我们感谢王大卫，阿纳托利Urisman，艾米奇石，Kael菲舍尔，邓健泓，和亚历山大Greninger的初步发展和优化的Virochip协议。这项工作是支持K08授予美国国立卫生研究院和雅培发现奖（消委会）和霍华德休斯医学研究所（JLD）。

Virochip检测的步骤包括:（1）从临床标本的核酸提取，（2）提取的RNA 逆转录和第二链cDNA合成，（3）随机引物的cDNA PCR扩增，（3）Cy3标记的荧光染料掺入（4）杂交标记的材料Virochip芯片，（5）扫描和分析（图1）。如下图所示的协议，将展示从儿童流感样病例鼻拭子样品的Virochip检测上使用。 引物序列引物5&#39; - GTTTCCCAGTCACGATA（N 9）-3" 引物乙5&#39; - GTTTCCCAGTCACGATA - 3" 1。核酸提取<ol><li>从临床标本的核酸提取应执行2级生物安全（BSL - 2）或更高的条件下，这些样本有潜在的传染性。 </li><li>鼻拭子样本运送病毒控股中期。分装200μL的样品至1.5毫升的Eppendorf离心管。 </li><li> 可选:通过样品通过0.22微米的过滤器（净化病毒颗粒） </li><li> 可选:用DNase（降解宿主基因组DNA和净化保护，encapsidated病毒核酸每制造商的说明使用的Turbo DNA酶试剂盒（Ambion公司）的预处理，样品） </li><li>提取的样品使用Zymo ZR的病毒RNA提取试剂盒或每Qiagen公司Ultrasens病毒试剂盒制造商的指示。 </li></ol> 2。逆转录和第二链cDNA合成（"回合一个"） <ol><li> 1 UL 40 pmol /μL底漆4 UL提取RNA。加热至65 °让C为5分钟在一个热循环仪（例如GeneAmp PCR系统9700），然后在室温× 5分钟冷却。 </li><li>添加5μL2微升5X RT缓冲液，1μL12.5毫米的dNTP，1μL水，0.5μL0.1M数码地面电视，0.5μL，SSIII RT（Invitrogen公司）组成的一个主混合。在42 ° C孵育× 60分钟。 </li><li>在一个可编程的热循环，加热至94 ° C X 2分钟（中止逆转录反应），冷却至10 ° C，脉冲离心机，然后加入5μLSequenase 1μL5X Sequenase缓冲液，3.8μL水组成的混合，和0.15μLSequenase（USB公司）。 第二链的合成，斜10 ° C至37 ° C，超过8分钟，保持8分钟，然后加热至94 ° C X 2分钟（灭活Sequenase），冷却至10 ° C（按住）。 </li><li>路可选 :一个样品的cDNA在这一点上可以存储在-20 ° C。 </li></ol> 3。随机引物PCR扩增的cDNA（"B轮"） <ol><li> 5路UL A样品添加45μL5μL的10X PCR缓冲液，1μL12.5毫米的dNTP，1μL100 pmol /μL引物b，1μL，KlenTaq LA（Sigma公司），以及37μL水组成的混合主。 </li><li>运行PCR协议如下:94 ° C，2分钟→（25周期94℃，30 S / 50 ° C，45秒/ 72 ° C，1分钟）→72 ° C，5分钟→10 ° C（持有） </li><li>路B瓶检查在1.5％琼脂糖凝胶电泳凝胶。涂片从约200 - 1000 BP应可视化（图2A） </li></ol> 4。 Cy3标记荧光染料法团<ol><li> 可选 :另一样品可与Cy5的荧光染料标记相同的芯片杂交。 </li><li>要结合AA -的dNTP，添加5号B瓶UL UL主组成的混合5 UL 10X PCR缓冲液，1 UL 12.5毫米AA -的dNTP（Invitrogen公司），1 UL 100 pmol / UL引物b，1至45， 1 UL KlenTaq洛杉矶（Sigma公司），和37 uL的水。运行PCR协议如下:94 ° C，2分钟→（15周期94℃，30 S / 50 ° C，45秒/ 72 ° C，1分钟）→72 ° C，5分钟→10 ° C（持有） </li><li>清洁道中的样本DNA清洁和每制造商的指示选矿厂5包（Zymo研究）。洗脱10微升。 </li><li>新增1 UL 1M碳酸氢钠（Sigma）和1 UL Cy5的（通用电气医疗集团）号彗星样本。 </li><li>孵育1个小时在黑暗中。 </li><li>清洁Cy3标记的样本DNA清洁和每制造商的指示选矿厂5包（Zymo研究）。洗脱12微升。 </li></ol> 5。杂交Virochip芯片<ol><li> 可选:使用1.5μL检查cDNA的浓度和染料的金额纳入上Nanodrop分光光度计（样本正常化） </li><li>在PCR地带管添加1μL的25X碎片缓冲区，5μL的5倍封闭液，9.5μLCy3标记，标记的样品（或金额正常化），和水的总体积至25μL（安捷伦） </li><li>加入25μL2X GEX杂交缓冲液（安捷伦）。离心简单，赶走气泡。 </li><li>加载到垫片幻灯片40μL的样品。 </li><li>广场安捷伦印Virochip阵列（Californi大学旧金山/安捷伦）（标有"安捷伦"下来方）垫片幻灯片上并拧紧螺钉紧。 </li><li>杂交一夜之间在65 ° C烘箱，在10转的速度设置。 </li><li>清洗阵列，预热缓冲区2（安捷伦）至37 ° C。除了在缓冲区1（安捷伦）垫片幻灯片/阵列夹心水下。在缓冲区1 × 1分钟洗净。洗，在预热的缓冲2 × 1分钟。慢慢地从缓冲区2中删除的幻灯片，要小心，以避免气泡（使用kimwipe到灯芯掉多余的水分，要小心，以避免直接接触阵列的主动方） </li><li>成幻灯片固定放置幻灯片和插入阵列扫描仪。 </li></ol> 6。 Virochip扫描和分析<ol><li>扫描Cy3标记在5微米或根据仪器的标准扫描协议（图2B）2微米阵列。 </li><li>通过目测，聚类分析，或自动化Virochip分析程序分析Virochip芯片，电子预测11。这三种方法，流感病毒很容易发现在鼻拭子样本（从流感样病例的孩子）（图2C） </li></ol> 7。代表性的成果: 是正确的协议时，应看到涂片，在琼脂糖凝胶电泳后的路乙一步（图2A）。安捷伦平台上Virochip芯片将难以直观分析（图2B），但如果病毒是目前应随时确定目视检查，聚类分析，和/或E -预测（图1C）。样品可以添加与测量，从已知的病毒（如烟草花叶病毒的MS2噬菌体）核酸作为一个检测的阳性对照的数量。 <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="图1" /> 图1。随机扩增示意图Virochip芯片处理和分析 ，从临床标本中提取核酸，用荧光染料标记，杂交Virochip芯片。在2微米分辨率的微阵列扫描和使用各种计算工具，包括电子预测分析。 <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="图2" /> 图2。在Virochip检测的步骤后随机，涂抹200 PCR扩增- 1000 BP可以用凝胶电泳观察（一）（二 ）三个Virochip微阵列8阵列/玻片上显示，与一个小区域。一吹，在右下角的插图芯片（C）使用E自动芯片病毒分析预测揭示了A型流感病毒的临床样本的存在。很高的相似性得分（S = 0.55）对应的p值接近于零，从而使这一极其显着的预测。

