作者希望认识到，发展援助和进行免疫组化米歇尔巴内斯的技术援助以及佩特里 - 汉森博士，博士鳗鱼和Timothy Brown评论。该项目是由美国农业部（USDA）CRIS项目＃MISV - 0801310和密西西比州立大学兽医学院。

<html> 1。细菌的挑战<ol><li>细菌在一夜之间成长为30 ° C摇脑心浸液肉汤。 </li><li>停止在鱼缸水的循环和添加肉汤100 1浓度（每升10毫升，例如）。 </li><li>一小时后孵化，在坦克重新启动的循环水，其余的细菌冲洗出来。 </li></ol> 2。采样<ol><li>采样时间和器官取决于研究的目的，但在我们的研究中，我们使用以下采样点：1，6，16，24，36，48，60，72，96，110，125和175小时后感染。 </li><li>在每一个时间点，安乐死六个鱼浸泡在水含MS222和样品（采样机关的选择是对什么是已知的感染过程中的）以下机关：鳃，沿侧线，肠，脾后的肌肉，肝，胃，心，头肾和躯干肾。 </li><li>采样后，立即组织学磁带加载机关和两个小时在1％福尔马林浸泡。 </li><li>固定两个小时后，从福尔马林50％的乙醇，在那里，直到嵌入保持磁带转移。 </li><li>石蜡嵌入机关通宵使用纸巾- Tek的贵宾3000组织的处理器（樱花TEK，托兰斯，加利福尼亚州）和部分在5μm的，使用了Leica RM2255 microtone（徕卡显微系统，班诺克本，伊利诺斯州）。 </li></ol> 3。切片脱蜡<ol><li>沉浸在明确的仪式，让所有的蜡溶解约5分钟，一个充满染色槽部分。电梯机架和排出多余的蜡溶剂。 </li><li>沉浸在一个明确的仪式5分钟，第二个容器的部分。经过几次的使用，取代溶剂之间的第一和第二的容器中的第一个容器和备用。 </li><li>沉浸在100％的酒精部分，除去蜡溶剂。 </li><li>沉浸在第二个100％的酒精槽部分。 </li><li>浸入70％的酒精槽部分。 </li><li>沉浸在运行的自来水，消除酒精的所有痕迹的章节。 </li></ol> 4。缓冲液的制备<ol><li>准备磷酸盐缓冲生理盐水溶解到1公升的蒸馏水和pH值8.0 g氯化钠，氯化钾0.2克，1.15克钠2 HPO 4和0.2克的KH 2 PO 4至7.4 。 </li><li> PBS稀释到0.9倍，加入900毫升的PBS到100毫升的蒸馏水。 </li><li>准备阻止加入2毫升小鼠血清的血清200μLTRITON - X 100和500μLBSA的50毫升的0.9倍PBS。 </li><li>准备50 mM的碳酸氢盐缓冲液是由溶解在500毫升蒸馏水H 2 O 2.1克碳酸氢钠3和4 g氯化钠和滴定，pH值8.2准备。 </li></ol> 5。 Immunhistochemistry <ol><li>补充水分在0.9倍PBS浸泡的部分。 </li><li>孵育30分钟，在阻断血清的幻灯片。 </li><li>孵育Ed9（100阻断血清稀释1）在一个不透明的湿盒内室温两小时的部分。 </li><li>冲洗部分PBS中4次，每次10分钟。 </li><li>在一个不透明的湿盒内室温孵育50μL二次抗体（羊抗鼠FITC标记的抗体，阻止血清稀释500倍）在两小时的部分。 </li><li>冲洗部分缓冲液中的碳酸氢盐的3倍。 </li><li>在去离子水冲洗部分简要。 </li></ol> 6。安装<ol><li>离开幻灯片，在黑暗中干燥几分钟。 </li><li>安装使用permafluor。 </li><li>安装介质来过夜，在黑暗阴凉的环境中，干燥，优先许可。 </li></ol> 7。显微镜<ol><li>组织切片观察，荧光显微镜下。在我们的实验中，我们使用奥林巴斯BX51显微镜配备了三重（FITC; MCA得克萨斯红）过滤器。相框软件（MicroFire，Goleta，加利福尼亚州）。 </li></ol> 8。代表性的成果： 由于鱼组织的自体荧光是如此之高，这些组织会出现下脐橙的三滤，方便地提供了一个细菌的背景。 在此背景下，个体FITC染色细菌会出现明亮的绿色圆点，在200X棒状将识别（图1）。 坏的部分，可以构成假阳性（由于在冲洗阶段的问题），在这种情况下，会出现大量的细菌统一目前在幻灯片上。 <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="图1"/> 图1一块肌肉的显微照片显示两个五部分。 ictaluri（箭头A和B）（200X）。 igure 2“/&gt; 图2实验的总体方案 。

