Double-color fluorescence in situ hybridization (FISH) for the detection of Bacillus anthracis spores in environmental samples with a novel permeabilization protocol.

For anti-bioterrorism measures against the use of Bacillus anthracis, a double-color fluorescence in situ hybridization (FISH) is proposed, for the rapid and specific detection of B. anthracis. The probes were designed based on the differences in 16S and 23S rRNA genes of B. cereus group. A new permeabilization protocol was developed to enhance the permeability of FISH probes into B. anthracis spores. The highest detection rate (90.8 ± 0.69) of B. anthracis spores by FISH was obtained with successive incubation steps with 50% ethanol at 80 °C, a mixture of SDS/DTT solution (10mg/ml SDS, 50mM DTT) at 65 °C and finally in a lysozyme solution (20mg/ml) at 37 °C for 30 min each. This protocol was evaluated for the detection of B. anthracis spores in soil and air samples after adding formalin-fixed spores at different densities. The results have proven the success of double-color FISH in detecting B. anthracis spores in air samples in the range of 10(3) spores/m(3) and above. Conversely, for detecting B. anthracis spores in a soil sample, the lowest detection limit was found to be 10(7) spores/g dry soils. These results confirm the applicability of the developed permeabilization protocol, combined with the double-color FISH technique in specific detection of B. anthracis in soil and air samples.