Name: in situ detection of bacteria within paraffin-embedded tissues using a digoxin-labeled dna probe targeting 16s rrna
Uploaded: 2015-05-21T13:00:00.000+00:00
Duration: 11 min 15 s
Description: Watch this Scientific Journal Video about In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA at JoVE.com

这项研究是由来自韩国国家研究基金会的资助（2013R1A1A3005669）和韩国保健技术研发项目，卫生部与福利的补助金（HI13C0016）的支持。

1.探针制备<ol><li>探针的PCR扩增<ol><li>为P.牙龈特异性探针，放大P的343 bp的DNA片段牙龈 16S通过PCR使用P的基因组DNA的rRNA 牙龈和下列引物:5&#39;-TGC AAC TTG CCT TAC AGA GG-3&#39;和5&#39;-ACT CGT ATC GCC CGT TAT TC-3&#39;10。用下面的循环条件进行扩增:35个循环的95℃30秒，60℃30秒，和72℃进行1分20秒，然后用5分钟延伸72℃，10,11。 </li><li>使用70-bp的DNA片段（5&#39;-CAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAG扩增真细菌探头 TCCCGCAACGAGCGCAACCC-3&#39;）合成的寡核苷酸和下列引物:5&#39;-CAG GTR CTG CMT GGY-3&#39;和5&#39;-AGG GTT GCG CTC GTT-3&#39;11。用下面的循环条件进行扩增:40个循环在9 4℃，30秒，60℃30秒和72℃1分钟20秒，然后用5分钟延伸72℃，11,12。 </li><li>并在-20℃下温育2小时，加入3M乙酸钠（pH 5.2）和100％乙醇（:1 2的比例在10）沉淀的PCR产物（≥500微升）。 </li><li>离心机将混合物在14000×g离心15分钟，重新悬浮于100μlTE缓冲液中的沉淀物，并测量260nm处光密度的DNA浓度。 </li></ol></li><li>使用DIG DNA标记和检测试剂盒DIG标签<ol><li>拌3微克探针与水的15微升的最终体积。 </li><li>使DNA变性在95℃下进行10分钟，并迅速变冷冰上。 </li><li>加入2微升10X六核苷酸组合，2微升的dNTP标记组合，和1微升克列诺酶以15微升的变性DNA。 </li><li>混合并在37℃进行24小时至48小时，并停止反应加热到65℃。 </li></ol></li><li>检查该DIG标记的探针使用DIG DNA标记和检测试剂盒的灵敏度<ol><li>手动加载1微升阳性对照探针的试剂盒中提供连续稀释和1ng对尼龙膜中的DIG标记的探针，并干燥5分钟，在室温。 </li><li>简要地浸泡的膜在2×SSC缓冲液（0.3M NaCl的30 mM柠檬酸钠，pH 7.0）中孵育该膜在80℃下1小时，以将DNA固定。 </li><li>简要地浸泡在膜中的马来酸缓冲液（MAB，0.1M马来酸，0.15M氯化钠，pH值7.5）中，用抗DIG碱性磷酸酶（AP）缀合的抗体稀释孵育（1:5,000）在6ml封闭溶液提供在试剂盒中，在室温搅拌30分钟。 </li><li>与MABT（含1％吐温20，MAB）洗膜，并应用40微升的NBT / BCIP溶液用2ml检测缓冲液（0.1M的Tris-HCl，0.1M氯化钠，0.05M的氯化镁 ，pH 9.5）中到混合的膜1分钟。如果该标记探针的灵敏度是不能让人满意，重复步骤从1.1.3到1.3.4。 </li><li>测量标记的探针（OD260）的DNA的浓度和浓度调整到100ng微升-1。 </li></ol></li><li>检查该DIG标记的探针的特异性<ol><li>变性的基因组DNA样品（25纳克3微升-1每每个样品）从各种细菌物种，包括由特定探针靶向的细菌，在95℃下进行10分钟，并迅速变冷冰上。 </li><li>手动加载在尼龙膜上，变性DNA和干燥20分钟，在室温。 </li><li>简要地浸泡在2×SSC缓冲液的膜孵育该膜在80℃下2小时，进行固定化的DNA。 </li><li>方框与在RT封闭液1小时的膜。 </li><li>稀释探头在1纳克微升-1杂交缓冲液，变性稀释探针，并应用到膜上。 </li><li>孵育该膜在45℃下1小时。 </li><li>洗膜3次2X SSC缓冲。 </li><li>简言之泡膜MAB。 </li><li>孵育抗DIG的AP缀合的抗体稀释（1:2,000）在6ml封闭溶液在RT 1小时的。 </li><li>与MABT洗膜，并应用40微升的NBT / BCIP溶液2毫升检测缓冲液混合。 </li><li>当探针未达到预期的特异性，增加杂交温度直到特异性观察。 </li></ol></li></ol> 2. 在原位杂交注意:为了避免标本和试剂的干燥，执行所有的孵化在湿盒内衬用湿纸巾。 <ol><li>去石蜡和组织切片补液<ol><li>从石蜡包埋块准备4微米厚的组织切片。福尔马林，多聚甲醛，以及锌系固定剂与本协议兼容。 </li><li>准备用DEPC处理过的水全部试剂。 </li><li>在干燥烘箱中在60℃下进行30分钟热幻灯片孵育载玻片在室温30分钟。 </li><li>沉浸在100％二甲苯幻灯片5分钟三次;用新鲜二甲苯各一次。 注:请去打蜡过程中的化学通风柜。 </li><li>沉浸在100％的乙醇5分钟载玻片两次，使用新鲜的乙醇，每次，和再水合样品以串行90％，80％和70％的乙醇溶液，每次5分钟。 </li><li>沉浸载玻片于DEPC处理水1分钟和在DEPC处理的PBS（pH 7.4）中6分钟洗幻灯片。 </li></ol></li><li>组织切片的预处理<ol><li>沉浸在0.1N盐酸载玻片20分钟，并于DEPC处理过的水冲洗载玻片30秒。 </li><li>周围绘制用蜡笔样品疏水屏障。 注意:所述疏水性屏障的大小取决于试样的大小。因此，在下面的步骤中使用的试剂的量取决于试样的大小，但必须填写的HYdrophobic屏障（50微升的小试件和400微升的大样本）。 </li><li>治疗的标本，并且在干燥烘箱50至400微升蛋白酶K（1至10微克毫升-1在DEPC处理的PBS）中于37℃进行30分钟，并在DEPC-处理的1×PBS，1分钟洗幻灯片。 </li><li>泡在4％低聚甲醛的幻灯片在DEPC-处理的PBS 10分钟，然后于DEPC处理过的PBS洗涤玻片，持续1分钟。 </li><li>浸泡载玻片在0.1M三乙醇胺 - 盐酸含0.5％乙酸酐进行20分钟（pH 8.0）中和在DEPC处理过的水冲洗载玻片30秒。 </li></ol></li><li>杂交探针和洗涤<ol><li>浸入2X SSC缓冲滑轨20分钟。 </li><li>稀释探针在1纳克微升-1在杂交缓冲液（4×SSC中，50％[体积/体积]甲酰胺，1×Denhardt溶液，10％[重量/体积]硫酸葡聚糖，0.1％[重量/体积]十二烷基硫酸钠， 0.4毫克毫升-1鲑鱼精子DNA）。对于阴性对照，用混合的标记探针10倍过量的非标记探针的。 </li><li>变性稀释探针和应用50〜400微升到组织切片。广场上的幻灯片盖玻片和密封用指甲油。 </li><li>热滑动PCR仪在90℃下进行10分钟，并孵育玻片在加湿室中O / N在45℃或最佳的温度，在步骤1.4.11确定。 </li><li>冷却该玻片在4℃下进行30分钟，除去盖玻片，并浸入滑动以串行SSC缓冲液如下:4×SSC中10分钟，预热（45℃）2×SSC中20分钟，2×SSC中10分钟，0.2×SSC中10分钟。 </li></ol></li><li>检测用抗DIG的AP缀合的抗体<ol><li>在MABT 5分钟洗幻灯片。 </li><li>拥有50至400微升阻断与1％吐温20溶液中20分钟的阻塞。 </li><li>加50至400微升抗DIG-AP抗体稀释在封闭溶液（1:1,000）anld孵育在37℃下90分钟。 </li><li>在MABT 10分钟洗幻灯片。 </li> &lt;利&gt;浸入滑动含有1％吐温20进行5分钟的检测缓冲液中。 </li><li>用50至400治疗μ1mM的左旋咪唑（在含有1％吐温20的检测缓冲液）中5分钟以灭活内源性碱性磷酸酶。 </li><li>分发50-400微升预混NBT / BCIP溶液（在1稀释于检测缓冲液:100）到每个样品孵育载玻片在潮湿的腔室在RT 2至3小时，直到开发出信号是令人满意的。 </li><li>冲洗载玻片用去离子水以停止可视化和应用50〜400微升的0.05％[重量/体积]甲基绿作为复染剂在37℃下进行10分钟。 </li><li>冲洗载玻片用去离子水，脱水在100％乙醇，并沉浸在二甲苯。 </li><li>应用80微升安装解决方案到每张幻灯片，并盖上盖玻片。 </li></ol></li></ol>

