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首页> 外文期刊>Food microbiology >Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment
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Detection of 5 CFU/g of Escherichia coli O157:H7 on lettuce using activated charcoal and real-time PCR without enrichment

机译：使用活性炭和实时PCR快速检测生菜中5 CFU / g大肠杆菌O157：H7

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摘要

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 off. coli O157:H7 without the use of the bentonite treated charcoal, the real-time PCR (Rti-PCR) failed to detect 1 x 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 x 10~0 to 1.0 x 10~4 CFU/g by using Rti-PCR.
机译：描述了一种样品处理方法，该方法从生菜中分离出大肠杆菌O157：H7并去除PCR抑制剂，从而可以使用实时PCR检测5 CFU / g靶细胞。用0.025％的十二烷基硫酸钠（SDS）冲洗接种了O157：H7大肠杆菌的莴苣叶。在这项研究中，有两个主要因素严重影响样品制备过程中大肠杆菌O157：H7的回收率，用于去除PCR抑制剂的膨润土涂层活性炭的量以及样品与涂层炭的搅拌接触时间。当将3.0 g涂覆有膨润土的活性炭与源自50 g生菜的目标细胞悬浮液（30 ml）混合时，可获得高回收率的O157：H7大肠杆菌（93％）。用膨润土包被的活性炭搅拌样品15分钟，可回收95％的大肠杆菌O157：H7。当使用市售的DNA纯化树脂进行检测时。大肠杆菌O157：H7不使用经膨润土处理的木炭，实时PCR（Rti-PCR）未能检测到1 x 10〜2 CFU / g。相反，结合使用膨润土包被的活性炭和商业化的DNA纯化树脂，Rti-PCR能够检测出5 CFU的大肠杆菌O157：H7 / g生菜，相当于2.8 CFU / Rti- PCR。如此成功的检测水平是由膨润土包被的活性炭吸收分离过程中从种子生菜释放的PCR抑制剂的能力的结果。通过将Ct值相对于目标细菌细胞的CFU的对数作图来生成标准曲线。通过Rti-PCR，DNA扩增的线性范围为5.0×10〜0至1.0×10〜4 CFU / g。

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