Rapid molecular detection of Bacillus anthracis spores

Bacillus anthracis as a spore-forming, aerobic, gram-positive bacterium is the causative agent of the anthrax disease. In recent years, it has been used in the bioterrorism attacks. Its spore is highly resistant to physical and chemical agents. For rapid detection of B.anthracis spore by molecular methods, it is necessary to extract genomic DNA by disrupting the spores. The aim of this study was rapid molecular detection of B.anthracis DNA by Multiplex PCR using rapid physical disruption of spore in a short time without extraction of the genome. Genome of non-virulent strain of B.anthracis spores (Stern Vaccine strain) was extracted by Mini Bead Beater-8 system by using 0.1 mm glass beads in 5000 RPM. The sample was directly used for PCR. Multiplex PCR technique was used for detection of bacterial DNA using 3 pairs of primers.Amplified PCR products generated 3 fragments of DNA that were analyzed by agarose gel electrophoresis.Detection limit and specificity of the test were determined at different spore dilutions by using other bacterial strains belonging to the same genus. By this method, B.anthracis spores could be disrupted within 3 minutes, and detected with specific primers by Multiplex PCR method. PCR products of 1083bp, 385bp, 164bp were analyzed in %2 agarose gel. By Hind III restriction enzyme, larger PCR product (1083bp) was confirmed. Detection limit by CFU/ml method was determined by 1/512 dilution of suspension with 7680/ml spore or 7 spore/μl. The results of optimization showed that the physical disruption method by glass bead and Bead Meal Homogenization has the proper speed of 3 minutes and there is no need for DNA extraction. By this method, we were able to rapidly disrupt, detect the spore of B.anthracis in samples specifically and with high sensitivity.