Detection For Bacillus Anthracis In Environment

As the Gram-positive endospore-forming bacterium, Bacillus anthracis is an infectious pathogenic bacterium with high epidemic potential. B. anthracis spores are stable for long periods in natural environment, they may be transmitted in human populations by the way of aerosolization with high morbidity and mortality causing great to public health,and the possibility of being used in a bioterrorism attack with spores of B. anthracis. America anthrax incidents had maked B. anthracis to cause the wide attention in the world on the autumn of 2001. Due to the interference of Bacillus cereus group, isolation and identification of B. anthracis from environmental samples is still a real challenge.The multiplex PCR was developed according to the three specific virulence genes of B. anthracis, including the protective antigen gene(pag), capsule gene(cap)and S-layer protein gene(sap). Using the PCR assay all the 11 B. anthracis strains were identified as positive by the PCR assay, and the other 29 Bacillus ssp. strains were identified as negative. The DNA of the simulated blood specimens was extracted and detected by the PCR, the detection limit was 2×106 CFU/mL(i.e., the lowest2×106B. anthracis strains per 1 mL blood specimen). After enrichment culture of bacteria, the detection limit was 2×102 CFU/mL. The simulated soil samples were also prepared and detected by the PCR assay. After enrichment culture, the detection limit was 3 ×104spores/g in soil. In addition, the PCR primers of spore surface glycoprotein gene(BclA) were designed, which could effectively distinguish the B.anthracis II vaccine strain and clinical isolates by PCR amplified fragment length and sequencing results.The rapid method for detection of anthrax spores from soil specimens was developed based on γ-phage amplification. After subculture, the titer of γ-phage is1.5×109PFU/mL. The lysis activity of γ-phage was analyzed for the three different anthrax strains, which demonstrated that the strain loss of the two virulent plasmids(BAV-) was more suitable for observing the plaques formation. The optimal growth temperature, pH stability and the one-step growth cure of γ-phage were obtained. As a result, the optimal growth condition of γ-phage was cultured at 37℃ for pH 7. The replication cycle of γ-phage is 4 h, which release about 200 progeny for lysis of the single bacterium. As the established method, the total operation time is only 6- 8 h,and the threshoid sensitivity is 50 CFU/g of the simulated soil samples.The pre-concentration method of anthrax spores was developed based on the immunomagnetic beads technique. Firstly, the polyclonal antibodies were produced by rabbits using formalin inactivated Bacillus anthracis strains and spores as antigens. Then the anti-spore antibody was used to couple magnetic beads after purified, to prepare the immunomagnetic beads for capturing the anthrax spores. The optimal condition of coupling reaction was 10 mg activated carboxylate-modified magnetic beads(resuspended by 500 μL PBS) with 500 μL antibody at 37℃ for 2 h.The sucrose plus 0.5% Triton X-100 PBS solution was screened for extracting anthrax spores from the soil specimens. As the established method of immunomagnetic beads enrichment anthrax spores, the detection limit was 19 CFU/mL in pure spore suspension, and the detection limit was 100 CFU/g in simulated soil samples.