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

该协议的细节在组织切片中的鲶鱼病原体爱德华ictaluri原位可视化技术。据我们所知，这是描述的第一个此类协议。 最关键的一步，在我们的经验，是在作为本议定书不足洗涤步骤4.4中所述的抗体冲洗，可能会导致假阳性。 这项技术的结果与解释的主要问题是组织的自体荧光。然而，人们发现，使用三滤，主要是解决问题的非特异性荧光绝?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td>脑灌注液</td><td>碧迪</td><td> 237200 </td></tr><tr><td> MS222 </td><td>银色实验室</td><td></td></tr><tr><td>全小鼠血清</td><td> Cappel </td><td> 55989 </td></tr><tr><td>羊抗小鼠FITC </td><td>南方生物技术</td><td> 1010年至1004年</td></tr><tr><td> Permafluor </td><td>实验室的愿景</td><td> TA - 030 - FM </td></tr><tr><td>氯化钾（KCl） </td><td>西格玛</td><td> P - 4504 </td></tr><tr><td> TRITON - X </td><td> Sigma - Aldrich公司</td><td> X100 - 6X500ML </td></tr><tr><td>牛血清白蛋白</td><td>西格玛</td><td> 85040C </td></tr><tr><td>磷酸钾（KH 2 PO 4） </td><td>西格玛</td><td> P - 5379 </td></tr><tr><td>小苏打（碳酸氢钠3） </td><td>西格玛</td><td> S - 5761 </td></tr><tr><td>氯化钠（NaCal） </td><td>西格玛</td><td> S - 9625 </td></tr><tr><td>钠磷酸氢二钠（以Na 2 HPO 4） </td><td>西格玛</td><td> S - 9390 </td></tr></tbody></table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

虽然爱德华ictaluri是斑点叉尾鮰毛虫的主要病原体，并已发现了近三十年前 1,2，到目前为止，这些作者的知识，没有方法已发展到允许在原位可视化细菌在组织学切片。 虽然细菌的本地化已在以前的研究使用 3，辐射标记 4平板计数，或发光细菌体内确定，这些研究大多只进行总值器官水平，但有一个例外 6 。此限制的特别关注，是因为大肠杆菌ictaluri有一个复杂的感染周期1,7，和它具有的各种致病因素8,9。 E.复杂的相互作用与它的主机ictaluri是在许多方面类似伤寒沙门氏菌10，这是在同一分类家庭。 在这里，我们描述的一种技术，允许检测细菌用间接免疫组织化学，使用单克隆Ed9抗体安斯沃思等11。 简单地说，一个封闭血清应用于石蜡包埋组织切片，以防止非特定招标。然后，部分孵育与初级抗体：E. ictaluri特异性单克隆抗体Ed9。多余的抗体冲洗，加入FITC标记的抗体。冲洗后，部分安装带有荧光的具体安装介质。 这使得对大肠杆菌的检测ictaluri鮰组织的组织切片原位。

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

在这里，我们描述了一个程序，允许标签<em爱德华ictaluri原位</em在斑点叉尾鮰的组织切片<em&gt;斑点叉尾毛虫</em&gt;作为一个小学，和荧光FITC标记抗体作为辅助用单克隆抗体间接免疫组化Ed9。这允许使用荧光显微镜观察细菌的检测。

使用荧光免疫化学检测爱德华ictaluri</em斑点叉尾鮰>（一毛虫）样品

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

use-fluorescent-immuno-chemistry-for-detection-edwardsiella-ictaluri

Research

JoVE Journal

Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

10.4K 次观看.  Mississippi State University. 在这里，我们描述了一个程序，允许标签

视频: Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

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

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&deg;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 (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;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 jalape&ntilde;o peppers.

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

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (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 ...

科研

免疫与感染

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

检测荧光纳米粒子的相互作用与原发性免疫细胞亚群流式细胞仪的

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

荧光目标阵列的T细胞反应评价中的应用<em&gt;在体内</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

对于检测磁力搅拌器方法<em&gt;旋毛虫</em&gt;幼虫肌肉样品

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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 ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

淋巴瘤组织学标本中多路荧光组织化学染色、成像与分析

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

病毒测试,水样中检测病毒的一种使用点方法

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

使用荧光免疫化学检测爱德华ictaluri（一毛虫）样品