为这项工作提供了资金增长爱荷华值基金奖BFBS。

1。表面取样用无菌胶带<ol><li>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。 </li><li>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。 </li><li>形成一个“C”形循环的磁带，粘方面临的表面进行采样。要做到这一点，对磁带的背面（非粘性的一面）（图1）的正方形，用拇指和中指和食指的位置举行的粘性末端。 </li><li>放置在磁带表面进行采样，并轻轻按压表面对标记的区域。不释放磁带的边缘，用食指，以确保磁带的粘端与样品表面的充分接触，避免气泡。 </li><li>使用更议案，慢慢拉磁带远离样品，身体表面结合的微生物提取。法尔胜细胞电胶带，粘边，使用通用透明办公磁带到玻璃显微镜幻灯片。确保适当的张力磁带/伸展，平坦，不皱面，使创建。这将有助于减少变形/磁带卷曲在随后的加热过程中杂交问题。 </li></ol> 2。固相浓缩和液体表面Miniculture <ol><li>固相富集朝下放置磁带木糖赖氨酸TERGITOL 4（XLT - 4）琼脂平板上，使采样细胞在琼脂表面的直接接触。磁带的接触部分模板/样本被置于与琼脂表面和磁带的一端平齐的是松散地坚持培养皿的侧墙，以方便磁带容易去除琼脂表面以下浓缩。 </li><li>倒板（以避免水汽凝结），孵育35 - 37 ° C至允许沙门氏菌足够的增长。需要丰富的细胞可检测水平的潜伏期的长短将取决于最初的沙门氏菌污染水平。我们观察到从低初始接种后8小时浓缩优秀microcolony形成。 </li><li>所需的浓缩铀期后，开放的琼脂平板上。磁带将保留其孵化过程中的粘性。轻轻按下磁带对琼脂，用食指，以确保在磁带琼脂界面形成microcolonies的最大恢复。从附着到培养皿壁的边缘抓住磁带和一个缓慢的，甚至动议删除。装载到载玻片的细胞收取的胶带，粘边，上文第1.5节所述。继续以“固定和脱水”一步。 </li><li>对于液体的表面miniculture，开始固定到载玻片采样上文第1.5节所述使用的磁带。下一步，包括磁带的模板/样本接触与非无菌硅胶灌注室（Coverwell，格雷斯生物实验室公司）的部分，形成一个密封腔，其底部是由细胞电磁带，朝上。牢固，但轻轻按入到位室，以确保防水密封。 </li><li>使用灵活的凝胶装载枪头，转移≤500μL胰酪胨大豆肉汤或其他合适的浓缩液通过商会的小进气道。透明办公胶带密封两个端口，以防止蒸发和孵化的幻灯片，在35 -37 ° C的需要足够的浓缩铀。虽然所有操作应根据良好的采样和处理方法，不育的端口密封胶带或灌注井不需要在液体表面miniculture。鱼和流式细胞仪的结合，将使沙门氏菌明确的歧视。从非目标细菌可能存在与预期来自样品本身的非目标植物的贡献最大，。继续以“固定和脱水”一步。 </li></ol> 3。固定和脱水<ol><li>对于直接表面取样或固相富集沙门氏菌 。 XLT - 4琼脂培养基上，进行磁带上的细胞固定在25分钟30 °彗星样本的接触面积覆盖500μL，10％中性福尔马林。 </li><li>丢弃到一个密封的容器（下化学罩，以尽量减少接触刺激性/有毒蒸气）的固定液。 </li><li>脱水乙醇（50％，80％和95％;每个浓度300μL/ 3分钟）。继续进行“杂交”的步骤下面。 </li><li> 500μL液体表面miniculture样品固定S，灌注商会启封，和一个灵活的凝胶装枪头是用来提取enrichate。用于快速向上和向下吹打，以帮助确保有效去除任何剩余磁带绑定细胞。 </li><li>接下来，整个500μLminiculture量转移到离心管和纺2,000 XG（5分钟）。丢弃上清液，沉淀振荡大力30-60小号，然后悬浮在同等体积的10％中性缓冲福尔马林。细胞是固定的25 ° C时为30分钟</li><li>接下来，固定液和样品悬浮细胞存储缓冲区，如下所示。该样本是2,000 XG（5分钟，25 ° C）被丢弃上清液，沉淀是振荡30大力 - 60秒，在同等体积的50％的非变性乙醇50％的重悬于磷酸盐降速，缓冲液。固定细胞，可以无限期地储存在-20 ° C（年）注：每当液体被改变（例如，当引入固定液或不同的缓冲区），重要的是彻底重悬（震荡）在一个最小的部分的细胞沉淀（约10 - 20μL）“传出”液体系统。这将有助于防止结块，并确保获得，甚至单个细胞悬浮液。继续进行“杂交”的步骤下面。 </li></ol> 4。杂交<ol><li>准备杂交/洗涤缓冲液使用的商业分子生物学级解决方案在材料表中注明。最后的缓冲组件的浓度为0.7 M氯化钠，0.1中号的TRIS pH值8.0，10 mM的EDTA，0.1％十二烷基硫酸钠，分子级的水和使用0.22微米的注射器或一杯过滤筛选，最终所需的量。使用相同的缓冲区，没有探头（见下文）此外，作为洗涤液。预热杂交和洗涤缓冲液至55 ° C。 </li><li>加入荧光标记的Sal3（Nordentoft 等，1997; 5&#39; -亚洲空运中心CAC TTC ACC交咨会的GTG - 3&#39;）的Salm - 63（Kutter 等，2006; 5&#39; - TCG ACT GAC TTC AGC TCC - 3“ ）寡核苷酸探针（材料表）预热杂交缓冲。一个5毫微微升-1的总探针浓度（如2.5毫微微升 -1每个探头;碧沙和布雷姆-施特彻，2009A） 。 </li><li>对于直接到磁带和固相浓缩样本，覆盖300μL杂交缓冲区包含探头鸡尾酒磁带的模板样本的接触面积，并在潮湿，密封的孵化室设置为55 ° C杂交磁带势必细胞如果直接接触的孵化器如幻灯片护城河仪器（材料表）用于杂交，样品为15分钟，可以同时处理&gt; 20张幻灯片。如果一个小孩的仪器（材料表），如旋转式杂交炉，幻灯片置于50 mL聚丙烯离心管中进行杂交，每管幻灯片。由于不是直接传热，这些样品是杂交更长的时间（最多30分钟）。 </li><li>杂交之后，被删除的幻灯片和含探针的杂交缓冲覆盖被丢弃。幻灯片然后预热洗涤液覆盖或预热洗涤液冲洗简单地，轻轻地吹打在一个倾斜的幻灯片（3 300μL，每冲洗）体积小的缓冲，或正式洗净（最多30分钟）浸泡在含有预热洗涤液50 mL聚丙烯离心管。根据我们的经验，虽然正式洗会提供更好的结果（未结合的探针阴霾），一个简单的冲洗是足够明确的检测沙门氏菌 。下一步，幻灯片移动到下面的“检测”步骤之前，空气干燥。 </li><li>纺纱2000 XG 5分钟的样品（新鲜固定或存储缓冲区保存在-20 ° C）是由液体表面miniculture样品的杂交，大力振荡的沉淀和再悬浮在100μL预热的杂交含有缓冲区探头的鸡尾酒。样品进行杂交，在55 ° C为30分钟加500μL预热的洗涤液，在热块或其他合适的孵化站（材料表）。