Indirect Detection Of Bacillus Anthracis (Anthrax) Using Amplified Gamma Phage-Based Assays

• Main Author: Reiman, Robert W
• Format: Report
• Language: English
• Subjects: AMPLIFICATION; ANTHRAX; BACILLUS ANTHRACIS; BACTERIOPHAGES; Biochemistry; BIOLOGICAL DETECTION; CELLS(BIOLOGY); CHAIN REACTIONS; CONTROL; DEOXYRIBONUCLEIC ACIDS; DETECTION; DISEASES; FLUORESCENCE; GAMMA RAYS; HAND HELD; IMMUNOASSAY; ISOLATION; LABORATORIES; Medicine and Medical Research; Microbiology; POLYMERIZATION; PRODUCTION; REAL TIME; VEGETATION; VIABILITY

The need for a simple, specific, sensitive, inexpensive, accurate, and rapid method to identify Bacillus anthracis became apparent during the Fall 2001 anthrax attacks which caused widespread panic and ultimately killed five individuals. The Centers for Disease Control and Prevention currently employs agar plate lysis by gamma phage and direct fluorescence assay to confirm the presence of Bacillus anthracis. These confirmatory methods require isolation of individual colonies from an overnight culture, are time consuming, and are best suited for laboratory environments. The research described in this dissertation focused on applying the highly specific gamma phage lytic replication cycle to indirectly detect Bacillus anthracis. The production of progeny gamma phage only occurs in the presence of a suitable host, so the detection of increasing concentrations, or amplification, of progeny gamma phage implies the presence of viable Bacillus anthracis cells. Four unique gamma phage-based detection assays using real-time polymerase chain reaction, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and two different designs of hand-held immunoassays based on this gamma phage amplification phenomenon were designed, developed, and experimentally tested for indirectly detecting viable Bacillus anthracis. This research demonstrated that vegetative Bacillus anthracis was required to produce the gamma phage amplification event. Real-time polymerase chain reaction was used to detect the increased amount of gamma phage DNA produced by gamma phage amplification, and thereby indirectly detect a starting concentration of four Bacillus anthracis cells in less than five hours. The other three gamma phage-based detection assays were unable to indirectly detect Bacillus anthracis, but three of the four assays demonstrated gamma phage detection capabilities. The original document contains color images.