InDevR承认NIH / NIAID的U01AI070276和R43AI077112为这项工作提供资金。

1。用RT - PCR样品扩增<ol><li>提取病毒RNA的临床物质或病毒隔离QIAcube全自动核酸提取平台一起使用Qiagen公司MinElute病毒自旋套件。拔牙是200μL标本与最终洗脱体积60μL。商店提取物在-70 ° C或较低，以供日后使用。 </li><li>在模板中自由区，准备上冰的RT - PCR扩增预混，根据制造商的协议。在RT - PCR技术结合生物素，使用制造商提供的dNTP混合液进行生物素化的dNTP混合物。使用生物素化的dNTP混合物的成本低于1美元/检测。也可用于替代方法，如使用生物素标记的引物生物素掺入。甲型流感混合添加引物的最终浓度为1.0μM，1.0微米流感乙，和内部控制的0.14微米。甲型流感引物放大矩阵基因片段（1032 NT产品），流感乙引物扩增非结构性的基因片段（811 NT产品）。每个流感集成电路芯片引物包含一个5&#39;磷酸基团，以方便通过的lambda以下PCR扩增的核酸外切酶消化产生的单链DNA的引物。由此产生的单链的产品需要更短的双链等效杂交倍，并大大缩短了整体检测时间。一个预先准备好这些引物的混合物以及内部控制模板RNA可从InDevR感兴趣的研究人员在有限的基础。 </li><li>简要涡和分发18μL成薄壁PCR管的主结构。 </li><li>转让冰管到一个合适的工作区模板此外，并添加到每个反应管中加入2μl模板。 </li><li> PCR管转移到热循环，并运行以下的温度曲线：逆转录50 ° 30分钟，激活酶失活/ 95 C ° 15分钟，30秒40 PCR周期为95 ° C，55 C ° C为30岁和72 ° C为1分，并在最后72℃延伸10分钟。 </li></ol> 2。低密度基因芯片杂交RT - PCR产物<ol><li>为了生成单一的双链DNA流感集成电路芯片杂交，1.0μL的lambda核酸外切酶，所附的反应缓冲液2.2μL，0.8μL无核酸酶水相结合的酶消化混合物准备。这些款项是一个单一的样本，但可以简单地被消化的样本总数的比例。删除从热循环仪的样本和4μL准备好的混合物添加到每个反应消化的PCR产物的磷酸化链。返回到摄氏15分钟，95摄氏度10分钟才能完成酶解法和热碎片步骤度至37度的热循环仪的热循环和程序的样本。 </li><li>自定义使用的流感芯片上印有醛官能玻片应用微阵列技术公司（坦佩，AZ）。 5&#39; -氨基终止捕获序列相结合，与优化斑点缓冲区，并在终浓度为20微米印刷（除另外有一个3&#39; -生物素的修改，并发现了500 Nm的终浓度为阳性对照序列） 。使用非接触斑点的方法是，用最佳的光斑直径300微米，中心，中心间距700微米。 </li><li>从存储中删除微阵列和删除的保护板和紧迫以及周边各地坚决周围的芯片适用于一次性使用的杂交井。 </li><li>含有110毫升的纯净水5分钟在60-90 RPM使用预杂交的轨道振动筛洗一洗斌地点的幻灯片。轻轻触摸组织雨刮器以及边缘，使水恶人远离干燥的阵列。 </li><li>结合每个零散的单链DNA产品22μL2X杂交缓冲液，吸管40μL杂交到芯片井的解决方案。 </li><li>允许的幻灯片在封闭的湿度室为60分钟杂交。 </li><li>从湿度试验箱中取出的幻灯片，简单地在冲洗瓶冲洗洗缓冲液D阵列前放置到幻灯片机架。通常的洗涤缓冲液D的冲洗量是2毫升每阵列。广场滑动机架包含幻灯片洗斌含110毫升清洗缓冲液A的轨道摇床在60-90 1分钟的转速下的bin。 </li><li>从洗涤缓冲液中取出一个滑动机架，简单冲洗洗缓冲液D，转移滑动机架的bin包含洗缓冲液B，在60-90 5分钟的转速和动摇。 </li><li>轻轻擦干​​每张幻灯片上的阵列，并放置在恒温恒湿ampliPHOX色度检测步骤准备干的幻灯片。 </li></ol> 3。 ampliPHOX：标签与ampliTAG杂交的产品和校准芯片<ol><li>结合<html> ampliTAG 10μL，20μL2X ampliTAG缓冲区，10μL纯化水为每个要处理的数组。试剂卷可以简单地为样本总数的比例。请一定要准备足够的标签混合物帐户所需的所有样品阵列和校准阵列。校准需要的芯片数量取决于您是否正在执行一个完整的校准（一个新的文书或新批号的试剂），或只是一个仪器检查。对于一个完整的校准，应标有3个校准芯片和一种试剂的检查，只是一个单一的校准芯片应标。请注意，校准阵列标记之前，他们应该去通过预杂交洗涤步骤以前流感阵列。 </li><li> 40μL的标签混合传输到每个阵列，并允许标记反应进行5分钟的一个封闭的恒温恒湿。 </li><li>立即用洗涤缓冲液D阵列前放置幻灯片在幻灯片机架的冲洗瓶。转移到含有110毫升的洗涤缓冲液C的bin的机架，并在60-90 RPM震动5分钟。 </li><li>使用第二个洗斌充满了纯净水，连续执行三个简短的水逢低删除盐渣。干井的边缘轻轻触摸组织雨刷阵列。 </li><li>该芯片现在适当的标签，ampliTAG ampliPHOX检测程序的其余部分可以执行。 Photoactivati​​on和标记阵列成像应在24小时内完成，和额外的阵列，可以存储在一个黑暗的幻灯片中，直到使用。 </li></ol> 4。 ampliPHOX：校准，信号放大和成像<ol><li>打开ampliPHOX读者和确保ampliVIEW软件photoactivati​​on准备。 </li><li>确定的最佳photoactivati​​on时间，使用校准芯片。另外的简要介绍如下，还简要介绍了此过程中详细的ampliPHOX操作手册。校准芯片包含一系列的生物素标记的控制序列，利用优化校准芯片产生一个积极的信号，由软件确定的行数最大化的目标检测的灵敏度，稀释。 </li><li>删除ampliPHY从4 °，让温暖的室温，涡旋简单组合。吸取3μLampliPHY增强剂ampliPHY小瓶，10秒和涡彻底。 </li><li>均匀地转移到40μLampliPHY解决方案以及含有ampliTAG标记阵列芯片，确保无气泡存在。关闭每一个应用程序之间ampliPHY的小瓶。插入ampliPHOX读者photoactivati​​on湾的芯片的幻灯片。 </li><li>对于第一次校准阵列，使用默认photoactivati​​on时间在软件的左侧面板上的“时代”框，然后按一下绿色的“开始”按钮启动的photoactivati​​on。一旦完成，删除阵列，并用纯净水，去除多余的ampliPHY冲洗。现在应该可以看到透明的聚合体形成的斑点。 </li><li>允许聚合物点干2分钟，然后分发两滴ampliRED的阵列上，并允许染色进行2分钟。 </li><li>接下来，快速冲洗芯片与纯净水，用纸巾擦拭干。 </li><li>插入成像湾ampliPHOX读者的芯片，在成像“选项卡，然后单击”捕捉新形象“按钮。一旦影像，调整作物和保存图像。 </li><li>在分析“选项卡，选择校准掩膜和”自动放置“按钮开始分析。该软件会自动产生一个“摘要记录”显示的量化结果。基于这些结果，photoactivati​​on时间由10秒调整为第二校准阵列，并重复这个过程。 </li><li>后的最佳photoactivati​​on时间已经确定，可以使用相同的信号放大和成像协议，用于校准阵列处理的样品阵列。 </li><li>一旦图像捕获样本阵列，为您特定的阵列布局，如这里所描述的流感阵列布局的面膜可以created.The ampliVIEW软件能够执行自动图像分析和产生的流感亚型对每一个样品的结果。 </li></ol> 5。代表性的成果： <img src="/files/ftp_upload/2682/2682fig1.jpg" alt="图1"/> 图1。ampliPHOX比色法检测方法的示意图。 （一）生物素标记的靶DNA杂交当场数组中的每个，及（B）与ampliTAG标记。 （三）ampliPHY解决方案，然后补充说，（四）暴露在光线下形成可见的聚合物点。 （五）形成的聚合物点，其后沾上一种无毒的染料，以提高对比度。 ontent“&gt; <img src="/files/ftp_upload/2682/2682fig2.jpg" alt="图2"/> 图2：（一）流感低密度芯片布局。序列1-7和10-13 A和序列，8，9目标乙型流感（二）ampliPHOX（三）2009年新型H1N1流感（“猪流感”），显示了相同的检测模式标本的荧光图像双方的目标流感方法。 <img src="/files/ftp_upload/2682/2682fig3.jpg" alt="图3"/> 图3：从左至右的权利，代表ampliPHOX为A型流感H3N2病毒，人类的起源H1N1，2009年新型H1N1（猪原籍），并负标本的图像。所有3种亚型显示阵列上的视觉不同的模式。注意，只有MS2的内部控制被认为是负，表明RT - PCR扩增没有受到抑制。