用胶带绑定的样品，这些样品可能是正式洗进一步孵化到30分钟，55 °在这个间歇振荡的洗涤缓冲液，C。此外，样品可能会彻底振荡后加入500μL预热的洗涤液，立即进行分析收获（下图）。 </li><li>在沙门氏菌检测的准备。通过流式细胞仪，液体表面miniculture样品纺在2000 XG 5分钟，取上清液丢弃，样品重悬于300μL室温（〜25 ° C）PBS。如果样品需要运输到遥远的设施，或延迟预期之间的杂交和分析，样品可冷藏或冰面上举行前以ANA裂解。此外，样品可能会转移到细胞储存的缓冲液（50:50 PBS和无水乙醇混合），在-20 ° C，没有明显的损失探头一个星期举行，赋予荧光（碧沙和布雷姆 - 施特彻，2009b） 。 </li></ol> 5。检测<ol><li>磁带上的沙门氏菌的检测。通过荧光显微镜，直接到磁带或固体阶段浓缩样本覆盖〜10μLVectashield的H - 1200安装含有核染液4的媒介“,6 - diamidino - 2 -苯基吲哚（DAPI）的安装，盖玻片，然后在10分钟的黑暗孵育。 </li><li>浸油放在盖玻片，使用高倍率的油浸物镜（63X或100X）和样品检验。的DAPI过滤器是用来将成为关注的焦点样品，然后切换到适当的过滤器（绿色或红色，取决于结束标签的探头使用的染料）和沙门氏菌细胞显微镜取得视觉根据其荧光（图2和3）。 </li><li>液体表面miniculture样品的流式细胞仪检测，各种仪器可以使用，这取决于当地的可用性。在我们的实验室，PBS暂停样品转移到5 mL圆底采样管（BD猎鹰），并用一个FACSCanto流式细胞仪在647纳米（材料表）的激发。对于丰富的样品含有高数量的细菌总数，使用“低流量”设置（10μL 分钟-1）和5,000-50,000事件收集。数据分析使用FlowJo软件（版本8.8.6，树星公司，亚什兰，或）或其他合适的分析软件（图4，面板A和B）。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/2308/2308fig1.jpg" alt="图1" /> 图1。胶带使用沙门氏菌抽样。从表面人为污染的番茄。 <img src="/files/ftp_upload/2308/2308fig2.jpg" alt="图2" /> 图2。直接到磁带的采样和一个西红柿的表面（100 ×油浸物镜）鱼鼠伤寒沙门氏菌ATCC 14028检测的典型结果。得克萨斯的两个探头鸡尾酒红色标记的探针（Sal3/Salm-63）标签，这些细胞。 <img src="/files/ftp_upload/2308/2308fig3.jpg" alt="图3" /> 图3。鼠伤寒沙门氏菌 ATCC 14028 Microcolonies木糖赖氨酸TERGITOL - 4（XLT - 4）琼脂表面上形成初始接种后8小时的磁带上浓缩在37 ° C是从人为污染的西红柿的表面采样。固相浓缩，增加检测细胞的数目，也增强了细胞的rRNA的内容。 <img src="/files/ftp_upload/2308/2308fig4.jpg" alt="图4" /> 图4。胶带使用S 的采样鼠伤寒沙门从表面通过鱼类和流式细胞仪（A组）或5 h的非选择性液体的表面，在与500μL，胰酪胨大豆肉汤（B组填补了灌注腔miniculture浓缩后直接分析，人为污染的番茄）。

