Reliable measurements of the concentration of Bacillus anthracis (BA) spore suspensions are essential for the production of useful reference materials that can be used to test detection devices and laboratory instruments. In this study, we have compared the results of classic microbiological methods (plate and microscope counts) to extracting DNA from the same samples, and measuring the genomic equivalents. A method based on chemical and enzymatic disruption of the spores was used to extract the DNA. The DNA recovered from this procedure had a high molecular weight and was sufficiently pure for the mass to be measured by agarose gel electrophoresis and for the concentration of the DNA markers to be measured using quantitative PCR (QPCR) assays. QPCR assays were used for markers on the chromosome (rpoB), the pXO1 plasmid (pag), and the pXO2 plasmid (capC). QPCR assays were calibrated using plasmids containing the cloned DNA sequences for the gene markers. Total spore DNA and extra-sporular DNA (prepared by milder extraction conditions) were measured on two different lots of BA (Sterne). The total DNA concentrations of the two lots were similar, but their extra-spore DNA contents were very different. We also examined the effect of gamma irradiation on the recovery of DNA for use with the QPCR assays. Gamma irradiation of a lot of BA (Sterne) resulted in an approximately two-fold lower concentration of spore genomic equivalents as determined by the QPCR assays. Gamma-irradiated BA (Ames) functioned as a satisfactory positive control for the pXO2 marker in QPCR assays.
Citation: Journal of Microbiological Methods
Pub Type: Journals
Bacillus anthracis, DNA extraction, gamma irradiation, genomic equivalents, inactivated, quantitative PCR, spores, Sterne