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作者什么都没有透露。

这里的协议来定位使用已经描述了DIG标记的DNA探针石蜡包埋组织中的细菌。探针靶向细菌16S rRNA基因的DNA或RNA分子，和16S rRNA基因靶向探针可设计为任意的种特异性或通用。在P的特异性杂交牙龈特异性探针P.牙龈而不是其他口腔内细菌先前已经证明10。与此相反，在真细菌探针杂交至所有细菌的基因组DNA进行测试（17种不同的种），尽管杂交效率变化12。的探针内的T核苷酸的数量决定DIG标记的效率。生产具有高灵敏度的DIG标记探针是成功实施所描述的协议中最关键的一步。通过提取蛋白和部分hydrolysi的标本用盐酸处理改进了信噪比的目标序列。在蛋白酶K的浓度的增加而增加探针的目标可访问，但降低组织保存。因此，建议以测试不同浓度的蛋白酶K，这取决于组织的类型。乙酰化步骤降低了带负电荷的探针通过酰化的带正电荷的氨基背​​景结合到组织的蛋白质。经过一系列的准备，这些步骤，组织切片容易撕裂，因此，必须谨慎处理。鱼相比，背景是最小的。然而，从组织中内源性高碱性磷酸酶活性，如骨骼和粘液腺的信号，应严格解释。 相比RNA探针PCR扩增的DNA探针是易产生和稳定。免疫组化检测杂交探针可以识别组织结构，通过与H＆E染色切片对比:上皮细胞，结缔组织，炎性浸润和血管是公区分。之一的描述的方案的局限性是不能同时应用多个探针。此外，信号的部分的表面上排除从细菌污染物是有限的。 这个协议可以适于内的组织切片16检测其他细菌转录。此外，该协议可以扩展到两倍与免疫组织化学检测的宿主细胞标记物染色。

