Submitted to: ASM Biodefense Research Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 2/8/2005
Publication Date: 3/20/2005
Citation: Zhu, P., Shelton, D.R., Higgins, J.A., Karns, J.S., Bull, R.L., Martin, P.A., Amstutz, P., Tang, C. 2005. A Biosensor Platform for Genetic Identification and Quantitation of Bacillus Anthracis. ASM Biodefense Research Meeting, March 20-23, 2005, Baltimore, MD. Interpretive Summary:
Technical Abstract: Real-time PCR assays for identification and quantitation of Bacillus anthracis were developed by Creatv MicroTech, Inc for detection of B. anthracis spores in environmental samples. Three genetic markers were detected in the PCR reactions, utilizing DNA extracted from B. anthracis spores and vegetative cells. A chromosomal marker rpoB encoding the RNA polymerase ß-subunit was used in real-time PCR to quantify genome copy numbers of B. anthracis spores and vegetative cells. Two plasmid markers, pagA and capA, which are involved in the biosynthesis of anthrax toxin and capsule, respectively, were used for the identification of virulent strains containing plasmids PXO1 and PXO2. In addition, an internal positive control (IPC) was also developed as a quality control of the PCR reaction and to detect potential PCR inhibitors in environmental samples. A standard curve was constructed for the quantitative real-time PCR. There was strong linear inverse relationship (R2=0.997) between threshold cycle (CT) and the log10 number of rpoB copies over 7 orders of magnitude. The detection limit was less than 10 copies of B. anthracis. Fifteen representative strains of Bacillus species were tested in this study. The results showed that the real-time PCR assays were specific to B. anthracis and did not cross-react with B. thuringiensis and B. cereus. By coupling this procedure with the immuno-capture and enrichment procedures in Creatv's biosensor system, the real-time PCR procedure established in this study provides high sensitivity and specificity for rapid identification and quantitation of B. anthracis in environmental samples.