Sedlackova V, Dziedzinska R, Babak V, Kralik P
Veterinary Research Institute, Brno, Czech Republic.
J Appl Microbiol. 2017 Jul;123(1):116-123. doi: 10.1111/jam.13445. Epub 2017 Jun 9.
To optimize the DNA isolation for the routine detection and quantification of bacillary spores in soil and swabs. The procedure is primarily intended for diagnostics of Bacillus anthracis spores, but due to its high pathogenicity, B. thuringiensis served as its surrogate organism.
Various commercial kits for soils and swabs in combination with quantitative PCR were tested with different results. The PowerSoil DNA kit and the Ultra Clean Microbial DNA kit gave the best results for the extraction from soil and swabs, respectively. Extra beating led to considerably higher yields of DNA. The effectiveness of isolation reached 23% for DNA isolation from soil and 13% from swabs. The limit of detection was assessed to be 8·85 × 10 from 250 mg of soil and 2·79 × 10 from a swab inoculated with 100 μl of spore suspension.
The optimized protocol is suitable for direct isolation and quantification of bacillary spores without any previous culturing.
In contrast to previous studies, the isolation and quantification of spores was performed directly from the sample, without previous culture of spores on plates. Therefore, the method is suitable for such conditions where previous culturing is not possible, such as in military installations under field conditions.
优化用于土壤和拭子中杆菌孢子常规检测和定量的DNA提取方法。该方法主要用于炭疽芽孢杆菌孢子的诊断，但由于苏云金芽孢杆菌具有高致病性，因此将其作为替代生物体。
对各种用于土壤和拭子的商业试剂盒结合定量PCR进行了测试，结果各异。PowerSoil DNA试剂盒和Ultra Clean微生物DNA试剂盒分别在从土壤和拭子中提取DNA方面取得了最佳效果。额外的振荡显著提高了DNA产量。从土壤中提取DNA的分离效率达到23%，从拭子中提取的效率为13%。评估出从250毫克土壤中检测的下限为8·85×10，从接种100微升孢子悬液的拭子中检测的下限为2·79×10。
优化后的方案适用于直接分离和定量杆菌孢子，无需事先培养。
与以往研究不同，孢子的分离和定量是直接从样品中进行的，无需事先在平板上培养孢子。因此，该方法适用于无法进行事先培养的情况，如野外条件下的军事设施。