菌发挥各种口腔疾病如牙周炎，牙髓炎，冠周炎，蜂窝组织炎，骨髓炎的病因中起作用。为了了解细菌在疾病的发病机制中的作用，并监测治疗的效果，在组织内的定位的细菌是重要的。细菌牙龈组织内与牙周炎的存在从患者已经使用各种方法被证明，包括使用荧光-电子显微镜1,2，免疫组织化学和免疫荧光细菌特异性抗体3-7，和荧光原位杂交（FISH）8标记的寡核苷酸探针靶向16S rRNA基因。然而，这些方法都没有得到广泛由于技术限制或困难使用。与抗体相比，靶向16S rRNA基因的探针是容易产生和实现的种特异性。鱼已被证明是一个极好的工具BACT的可视化ERIA在其天然环境中，如菌斑生物膜。然而，应用FISH对组织样品是由于各种组织成分的自体荧光的限制。例如，红血细胞的强的自体荧光常常妨碍荧光技术对发炎组织的应用程序时，它们涉及出血9。 为了在发炎的牙龈组织内细菌定位，因此，使用地高辛原位杂交法（DIG）标记的DNA探针已经开发并成功应用10,11。这里细菌利用P.石蜡包埋组织内的本地化的详细协议牙龈特异性和通用的真细菌的探针进行了说明。它特别着重于方法，使得类似的结果可以在其他实验室被再现的标准化。该协议允许他们组织上下文和RESU内细菌的定位LTS是高度可重复的。所描述的协议可以用于无论是在各种组织中的一种特异性或通用方式细菌本地化。的通用探针是特别有用的，以检测多微生物疾病的细菌，并研究细菌的潜在作用，在疾病，其中具体的细菌的作用是未知的。

<table><tbody><tr><td>Acetic anhydride</td><td>Sigma</td><td>6404</td><td></td></tr><tr><td>50% Dextran sulfate solution</td><td>Millipore</td><td>S4030</td><td></td></tr><tr><td>50X Denhardt&amp;rsquo;s solution</td><td>Sigma</td><td>D2532</td><td></td></tr><tr><td>DEPC</td><td>Sigma</td><td>P159220</td><td></td></tr><tr><td>DIG DNA labeling and detection kit</td><td>Roche</td><td>11 093 657 910</td><td></td></tr><tr><td>Formamide</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>ImmEdge&amp;trade; Pen</td><td>Dako</td><td>H-400</td><td></td></tr><tr><td>Levamisole</td><td>Vector</td><td>SP-5000</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>246964</td><td></td></tr><tr><td>Maleic acid</td><td>Sigma</td><td>M0375</td><td></td></tr><tr><td>Methyl green</td><td>Sigma</td><td>M6776</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P1648</td><td></td></tr><tr><td>Permount</td><td>Fisher</td><td>SP15-500</td><td></td></tr><tr><td>Salmon sperm DNA solution</td><td>Invitrogen</td><td>#15632-011</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>Sodium citrate</td><td>Duksan</td><td>D1420</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Amresco</td><td>227</td><td></td></tr><tr><td>Triethanolamine-HCl</td><td>Sigma</td><td>90279</td><td></td></tr><tr><td>Tris-HCl</td><td>Research organics</td><td>3098T</td><td></td></tr></tbody></table>

in situ detection of bacteria within paraffin-embedded tissues using a digoxin-labeled dna probe targeting 16s rrna