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

此处所述的Virochip协议是复杂的，需要细致和熟练的科研技术人员。正确的试剂浓度和PCR，标签条件，和杂交是至关重要的。 Virochip协议可以很容易地修改，以适应分析病变组织的组织提取方法，如TRIZOL（Invitrogen）的核酸提取使用。探头可以在必要时添加或删除，以获得所需的频谱和覆盖的广度，例如，可以设计和探针检测的细菌和真菌，如非病毒的目标"飞"的补充。 Virochip检测目前正在开发的一些应用，包括在临床实验室，疫情调查，纯度的药物和疫苗的筛选，和新的病毒病原发现的广谱病毒诊断。

<table border="1"><tbody><tr><th>试剂/材料名称</th><th>试剂/材料名称</th></tr><tr><td>西甲</td><td> 5&#39; - GTTTCCCAGTCACGATA（N9）-3" </td></tr><tr><td>引物b </td><td> 5&#39; - GTTTCCCAGTCACGATA - 3&#39; </td></tr><tr><td rowspan="5"> RT的master mix（5微升） </td><td> 2μL，5X RT缓冲</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL水</td></tr><tr><td> 0.5微升0.1M数码地面电视</td></tr><tr><td> 0.5μL，SSIII RT（Invitrogen公司） </td></tr><tr><td rowspan="3"> Sequenase混合（5微升） </td><td> 1μL5X Sequenase缓冲区</td></tr><tr><td> 3.8μL水</td></tr><tr><td> 0.15微升Sequenase（USB公司） </td></tr><tr><td rowspan="5">路乙PCR扩增预混试剂（45微升） </td><td> 5μL的10X PCR缓冲液</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL，100 pmol /μL的引物b </td></tr><tr><td> 1μLKlenTaq洛杉矶（Sigma公司） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="5">道中PCR的master mix（45微升） </td><td> 5 UL 10X PCR缓冲液</td></tr><tr><td> 1μL，12.5毫米的dNTP（Invitrogen公司） </td></tr><tr><td> 1μL，100 pmol /μL的引物b </td></tr><tr><td> 1μLKlenTaq洛杉矶（Sigma公司） </td></tr><tr><td> 37μL水</td></tr><tr><td rowspan="4">杂交缓冲液（25微升） </td><td> 1μL25X碎片缓冲液（安捷伦） </td></tr><tr><td> 5μL的5倍封闭液（安捷伦） </td></tr><tr><td> Cy3标记，标记的样品9.5μL（或金额正常化） </td></tr><tr><td>加水到总体积25μL </td></tr><tr><td>安捷伦Virochip芯片</td><td>待许可证;邱实验室，加州大学旧金山分校</td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

该病毒会导致许多传染病的诊断，由于固有的病毒序列的多样性以及持续出现新型病毒的病原体，如SARS冠状病毒和2009年H1N1新型流感病毒，是不是用传统的方法检测，很难。为了应对这些挑战，我们以前开发和验证一个泛病毒的基因芯片平台与检测能力上的保守序列的同源性1的基础上所有已知的病毒以及新颖变种Virochip。使用的Virochip，我们已经确定了相关病毒与呼吸道感染，包括原因不明的重大疾病住院的患者案件，灵敏度相当于或优于传统的临床试验 2-5，全方位。 Virochip也被用来确定新的病毒，包括SARS冠状病毒6,7，一种新型的鼻病毒分支5，XMRV病毒（逆转录病毒与前列腺癌）8，禽流bornavirus（消耗性疾病在鹦鹉的原因）9 ，小说和儿童呼吸道疾病和腹泻病10 cardiovirus。 Virochip目前的版本已经被移植到安捷伦的芯片平台，并根据截至2009年12月从GenBank中的病毒超过1500〜〜36000探头组成。在这里，我们展示了在处理Virochip检测从开始涉及的步骤来完成（〜24小时的周转时间），包括样品核酸的提取，使用随机引物的PCR扩增，荧光染料掺入，与芯片杂交，扫描，并分析。

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Virochip是一个泛病毒的芯片设计，同时检测保守序列同源性的基础上所有已知的病毒，以及新颖的病毒。在这里，我们演示了如何运行的Virochip检测，分析存在的已知和未知病毒的临床样本。

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.7K 次观看.  University of California, San Francisco. Virochip是一个泛病毒的芯片设计，同时检测保守序列同源性的基础上所有已知的病毒，以及新颖的病毒。在这里，我们演示了如何运行的Virochip检测，分析存在的已知和未知病毒的临床样本。

视频: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

科研

免疫与感染

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, it is therefore important to have a quick and reliable way of measuring transcriptional activity in infected cells. Traditionally, transcription has been measured either by incorporation of radioactive nucleosides such as 3H-uridine followed by detection via autoradiography or scintillation counting, or incorporation of halogenated uridine analogs such as 5-bromouridine (BrU) followed by detection via immunostaining. The use of radioactive isotopes, however, requires specialized equipment and is not feasible in a number of laboratory settings, while the detection of BrU can be cumbersome and may suffer from low sensitivity.
The recently developed click chemistry, which involves a copper-catalyzed triazole formation from an azide and an alkyne, now provides a rapid and highly sensitive alternative to these two methods. Click chemistry is a two step process in which nascent RNA is first labeled by incorporation of the uridine analog 5-ethynyluridine (EU), followed by detection of the label with a fluorescent azide. These azides are available as several different fluorophores, allowing for a wide range of options for visualization.
This protocol describes a method to measure transcriptional suppression in cells infected with the Rift Valley fever virus (RVFV) strain MP-12 using click chemistry. Concurrently, expression of viral proteins in these cells is determined by classical intracellular immunostaining. Steps 1 through 4 detail a method to visualize transcriptional suppression via fluorescence microscopy, while steps 5 through 8 detail a method to quantify transcriptional suppression via flow cytometry. This protocol is easily adaptable for use with other viruses.

