Mölsä Markos, Kalin-Mänttäri Laura, Tonteri Elina, Hemmilä Heidi, Nikkari Simo
Centres for Military Medicine and for Biological Threat Preparedness, Helsinki, Finland.
National Institute for Health and Welfare (THL), Helsinki, Finland.
J Microbiol Methods. 2016 Sep;128:69-73. doi: 10.1016/j.mimet.2016.07.013. Epub 2016 Jul 17.
Bacillus spp. include human pathogens such as Bacillus anthracis, the causative agent of anthrax and a biothreat agent. Bacillus spp. form spores that are physically highly resistant and may remain active over sample handling. We tested four commercial DNA extraction kits (QIAamp DNA Mini Kit, RTP Pathogen Kit, ZR Fungal/Bacterial DNA MiniPrep, and genesig Easy DNA/RNA Extraction kit) for sample inactivation and DNA recovery from two powders (icing sugar and potato flour) spiked with Bacillus thuringiensis spores. The DNA was analysed using a B. thuringiensis-specific real-time PCR assay. The detection limit was 3×10(1)CFU of spiked B. thuringiensis spores with the QIAamp DNA Mini, RTP Pathogen, and genesig Easy DNA/RNA Extraction kits, and 3×10(3)CFU with the ZR Fungal/Bacterial DNA MiniPrep kit. The results showed that manual extraction kits are effective and safe for fast and easy DNA extraction from powder samples even in field conditions. Adding a DNA filtration step to the extraction protocol ensures the removal of Bacillus spp. spores from DNA samples without affecting sensitivity.
芽孢杆菌属包括人类病原体，如炭疽芽孢杆菌，它是炭疽病的病原体以及一种生物威胁因子。芽孢杆菌属形成的孢子具有很强的物理抗性，在样品处理过程中可能保持活性。我们测试了四种商用DNA提取试剂盒（QIAGEN QIAamp DNA Mini试剂盒、RTP病原体试剂盒、ZR真菌/细菌DNA微量制备试剂盒和genesig Easy DNA/RNA提取试剂盒），用于从两种添加了苏云金芽孢杆菌孢子的粉末（糖粉和土豆粉）中进行样品灭活和DNA回收。使用苏云金芽孢杆菌特异性实时PCR测定法对DNA进行分析。使用QIAGEN QIAamp DNA Mini试剂盒、RTP病原体试剂盒和genesig Easy DNA/RNA提取试剂盒时，加标苏云金芽孢杆菌孢子的检测限为3×10¹CFU，而使用ZR真菌/细菌DNA微量制备试剂盒时检测限为3×10³CFU。结果表明，手动提取试剂盒对于在野外条件下从粉末样品中快速简便地提取DNA是有效且安全的。在提取方案中增加一个DNA过滤步骤可确保从DNA样品中去除芽孢杆菌属孢子，而不影响灵敏度。
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