National Institute for Public Health and the Environment (RIVM), Centre for Infectious Disease Control (CIb), Laboratory for Zoonoses and Environmental Microbiology (LZO), Antonie van Leeuwenhoeklaan 9, P.O. Box 1, Bilthoven, The Netherlands.
Int J Food Microbiol. 2011 Nov 1;150(2-3):122-7. doi: 10.1016/j.ijfoodmicro.2011.07.023. Epub 2011 Jul 29.
The DNA extraction efficiency from milk, whey, soy, corn gluten meal, wheat powders and heat-treated corn grain that were spiked with Bacillus anthracis and Bacillus thuringiensis spores was determined. Two steps were critical: lysis of the spores and binding of the free DNA to the DNA binding magnetic beads in the presence of the interfering powders. For the guanidine-thiocyanate based Nuclisens lysis buffer from Biomerieux we found that between 15 and 30% of the spores survived the lysis step. As most lysis buffers in DNA/RNA extraction kits are guanidine based it is likely that other lysis buffers will show a similar partial lysis of the Bacillus spores. Our results show that soybean flour and wheat flour inhibited the DNA extraction process strongest, leading to unreliable DNA extractions when using too much of the matrix. For corn gluten meal, heat-treated corn grain and milk powders, DNA extraction efficiencies in the presence of 100mg and 10mg of powder resulted in 70%-95% reduced DNA recoveries. The inhibition was, however, reliable and intermediate compared to the inhibition by soy and wheat. Whey powder had the lowest inhibitory effect on DNA-extraction efficiency and recoveries of 70-100% could be reached when using 10mg of powder. The results show that reducing the amount of matrix leads to better DNA-extraction efficiencies, particularly for strongly inhibiting powders such as soy and wheat. Based on these results, a standard protocol to directly isolate DNA from micro-organisms present in complex matrixes such as food and feed powders was designed.
从牛奶、乳清、大豆、玉米蛋白粉、小麦粉和经过热处理的玉米粒中提取炭疽芽孢杆菌和苏云金芽孢杆菌孢子的 DNA 提取效率。有两个步骤至关重要：孢子的裂解以及在存在干扰粉末的情况下将游离 DNA 结合到 DNA 结合磁珠上。对于 Biomerieux 的基于异硫氰酸胍的 Nuclisens 裂解缓冲液，我们发现有 15%至 30%的孢子在裂解步骤中存活下来。由于大多数 DNA/RNA 提取试剂盒中的裂解缓冲液都是基于胍的，因此其他裂解缓冲液可能也会对芽孢杆菌孢子进行类似的不完全裂解。我们的结果表明，大豆粉和小麦粉对 DNA 提取过程的抑制作用最强，当使用过多基质时，会导致不可靠的 DNA 提取。对于玉米蛋白粉、经过热处理的玉米粒和乳粉，当存在 100mg 和 10mg 粉末时，DNA 提取效率会导致 70%-95%的 DNA 回收率降低。然而，与大豆和小麦的抑制作用相比，这种抑制作用是可靠的且处于中间水平。乳清粉对 DNA 提取效率的抑制作用最低，当使用 10mg 粉末时，回收率可达 70-100%。结果表明，减少基质的量会导致更好的 DNA 提取效率，特别是对于大豆和小麦等强烈抑制粉末。基于这些结果，设计了一种从食品和饲料粉末等复杂基质中直接分离存在的微生物 DNA 的标准方案。
