胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品
Summary
本协议描述了简单的基于磁带的粘合剂为番茄等新鲜农产品表面取样的方法，全细胞快速检测<em>沙门氏菌</em>用荧光<em>原位</em>杂交（FISH）。
Abstract
本协议描述了一个简单的基于磁带的粘合剂番茄等新鲜农产品表面取样的方法，沙门氏菌快速培养独立检测原位杂交（FISH） 技术在磁带上的荧光。细胞收取磁带也可以正面朝下放置在选择性琼脂固相富集前检测。另外，小批量的液体浓缩（液体表面miniculture）上可以执行的磁带表面，其次是鱼，并通过流式细胞仪分析，在非选择性的肉汤。首先，无菌胶带带来接触到新鲜农产品，应用温和的压力，并删除磁带，身体中提取这些表面上的微生物。磁带安装到玻璃显微镜玻片上粘端和采样细胞固定，10％的福尔马林（30分钟），脱水梯度乙醇系列（50，80，和95％;每个浓度3分钟）。下一步，细胞电磁带发现含有沙门氏菌针对性的DNA探针鸡尾酒缓冲区和杂交为15 – 30分钟，在55℃，其次是在洗涤液去除未结合的探针的一个简短的冲洗。贴壁，鱼标记的细胞，然后counterstained DNA染料4',6 – diamidino – 2 -苯基吲哚（DAPI）的结果与用荧光显微镜观看。固相浓缩，细胞带电磁带正面朝下放置在一个合适的选择性琼脂表面，培养沙门氏菌 microcolonies鱼和显微镜如上所述，允许在原位生长。液体表面miniculture，细胞带电磁带放置粘边和硅胶灌注室应用，使磁带和显微镜幻灯片的形式融入其中一个防水室底部的胰酪胨大豆体积小（≤500微升）肉汤（TSB）的介绍。密封进气道和商会孵育在35 – 37℃，允许的磁带中提取的微生物的增长为基础的放大。潜伏期之后，进气道被启封，细胞分离和混合大力回来回移液，通过离心收获并固定在10％中性缓冲福尔马林。最后，样品杂交，并通过流式细胞仪检测， 以揭示沙门氏菌的存在。这里描述的，我们的“大盘鱼”的做法可以提供番茄表面的简单和快速采样和检测沙门氏菌。我们也用这种方法进行取样其他类型的新鲜蔬菜，包括菠菜和墨西哥辣椒。
Protocol
1。表面取样用无菌胶带选择磁带使用采样。市售真菌磁带或Con轻触采样磁带无菌和易用性，特殊包装。然而，我们发现也可用于透明（透明）通用办公磁带。 使用永久性记号笔画出1厘米2广场上的胶带10厘米的片（一纸模板可用于）非粘性的一面。这将作为一个注意到的磁带部分已用于样品的食物或环境表面的视觉引导。 形成一个“C”形循环的磁带，粘方面临的表?…
Discussion
检测产生表面上的病原体的简单快速的方法可以帮助减轻食源性疾病，提供及时的和可操作的数据。胶粘剂基于磁带的抽样方法已用于环境，临床和食品微生物学自1950年以来涉及按下“苏格兰”式磁带的表面去除的微生物，由直接的微观考试或附着的微生物为固体介质转移增长（Barnetson和米尔恩，1973年，爱德华兹和哈特曼，1952年; Evancho等，2001;丰等，1980; Lakshmanan提供夏弗纳?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
为这项工作提供了资金增长爱荷华值基金奖BFBS。
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Fungi-Tape sampling tape | Scientific Device Laboratory, Des Plaines, IL | 745 | http://www.scientificdevice.com/ | |
| Con-Tact-It sampling tape | Birko Corporation, Denver, CO | http://www.birkocorp.com/ | ||
| Clear office tape, generic | Various suppliers | Should be optically clear, have low intrinsic fluorescence | ||
| Food surface | Local grocery | Tomatoes (red tomatoes on the vine, not waxed or oiled) used here | ||
| Trypticase Soy Broth | Difco, Sparks, MD | 211768 | For non-selective liquid surface miniculture enrichment | |
| Xylose-lysine-Tergitol 4 agar base | Difco, Sparks, MD | 223420 | For Salmonella-selective agar (XLT-4) | |
| Xylose-lysine-Tergitol 4 agar supplement | Difco, Sparks, MD | 235310 | For Salmonella-selective agar (XLT-4) | |
| Formalin solution | Sigma-Aldrich, St. Louis, MO | HT5011 | 10% solution, neutral, buffered (cell fixative) | |
| Absolute ethanol | Sigma-Aldrich, St. Louis, MO | E7023 | Molecular biology grade (pre-hybridization dehydration) | |
| 1.5 ml microcentrifuge tubes | Various suppliers | RNase- and DNase-free | ||
| Microscope slides and cover slips | Thermo Fisher Scientific, Waltham, MA | |||
| NaCl solution | Sigma-Aldrich, St. Louis, MO | S5150 | Molecular biology grade, 5M solution (hybridization buffer component) | |
| Tris-EDTA buffer solution (100X concentrate) | Sigma-Aldrich, St. Louis, MO | T9285 | 1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component) | |
| Sodium dodecyl sulfate solution | Sigma-Aldrich, St. Louis, MO | L4522 | 10% solution in 18 megohm water (hybridization buffer component) | |
| Sal3 and Salm-63 oligonucleotide probes | Integrated DNA Technologies, Coralville, IA | 5’-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified | ||
| Variable speed microcentrifuge | Various suppliers | Use rotor diameter to calculate RPM needed for RCF values described in protocol | ||
| CoverWell perfusion chamber | Grace Bio-Labs Inc., Bend, OR | PC1R-2.0 | Non-sterile | |
| Gel loading pipette tips (FS MultiFlex) | Thermo Fisher Scientific, Waltham, MA | 05-408-151 | Long, thin tips for easy access to small sampling ports and maneuverability within chamber | |
| Aluminum heat block or precision-controlled heating station | Various suppliers | Eppendorf Thermomixer R dry block heating and cooling shaker used here | ||
| Bambino mini hybridization oven | Boekel Scientific, Feasterville, PA | Model 230300 | Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct | |
| Slide Moat slide hybridizer | Boekel Scientific, Feasterville, PA | Model 240000 | Provides rapid, direct transmission of heat through glass slide | |
| Vectashield H-1200 mounting medium with 4’,6-diamidino-2-phenylindole (DAPI) | Vector Laboratories, Inc., Burlingame, CA | H-1200 | Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain | |
| Fluorescence microscope | Various suppliers | Leitz Laborlux S used here | ||
| Digital camera | Various suppliers | Canon PowerShot A640 camera used here | ||
| Image acquisition software | Various suppliers | Axiovision software v. 4.6 (Carl Zeiss) used | ||
| Adobe Photoshop | Adobe Inc. | For minimal processing of images (overlay of images taken in different channels) | ||
| Flow cytometer | Various suppliers | FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used | ||
| Flow cytometry analysis software | Various suppliers | FlowJo software v. 8.7.1 (Tree Star, Inc.) used |
Referencias
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Citar este artículo
Bisha, B., Brehm-Stecher, B. F. Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce. J. Vis. Exp. (44), e2308, doi:10.3791/2308 (2010).