods have been developed for the rapid detection of anthrax spores at high concentrations, consistent with a bioterrorism attack. However, detection at lower concentrations is complicated by the fact that closely related Bacillus strains, that are harmless and normally present in the environment, may also be detected. Additional genetic characterization is required to distinguish the different Bacillus strains. We developed a method in which presumptive Bacillus anthracis spores can be detected at low concentrations (1,000 spores) using antibodies, followed by rapid transformation of spores to vegetative cells, allowing for rapid confirmation using genetic techniques.

Technical 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. 2000 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 release, germination and outgrowth of spores, resulting in vegetative cells for PCR analysis.