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

图1显示了斑点印迹DIG标记的探针，在所述试剂盒，以确定其灵敏度提供的阳性对照探针进行比较。 343 bp的P.牙龈特异性探针是25倍，比70 bp的真细菌探针更敏感。 图2示出了在从慢性牙周炎患者检测P的获得牙龈组织的原位杂交牙龈和真细菌。细菌的信号，在紫所示，分别位于组织外的上皮，固有层，并且生物膜内检测。但是，P的分布牙龈和内齿龈组织真细菌是不同的。在细菌的高放大倍率（1000倍），各种形状（球菌，棒状，纺锤形，有毛的形式）均可见用真细菌探头，而只有杆状细菌的检测P.牙龈特异性探针。细菌信号的形式分散还观察到。 Hajishengallis 等 。证实共生细菌在P的发展中的重要作用诱发牙龈牙周炎实验小鼠14。葡聚糖硫酸钠的应用（DSS），紧密连接（TJ）-disrupting化学，上龈粘膜诱导牙周炎和牙槽骨丧失的小鼠13。在真细菌探针成功地检测细菌从DSS组不是由P.检测到牙龈组织内牙龈特异性探针（ 图3）。 的真细菌的探针是用于细菌的其他疾病的潜在参与的研究是有用的。由于国际癌症研究机构指定的幽门螺杆菌为I级致癌物质胃癌15，细菌在其他癌症的发展潜力参与已被广泛研究。当SE肺活检ctions诊断患有鳞状细胞癌是原位杂交的真细菌的探针，细菌内的肿瘤细胞和周围的间质中检测两者/使用真细菌探针浸润细胞（ 图4）。 原位杂交也适用于探索细菌在口腔扁平苔藓，炎症免疫疾病，病因不明病变的存在。有趣的是，检测到不仅在上皮，而且在固有层何处观察带状浸润细菌信号。在高倍率下，细菌信号在许多细胞中（ 图5）的细胞核中检测到。 <img alt="图1" src="/files/ftp_upload/52836/52836fig1.jpg" /> 该DIG标记的探针的图1.敏感性测试。连续稀释的定制标记的探针和正对照试剂盒中提供醇探针进行斑点印迹用抗DIG-AP缀合的抗体。加入底物，使印迹与P.后牙龈特异性探针30秒研发，同时与真细菌探针的印迹1分钟开发的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52836/52836fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52836/52836fig2.jpg" /> 图2.检测从慢性牙周炎患者获得牙龈组织内的细菌。从健康位点（A）和牙周炎患者的患病部位（B）的牙龈活检切片进行苏木精-曙红（H＆E）染色或在任一体育原位杂交牙龈特异性探针或真细菌探头。是负面Ë对照与探针杂交以10倍过量的未标记探针混合。放大的区域被指示具有正方形框。代表积极信号，在紫色所示，标有细箭头。粗箭头指示组织以外的菌斑生物膜。菌斑生物膜研究的1,000X放大显示在插图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52836/52836fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52836/52836fig3.jpg" /> 图中的鼠标的牙龈组织口服共生细菌的检测3.实验牙周炎诱导在BALB / c小鼠口服接种P的牙龈 （PG）或应用DSS的牙龈上黏膜。从小鼠牙龈切片进行H＆E染色或原位杂交无 ​​论是&lt;<html> EM&gt; P上。牙龈特异性探针或真细菌探头。代表正信号，在紫所示，以箭头标示。原来的放大倍数为400倍。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52836/52836fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/52836/52836fig4.jpg" /> 图4.在检测癌症病灶的细菌。从患者确诊为鳞状细胞癌肺活检的切片用H＆E或原位杂交无 ​​论是通用的真细菌探头或阴性对照探针。放大的区域被指示具有正方形框。代表正信号，在紫所示，以箭头标示。肿瘤细胞的边框以表示虚线。ARGET ="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图5" src="/files/ftp_upload/52836/52836fig5.jpg" /> 图5.检测口腔扁平苔藓病损内的细菌。活检从颊粘膜诊断为口腔扁平苔藓的切片用H＆E或原位杂交无 ​​论是通用的真细菌探头或阴性对照探针。放大的区域被指示具有正方形框。代表积极信号，在紫色所示，标有箭头。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52836/52836fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA at JoVE.com

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

in-situ-detection-bacteria-within-paraffin-embedded-tissues-using

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

10.9K 次观看.  School of Dentistry and Dental Research Institute, Seoul National University. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