This method describes the use of click chemistry to measure changes in host cell transcription after infection with the Rift Valley fever virus (RVFV) strain MP-12. Results can be visualized qualitatively via fluorescence microscopy or obtained quantitatively through flow cytometry. This method is adaptable for use with other viruses.

使用点击化学测量的影响，病毒感染宿主细胞RNA合成

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

小型化聚糖微阵列测定评定A型流感病毒血凝素的亲和力和特异性

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most biological materials do not contain dense elements capable of scattering electrons to create an image; therefore, a negative stain, which places dense heavy metal salts around the sample, is required. In order to visualize viruses in suspension under the TEM they must be applied to small grids coated with a transparent surface only nanometers thick. Due to their small size and fragility, these grids are difficult to handle and easily moved by air currents. The thin surface is easily damaged, leaving the sample difficult or impossible to image. Infectious viruses must be handled in a biosafety cabinet (BSC) and some require a biocontainment laboratory environment. Staining viruses in biosafety levels (BSL)-3 and -4 is especially challenging because these environments are more turbulent and technicians are required to wear personal protective equipment (PPE), which decreases dexterity. 
In this study, we evaluated a new device to assist in negative staining viruses in biocontainment. The device is a capsule that works as a specialized pipette tip. Once grids are loaded into the capsule, the user simply aspirates reagents into the capsule to deliver the virus and stains to the encapsulated grid, thus eliminating user handling of grids. Although this technique was designed specifically for use in BSL-3 or -4 biocontainment, it can ease sample preparation in any lab environment by enabling easy negative staining of virus. This same method can also be applied to prepare negative stained TEM specimens of nanoparticles, macromolecules and similar specimens.

This protocol provides instruction for negative staining virus samples which can easily be used in BSL-2, -3, or -4 laboratories. It includes the use of an innovative processing capsule, which protects the transmission electron microscopy grid and provides the user easier handling in the more turbulent environments within biocontainment.

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

评估细菌病原体免疫反应的声吞噬杀灭试验

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

使用基于路西法抹去的生长测定，确定化学抑制剂对细胞内弓形体贡迪生长的抑制效率

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by naturally acquired IgG antibodies specific for VAR2CSA. VAR2CSA is the parasite antigen that mediates the selective sequestration of IEs in the placenta that can cause a severe form of malaria in pregnant women, called placental malaria (PM). Protection from PM is mediated by VAR2CSA-specific antibodies that are believed to function by inhibiting placental sequestration and/or by opsonizing IEs for phagocytosis. The assay employs late-stage-synchronized IEs that have been selected in vitro to express VAR2CSA, plasma/serum-antibodies from women with naturally acquired PM-specific immunity, and the phagocytic cell line THP-1. However, the protocol can easily be modified to assay the functionality of antibodies to any parasite antigen present on the IE surface, whether induced by natural exposure or by vaccination. The assay offers simple and high-throughput evaluation, with good reproducibility, of an important functional aspect of antibody-mediated immunity in malaria. It is, therefore, useful when evaluating clinical immunity to P. falciparum malaria, a major cause of morbidity and mortality in the tropics, particularly in sub-Saharan Africa.

Measuring Naturally Acquired Phagocytosis-Inducing Antibodies to Plasmodium falciparum Parasites by a Flow Cytometry-Based Assay

The overall goal of this protocol is to provide instruction on how to measure the capacity of antibodies present in sera or plasma of individuals, naturally exposed to Plasmodium falciparum infection, to opsonize and induce phagocytosis of the parasite-infected erythrocytes (IEs).

通过流细胞学测定，测量自然获得的噬菌体诱导恶性疟Plasmodium falciparum原虫抗体

The protocol describes how to set up and run a flow cytometry-based phagocytosis assay of Plasmodium falciparum-infected erythrocytes (IEs) opsonized by ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...