Bisha Bledar, Brehm-Stecher Byron F
Center for Meat Safety and Quality, Department of Animal Sciences, Colorado State University, USA.
J Vis Exp. 2010 Oct 18(44):2308. doi: 10.3791/2308.
This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (≤ 500 μL) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeño peppers.
本方案描述了一种基于胶带对番茄及其他新鲜农产品表面进行采样的简单方法，随后进行胶带原位荧光杂交（FISH），以快速、无需培养地检测沙门氏菌属。带菌的胶带也可面朝下放置在选择性琼脂上进行固相富集，然后再进行检测。或者，可在非选择性肉汤中对胶带表面进行低体积液体富集（液体表面微量培养），随后进行FISH并通过流式细胞术分析。开始时，将无菌胶带与新鲜农产品接触，施加轻柔压力，然后取下胶带，从而物理提取这些表面存在的微生物。将胶带粘性面朝上安装在玻璃显微镜载玻片上，用10%福尔马林固定采样的细胞（30分钟），并使用梯度乙醇系列（50%、80%和95%；每种浓度3分钟）进行脱水。接下来，在带菌的胶带上点样含有沙门氏菌靶向DNA探针混合物的缓冲液，在55°C杂交15 - 30分钟，随后在洗涤缓冲液中短暂冲洗以去除未结合的探针。然后用DNA染料4',6-二脒基-2-苯基吲哚（DAPI）对附着的、FISH标记的细胞进行复染，并使用荧光显微镜观察结果。对于固相富集，将带菌的胶带面朝下放置在合适的选择性琼脂表面上，进行培养以使沙门氏菌小菌落原位生长，随后如上所述进行FISH和显微镜观察。对于液体表面微量培养，将带菌的胶带粘性面朝上放置，并应用硅胶灌注室，使胶带和显微镜载玻片形成一个水密腔室的底部，向其中加入少量（≤500μL）胰蛋白胨大豆肉汤（TSB）。密封入口，将腔室在35 - 37°C培养，使胶带提取的微生物基于生长进行扩增。培养后，打开入口密封，通过剧烈来回吹打使细胞脱离并混合，通过离心收获细胞并固定在10%中性缓冲福尔马林中。最后，对样品进行杂交并通过流式细胞术检查以揭示沙门氏菌属的存在。如此处所述,我们的“胶带-FISH”方法可提供对番茄表面沙门氏菌的简单快速采样和检测。我们也已将此方法用于对其他类型新鲜农产品的采样，包括菠菜和墨西哥胡椒。
