RAPID PCR OF BACILLUS ANTHRACIS DNA

DESCRIPTION (provided by applicant): Anthrax or Woolsorter's Disease is a zoonotic infection caused by Bacillus anthracis. During the peak of the 'anthrax letter scare of 2001', the US Army Medical Research Institute for Infectious Diseases was testing over 2000 samples per day. At the present time, nearly all anthrax testing at USAMRIID relies on microbiological methods, which require one to two days to carry out. Much faster and more informative diagnosis is possible using PCR-based methods; in particular using new machines designed for high-speed amplification of DNA fragments 100 to 500 base pairs long. The fastest PCR machine employed to date is a pressurized He/CO2 gas thermocycler with digitally programmable electronic valves. Using this gas burst device, a 368 b.p. DNA fragment from B. anthracis Sterne (vaccine) strain was amplified 108-fold through 30 PCR cycles in <4 minutes. Even faster (1.5 to 3.0 minute) PCR-amplification of B. anthracis plasmid-encoded virulence factors (pag, lef, cya, capA, capC) is possible using shorter (151 -288 b.p.) amplicons and/or fewer (20-25) PCR cycles. The present experiments seek to improve the ease-of-use of this high performance He/CO2 gas thermocycler, for deployment at USAMRIID within the next year.