Preparation of a positive control DNA for molecular diagnosis of Bacillus anthracis

Bacillus anthracis is considered to be one of the most potent biological weapons because of its highly pathogenic nature and efficiency of transmission. Routinely, a presumptive diagnosis of anthrax is achieved if the bands with predicted sizes are detected after the PCR targeted to the pag and cap genes residing on pXO1 and pXO2 plasmids, respectively. A positive control DNA prepared from the standard strains of B. anthracis (PAI and PAII) is usually included in the PCR tests. The handling of living B. anthracis, however, requires physical containment. The inclusion of DNA from B. anthracis as a positive control in the PCR test also has a potential risk of cross contamination that may confuse the results. In order to circumvent such problems, we attempted to construct a recombinant plasmid harboring the fragments of the pag and cap genes that could be distinguished from authentic sequences by the presence the restriction-enzyme site, the EcoRV site for the pag gene and the BamHI site for the cap gene, respectively, which were newly introduced. The strategy reported here provides a safe and reproducible positive-control DNA template. It also allows the detection of possible cross contamination, indicating that this strategy would be useful and convenient for the molecular identification of not only B. anthracis but also other highly pathogenic microbes.