Division of Soil Science, Faculty of Agriculture, Saga University, Japan.
J Microbiol Methods. 2013 Jun;93(3):177-84. doi: 10.1016/j.mimet.2013.03.007. Epub 2013 Mar 20.
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.
为了对抗炭疽杆菌的生物恐怖主义措施，提出了一种双色荧光原位杂交（FISH）技术，用于快速和特异性检测炭疽杆菌。探针是根据芽孢杆菌群的 16S 和 23S rRNA 基因的差异设计的。开发了一种新的渗透处理方案，以增强 FISH 探针进入炭疽杆菌孢子的通透性。通过在 80°C 下用 50%乙醇连续孵育步骤、在 65°C 下用 SDS/DTT 溶液（10mg/ml SDS，50mM DTT）和最后在 37°C 下用溶菌酶溶液（20mg/ml）孵育 30 分钟，获得了最高的炭疽杆菌孢子检测率（90.8±0.69）。该方案用于检测在不同密度下添加福尔马林固定孢子后的土壤和空气样品中的炭疽杆菌孢子。结果证明，双色 FISH 成功地检测到空气中的炭疽杆菌孢子，其浓度范围在 10(3)孢子/m(3)及以上。相反，对于检测土壤样品中的炭疽杆菌孢子，最低检测限为 10(7)孢子/g 干土。这些结果证实了所开发的渗透处理方案与双色 FISH 技术相结合，在土壤和空气样品中特异性检测炭疽杆菌的适用性。
