Division of Molecular Biology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, Maryland, USA.
Appl Environ Microbiol. 2012 Aug;78(15):5297-304. doi: 10.1128/AEM.00794-12. Epub 2012 May 25.
The goal of this study was to develop a sensitive, specific, and accurate method for the selective detection of viable Escherichia coli O157:H7 cells in foods. A unique open reading frame (ORF), Z3276, was identified as a specific genetic marker for the detection of E. coli O157:H7. We developed a real-time PCR assay with primers and probe targeting ORF Z3276 and confirmed that this assay was sensitive and specific for E. coli O157:H7 strains (n = 298). Using this assay, we can detect amounts of genomic DNA of E. coli O157:H7 as low as a few CFU equivalents. Moreover, we have developed a new propidium monoazide (PMA)-real-time PCR protocol that allows for the clear differentiation of viable from dead cells. In addition, the protocol was adapted to a 96-well plate format for easy and consistent handling of a large number of samples. Amplification of DNA from PMA-treated dead cells was almost completely inhibited, in contrast to the virtually unaffected amplification of DNA from PMA-treated viable cells. With beef spiked simultaneously with 8 × 10(7) dead cells/g and 80 CFU viable cells/g, we were able to selectively detect viable E. coli O157:H7 cells with an 8-h enrichment. In conclusion, this PMA-real-time PCR assay offers a sensitive and specific means to selectively detect viable E. coli O157:H7 cells in spiked beef. It also has the potential for high-throughput selective detection of viable E. coli O157:H7 cells in other food matrices and, thus, will have an impact on the accurate microbiological and epidemiological monitoring of food safety and environmental sources.
本研究的目的是开发一种灵敏、特异和准确的方法，用于选择性检测食品中的存活大肠杆菌 O157:H7 细胞。鉴定了一个独特的开放阅读框（ORF），Z3276，作为检测大肠杆菌 O157:H7 的特异性遗传标记。我们开发了一种针对 ORF Z3276 的实时 PCR 检测方法，并证实该检测方法对大肠杆菌 O157:H7 菌株（n = 298）具有敏感性和特异性。使用该检测方法，我们可以检测到低至几个 CFU 当量的大肠杆菌 O157:H7 基因组 DNA 量。此外，我们已经开发了一种新的吖啶橙单偶氮（PMA）-实时 PCR 方案，可明确区分活细胞和死细胞。此外，该方案已适用于 96 孔板格式，便于大量样本的轻松和一致处理。与未处理的死细胞相比，PMA 处理的死细胞的 DNA 扩增几乎完全被抑制，而 PMA 处理的活细胞的 DNA 扩增几乎不受影响。在牛肉中同时掺入 8×10(7)个死细胞/g 和 80 CFU 活细胞/g 的情况下，我们能够在 8 小时的富集后选择性地检测到存活的大肠杆菌 O157:H7 细胞。总之，该 PMA-实时 PCR 检测方法提供了一种灵敏和特异的方法，用于选择性检测牛肉中掺入的存活大肠杆菌 O157:H7 细胞。它还有潜力用于高通量选择性检测其他食品基质中的存活大肠杆菌 O157:H7 细胞，从而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。
