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Title: REAL-TIME PCR DETECTION OF B. ANTHRACIS: LESSONS FROM THE REAL WORLD

Author
item HIGGINS, JAMES

Submitted to: Food Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 10/9/2003
Publication Date: 11/10/2003
Citation: Higgins, J.A. 2003. Real-time PCR detection of B. anthracis: Lessons from the real world [Abstract]. Food Microbiology. p.2.

Interpretive Summary:

Technical Abstract: During the anthrax bioterror outbreak which occurred in the Washington, DC area in October 2001, the USDA-ARS set up a mobile laboratory on the grounds of the SE Federal Facility for the purpose of testing suspicious envelopes, parcels, and mail room swab and air samples, for the presence of spores of Bacillus anthracis, via real-time PCR. Samples were also forwarded to the USDA-APHIS National Veterinary Services Laboratory in Ames, Iowa, for culture of B. anthracis. Six samples (out of > 3,000) were positive by culture, none by real-time PCR. The reasons for the negative real-time PCR tests were: low numbers of dispersed spores, and excessive 'dirtiness' of the samples (even mail room air samples), which taxed the ability of the DNA extraction protocols to remove inhibitors and led to reduced sensitivity of subsequent PCR assays. Our experiences with this large-scale environmental testing for threat agent(s) have convinced us that ultimately, determination of assay sensitivity and specificity should use samples strongly representative of those that may be encountered in bioterrorism scenarios, rather than the arguably more contrived sample types commonly encountered in published protocols. With this in mind, we have been evaluating new instrumentation from Idaho Technology, Inc., the SP1 robot, for automated extraction of DNA from cow blood, nasal swabs, and water spiked with spores of B. anthracis Sterne strain. The SP1 is capable of delivering PCR-quality DNA from these sample types in under 1 hr (for up to 16 samples). Detection limits for nasal swabs are: ~ 260 spores / swab, and for cow blood, ~ 1 x 10(4) genomes per 200 l. In tap water, the detection limit is ~ 500 spores per 100 ml. Automated sample preparation platforms such as the SP1 (and those pending from other companies, such as Xtrana, Cepheid, and MicroFluidics, Inc.) have the potential to offer improved assay speed, sensitivity, and specificity with regard to threat agent detection.