Food and Drug Administration, Retired, Poulsbo, WA, USA.
Int J Food Microbiol. 2011 Oct 3;149(3):209-17. doi: 10.1016/j.ijfoodmicro.2011.06.008. Epub 2011 Jul 5.
Escherichia coli O157:H7 has been linked to foodborne disease outbreaks with alfalfa sprouts. Detection of the organism in sprouts by standard cultural methods can be difficult due to the high background microflora. The objective of this study was to develop and optimize an enrichment protocol with and without post-enrichment immunomagnetic separation (IMS) for the rapid detection by real-time PCR (RTiPCR) and cultural recovery of E. coli O157:H7 from artificially contaminated alfalfa sprouts. Initially we found that the FDA BAM procedure, enriching samples in modified buffered peptone water with pyruvate and at 37°C for 5h, followed by the addition of acriflavin, cefsulodin and vancomycin (mBPWp+ACV) and static incubation at 42°C gave poor results for both PCR detection and isolation for alfalfa sprouts artificially contaminated at 0.2cfu/g. The addition of post-enrichment IMS improved detection but not isolation. This procedure was modified and optimized by changing to mBPWp with cefsulodin and vancomycin at 42°C and shaking for 24h with and without IMS prior to PCR detection and cultural isolation. Using the resulting protocol we were able to detect E. coli O157:H7 in 100% of samples of alfalfa sprouts contaminated at 0.2cfu/g. This was validated for five strains of E. coli O157:H7. Isolation was 84% without added post-enrichment IMS and 100% with IMS. The optimized procedure was effective for detection and isolation of E. coli O157:H7 from this difficult food matrix.
大肠杆菌 O157:H7 与豆芽引起的食源性疾病爆发有关。由于背景微生物区系较高，标准培养方法很难检测到豆芽中的该生物体。本研究旨在开发和优化一种富集方案，包括和不包括免疫磁分离（IMS）后处理，用于通过实时 PCR（RTiPCR）快速检测和文化回收人工污染的苜蓿芽中的大肠杆菌 O157:H7。最初，我们发现 FDA BAM 程序，在含有丙酮酸盐的改良缓冲蛋白胨水中富集样品，在 37°C 下孵育 5 小时，然后加入吖啶黄素、头孢磺啶和万古霉素（mBPWp+ACV），并在 42°C 下进行静态孵育，对人工污染 0.2cfu/g 的苜蓿芽进行 PCR 检测和分离的效果都很差。添加 IMS 后处理可提高检测率，但不能提高分离率。通过将程序修改为在 42°C 下使用含头孢磺啶和万古霉素的 mBPWp，并在进行 PCR 检测和文化分离之前进行 24 小时摇动（带或不带 IMS），对该程序进行了修改和优化。使用该方法，我们能够检测到人工污染 0.2cfu/g 的苜蓿芽中 100%的大肠杆菌 O157:H7。该方法经过了五种大肠杆菌 O157:H7 菌株的验证。不添加 IMS 后处理的分离率为 84%，添加 IMS 的分离率为 100%。该优化程序对于从这种困难的食品基质中检测和分离大肠杆菌 O157:H7 是有效的。