<ol>
	<li>Almeida, C., Azevedo, N. F., Fernandes, R. M., Keevil, C. W., Vieira, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization+method+using+a+peptide+nucleic+acid+probe+for+the+identification+of+Salmonella+spp.+in+a+broad+spectrum+of+samples.">Fluorescence in situ hybridization method using a peptide nucleic acid probe for the identification of Salmonella spp. in a broad spectrum of samples.</a> Appl. Environ. Microbiol. 76, 4476-4485 (2010).</li><li>Barnetson, R. S., Milne, L. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+sampling+for+Candida+with+adhesive+tape.">Skin sampling for Candida with adhesive tape.</a> Br. J. Dermatol. 88, 487-491 (1973).</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adhesive-tape-based+sampling+of+tomato+surfaces+combined+with+rapid+fluorescence+in+situ+hybridization+for+Salmonella+detection.">Simple adhesive-tape-based sampling of tomato surfaces combined with rapid fluorescence in situ hybridization for Salmonella detection.</a> Appl. Environ. Microbiol. 75, 1450-1455 .</li><li>Bisha, B., Brehm-Stecher, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-through+imaging+cytometry+for+characterization+of+Salmonella+subpopulations+in+alfalfa+sprouts,+a+microbiologically+complex+food+system.">Flow-through imaging cytometry for characterization of Salmonella subpopulations in alfalfa sprouts, a microbiologically complex food system.</a> Biotechnol. J. 4, 880-887 (2009).</li><li>Edwards, R. 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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?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Fungi-Tape sampling tape</td>
<td>&nbsp;</td>
<td>Scientific Device Laboratory, Des Plaines, IL</td>
<td>745</td>
<td>http://www.scientificdevice.com/</td>
</tr>
<tr>
<td>Con-Tact-It sampling tape</td>
<td>&nbsp;</td>
<td>Birko Corporation, Denver, CO</td>
<td>&nbsp;</td>
<td>http://www.birkocorp.com/</td>
</tr>
<tr>
<td>Clear office tape, generic</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Should be optically clear, have low intrinsic fluorescence</td>
</tr>
<tr>
<td>Food surface</td>
<td>&nbsp;</td>
<td>Local grocery</td>
<td>&nbsp;</td>
<td>Tomatoes (red tomatoes on the vine, not waxed or oiled) used here</td>
</tr>
<tr>
<td>Trypticase Soy Broth</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>211768</td>
<td>For non-selective liquid surface miniculture enrichment</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar base</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>223420</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Xylose-lysine-Tergitol 4 agar supplement</td>
<td>&nbsp;</td>
<td>Difco, Sparks, MD</td>
<td>235310</td>
<td>For Salmonella-selective agar (XLT-4)</td>
</tr>
<tr>
<td>Formalin solution </td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>HT5011</td>
<td>10% solution, neutral, buffered (cell fixative)</td>
</tr>
<tr>
<td>Absolute ethanol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>E7023</td>
<td>Molecular biology grade (pre-hybridization dehydration)</td>
</tr>
<tr>
<td>1.5 ml microcentrifuge tubes</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>RNase- and DNase-free</td>
</tr>
<tr>
<td>Microscope slides and cover slips</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NaCl solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>S5150</td>
<td>Molecular biology grade, 5M solution (hybridization buffer component)</td>
</tr>
<tr>
<td>Tris-EDTA buffer solution (100X concentrate)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>T9285</td>
<td>1M Tris [pH 8.0], 0.1M EDTA (hybridization buffer component)</td>
</tr>
<tr>
<td>Sodium dodecyl sulfate solution</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>L4522</td>
<td>10% solution in 18 megohm water (hybridization buffer component)</td>
</tr>
<tr>
<td>Sal3 and Salm-63 oligonucleotide probes</td>
<td>&nbsp;</td>
<td>Integrated DNA Technologies, Coralville, IA</td>
<td>&nbsp;</td>
<td>5&#x2019;-labeled with 6-carboxyfluorescein (FAM) or Texas Red (for microscopy) or Cy5 (for cytometry), HPLC-purified</td>
</tr>
<tr>
<td>Variable speed microcentrifuge</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Use rotor diameter to calculate RPM needed for RCF values described in protocol</td>
</tr>
<tr>
<td>CoverWell perfusion chamber </td>
<td>&nbsp;</td>
<td>Grace Bio-Labs Inc., Bend, OR</td>
<td>PC1R-2.0</td>
<td>Non-sterile</td>
</tr>
<tr>
<td>Gel loading pipette tips (FS MultiFlex)</td>
<td>&nbsp;</td>
<td>Thermo Fisher Scientific, Waltham, MA</td>
<td>05-408-151</td>
<td>Long, thin tips for easy access to small sampling ports and maneuverability within chamber</td>
</tr>
<tr>
<td>Aluminum heat block or precision-controlled heating station</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Eppendorf Thermomixer R dry block heating and cooling shaker used here</td>
</tr>
<tr>
<td>Bambino mini hybridization oven</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 230300</td>
<td>Slides are placed in 50 ml polypropylene centrifuge tubes for hybridization, heat transfer not direct</td>
</tr>
<tr>
<td>Slide Moat slide hybridizer</td>
<td>&nbsp;</td>
<td>Boekel Scientific, Feasterville, PA</td>
<td>Model 240000</td>
<td>Provides rapid, direct transmission of heat through glass slide</td>
</tr>
<tr>
<td>Vectashield H-1200 mounting medium with 4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Vector Laboratories, Inc., Burlingame, CA</td>
<td>H-1200</td>
<td>Minimizes quenching of fluorescence during microscopy, provides DAPI counterstain</td>
</tr>
<tr>
<td>Fluorescence microscope</td>
<td>&nbsp;</td>
<td>Various suppliers </td>
<td>&nbsp;</td>
<td>Leitz Laborlux S used here</td>
</tr>
<tr>
<td>Digital camera</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Canon PowerShot A640 camera used here</td>
</tr>
<tr>
<td>Image acquisition software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>Axiovision software v. 4.6 (Carl Zeiss) used</td>
</tr>
<tr>
<td>Adobe Photoshop</td>
<td>&nbsp;</td>
<td>Adobe Inc.</td>
<td>&nbsp;</td>
<td>For minimal processing of images (overlay of images taken in different channels)</td>
</tr>
<tr>
<td>Flow cytometer</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FACSCanto flow cytometer (BD Biosciences, San Jose, CA) with red (647 nm) excitation used</td>
</tr>
<tr>
<td>Flow cytometry analysis software</td>
<td>&nbsp;</td>
<td>Various suppliers</td>
<td>&nbsp;</td>
<td>FlowJo software v. 8.7.1 (Tree Star, Inc.) used</td>
</tr>		</tbody>
		</table>
		</div>