视频: In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

科研

遗传学

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫与感染

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ocean temperatures and ocean acidification, corals are increasingly under threat. To tackle these challenges, advances in cell and molecular biology have proven to be crucial for diagnosing the health of corals. Modifying some of the techniques commonly used in human medicine could greatly improve researchers&#39; ability to treat and save corals. To address this, a protocol for in situ hybridization used primarily in human medicine and evolutionary developmental biology has been adapted for use in adult corals under stress.
The purpose of this method is to visualize the spatial expression of an RNA probe in adult coral tissue that has been embedded in paraffin and sectioned onto glass slides. This method focuses on removal of the paraffin and rehydration of the sample, pretreatment of the sample to ensure permeability of the sample, pre-hybridization incubation, hybridization of the RNA probe, and visualization of the RNA probe. This is a powerful method when using non-model organisms to discover where specific genes are expressed, and the protocol can be easily adapted for other non-model organisms. However, the method is limited in that it is primarily qualitative, because expression intensity can vary depending on the amount of time used during the visualization step and the concentration of the probe. Furthermore, patience is required, as this protocol can take up to 5 days (and in many cases, longer) depending on the probe being used. Finally, non-specific background staining is common, but this limitation can be overcome.

In Situ Hybridization Techniques for Paraffin-Embedded Adult Coral Samples

The goal of this protocol is to perform in situ hybridization on adult coral samples that have been embedded in paraffin and sectioned onto glass slides. This is a qualitative method used to visualize the spatial expression of an RNA anti-sense probe in paraffin-embedded tissues.

就地石蜡镶嵌成人珊瑚样品的杂交技术

Corals are important ocean invertebrates that are critical for overall ocean health as well as human health. However, due to human impacts such as rising ...

环境科学

增强癌症组织样本中致癌驱动因素的 FISH 分析

Robust Detection of Gene Amplification in Formalin-Fixed Paraffin-Embedded Samples by Fluorescence In Situ Hybridization

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

作者聚焦：FISH 作为癌症标本中精确基因扩增评估的工具

This protocol provides a reproducible method to visualize gene amplification in formalin-fixed paraffin-embedded (FFPE) tissue specimens.

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While ...

癌症研究

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

生物学

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines

ddPCR is a highly sensitive PCR method that utilizes a water-oil emulsion system. Using a droplet generator, an extracted nucleic acid sample is partitioned into ~20,000 nano-sized, water-in-oil droplets, and PCR amplification occurs in individual droplets. The ddPCR approach is in identifying sequence mutations, copy number alterations, and select structural rearrangements involving targeted genes. Here, we demonstrate the use of ddPCR as a powerful technique for precisely quantitating rare BRAF V600E mutations in FFPE reference standard cell lines, which is helpful in identifying individuals with cancer. In conclusion, ddPCR technique offers the potential to precisely profile the specific rare mutations in different genes in various types of FFPE samples.

The goal of this video is to demonstrate how to perform&#160;automated DNA extraction from formalin-fixed paraffin-embedded (FFPE) reference standard cell lines and digital droplet PCR&#160;(ddPCR) analysis to detect rare mutations in a clinical setting. Detecting mutations in FFPE samples demonstrates the clinical utility of ddPCR in FFPE samples.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

摘要

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此视频中的章节

全挂载原位杂交法可视化蜱内脏微生物群

检测使用荧光脱氧核酶的细菌

就地石蜡镶嵌成人珊瑚样品的杂交技术

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

全山RNA荧光

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

摘要

探索更多视频

此视频中的章节

全挂载原位杂交法可视化蜱内脏微生物群

检测使用荧光脱氧核酶的细菌

就地石蜡镶嵌成人珊瑚样品的杂交技术

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

通过荧光原位杂交对福尔马林固定石蜡包埋样品中的基因扩增进行稳健检测

全山RNA荧光

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光 原位 杂交（FISH）可视化 秀丽隐杆线虫 肠道中的微生物定植和感染

采用数字液滴PCR技术检测BRAF V600E突变的福尔马林固定石蜡包埋的参考标准细胞系

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

RNA荧光原位杂交（FISH）可视化秀丽隐杆线虫肠道中的微生物定植和感染