Department of Human Ecology, College of Agriculture & Related Sciences, Delaware State University, Dover, DE 19901, USA.
Food Microbiol. 2011 May;28(3):562-7. doi: 10.1016/j.fm.2010.11.007. Epub 2010 Nov 22.
A sample treatment method which separates Escherichia coli O157:H7 from lettuce and removes PCR inhibitors allowing 5 CFU/g of target cells to be detected using real-time PCR is described. Lettuce leaves inoculated with E. coli O157:H7 were rinsed with 0.025% sodium dodecyl sulfate (SDS). In this study, there were two major factors that strongly affected the recovery of E. coli O157:H7 during sample preparation, the amount of bentonite coated activated charcoal used to remove PCR inhibitors and the agitated contact time of the samples with the coated charcoal. When 3.0 g of activated carbon coated with bentonite were mixed with target cell suspensions (30 ml) derived from 50 g of lettuce, a high recovery of E. coli O157:H7 (93%) was obtained. Sample agitation with bentonite coated activated charcoal for 15 min resulted in 95% recovery of E. coli O157:H7. When a commercial DNA purification resin was used for detection of E. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 × 10(2) CFU/g. In contrast, with the use of use of bentonite coated activated charcoal and a commercial DNA purifying resin together, Rti-PCR was able to detect 5 CFU of E. coli O157:H7/g of lettuce which was equivalent to 2.8 CFU/Rti-PCR. Such a successful detection level was the result of the bentonite coated activated charcoal's ability to absorb the PCR inhibitors released from seeded lettuce during detachment. A standard curve was generated by plotting the Ct values against the log of CFU of target bacterial cells. A linear range of DNA amplification was exhibited from 5.0 × 10(0) to 1.0 × 10(4) CFU/g by using Rti-PCR.
描述了一种从生菜中分离大肠杆菌 O157:H7 并去除 PCR 抑制剂的样品处理方法，该方法允许使用实时 PCR 检测到 5 CFU/g 的目标细胞。将接种有大肠杆菌 O157:H7 的生菜叶片用 0.025%十二烷基硫酸钠（SDS）冲洗。在这项研究中，有两个主要因素强烈影响了样品制备过程中大肠杆菌 O157:H7 的回收，即用膨润土涂覆的活性炭去除 PCR 抑制剂的量和样品与涂覆的活性炭的搅拌接触时间。当 3.0 g 膨润土涂覆的活性炭与源自 50 g 生菜的目标细胞悬浮液（30 ml）混合时，可获得大肠杆菌 O157:H7 的高回收率（93%）。用膨润土涂覆的活性炭搅拌样品 15 分钟可使大肠杆菌 O157:H7 的回收率达到 95%。当不使用经膨润土处理的活性炭而使用商用 DNA 纯化树脂检测大肠杆菌 O157:H7 时，实时 PCR（Rti-PCR）未能检测到 1 × 10(2) CFU/g。相比之下，当同时使用膨润土涂覆的活性炭和商用 DNA 纯化树脂时，Rti-PCR 能够检测到 5 CFU/g 的大肠杆菌 O157:H7，相当于 2.8 CFU/Rti-PCR。如此成功的检测水平是由于膨润土涂覆的活性炭能够吸收从接种的生菜中脱落时释放的 PCR 抑制剂。通过绘制 Ct 值与目标细菌细胞 CFU 的对数之间的关系生成标准曲线。使用 Rti-PCR 展示了从 5.0 × 10(0)到 1.0 × 10(4) CFU/g 的 DNA 扩增线性范围。