combination of adhesive-tape-based sampling and fluorescence in situ hybridization for rapid detection of salmonella on fresh produce

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&#39;,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。

Watch this Scientific Journal Video about Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce at JoVE.com

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

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&gt;沙门氏菌</em&gt;用荧光<em&gt;原位</em&gt;杂交（FISH）。

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

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

combination-adhesive-tape-based-sampling-fluorescence-situ

Research

JoVE Journal

Immunology and Infection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

12.9K 次观看.  Colorado State University. 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，全细胞快速检测沙门氏菌用荧光原位杂交（FISH）。

视频: Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

锁核酸流式细胞仪，荧光原位杂交（LNA流FISH）：细菌小RNA检测方法

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

科研

免疫与感染

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

杂交<em&gt;原位</em&gt;和蚊虫唾液腺，卵巢，胚胎

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

荧<em在原位</em&gt;蚊虫的有丝分裂染色体上的杂交

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Fluorescence in situ Hybridizations (FISH) for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms&#160;and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5&#39; or 3&#39; ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell.

Fluorescence in situ Hybridizations FISH for the Localization of Viruses and Endosymbiotic Bacteria in Plant and Insect Tissues

We describe here a simple fluorescence in situ hybridization (FISH) method for the localization of viruses and bacteria in insect and plant tissues. This protocol can be extended for the visualization of mRNA in whole mount and microscopic sections.

荧光<em&gt;原位</em&gt;杂交（FISH）技术对病毒和植物和昆虫组织内共生细菌的本土化

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV感染细胞中特定基因产物的定量原位荧光杂交（FISH）和免疫荧光（IF）

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...