IDENTIFICATION OF CAPSULE-FORMING BACILLUS-ANTHRACIS SPORES WITH THE PCR AND A NOVEL DUAL-PROBE HYBRIDIZATION FORMAT

Anthrax is a fatal infection of humans and livestock that is caused by the gram-positive bacterium Bacillus anthracis. The virulent strains of B. anthracis are encapsulated and toxigenic. In this paper we descr ibe the development of a PCR technique for identifying spores of B. an thracis. Two 20-mer oligonucleotide primers specific for the capB regi on of 60-MDa plasmid pX02 were used for amplification. The amplificati on products were detected by using biotin- and fluorescein-labeled pro bes in a novel dual-probe hybridization format. Using the combination of PCR amplification and dual-probe hybridization, we detected two cop ies of the bacterial genome. Because the PCR assay could detect a mini mum of 100 unprocessed spores per PCR mixture, we attempted to facilit ate the release of DNA by comparing the effect of limited spore germin ation with the effect of mechanical spore disruption prior to PCR ampl ification. The two methods were equally effective and allowed us to id entify single spores of B. anthracis in PCR mixtures.