Li Baoguang, Hu Zonglin, Elkins Christopher A
Center for Food Safety and Applied Nutrition, Division of Molecular Biology, Food and Drug Administration.
J Vis Exp. 2014 Feb 1(84):e50967. doi: 10.3791/50967.
A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.
一个独特的开放阅读框（ORF）Z3276被鉴定为大肠杆菌O157:H7的特异性遗传标记。通过靶向ORF Z3276开发了一种qPCR检测方法用于检测大肠杆菌O157:H7。利用该检测方法，我们能够检测低至几个拷贝的大肠杆菌O157:H7基因组DNA。通过对大量大肠杆菌O157:H7菌株（n = 369）和非O157菌株（n = 112）进行深入的验证试验，证实了该检测方法的灵敏度和特异性。此外，我们将单叠氮化丙锭（PMA）处理程序与新开发的qPCR方案相结合，用于从死细胞中选择性检测活细胞。与PMA处理的活细胞DNA扩增几乎不受影响形成对比，PMA处理的死细胞DNA扩增几乎完全受到抑制。此外，该方案已被修改并适用于96孔板形式，以便轻松、一致地处理大量样品。预计该方法将对食品安全和环境源的准确微生物学和流行病学监测产生影响。