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所有的作者是InDevR，公司，营利性实体的雇员。 InDevR是ampliPHOX本文所述的技术商业化。

这里介绍ampliPHOX比色法检测技术是一种快速，廉价的替代单一的颜色，密度更低的芯片应用荧光检测。示意图如图1所示，检测原理的基础上使用了光引发剂的标签（1B） 。含有一种单体溶液（1C）的存在，光线照射引起的光引发剂（ampliTAG），只有在标示的地区引发的聚合反应（1D）。虽然表现为流感的识别和亚型的DNA阵列，该技术可以扩展到检测到任何一个芯片上捕获生物素化的产?...

<table border="1"><tbody><tr><th>试剂/设备</th><th>制造商</th><th>目录＃ </th><th>评论</th></tr><tr><td> Qiagen公司MinElute病毒自旋套件</td><td> Qiagen公司</td><td> 57704 </td><td>单60μL洗脱</td></tr><tr><td> QIAcube </td><td> Qiagen公司</td><td> 9001292 </td><td>可选</td></tr><tr><td> ABI 9800快速热循环仪</td><td>美国应用生物系统公司</td><td> 4441166 </td><td></td></tr><tr><td> Qiagen公司OneStep RT - PCR检测试剂盒</td><td> Qiagen公司</td><td> 210210 </td><td>不使用的试剂盒dNTPs </td></tr><tr><td> 2X合模缓冲区</td><td> InDevR公司</td><td> MI - 5007 </td><td></td></tr><tr><td>生物素标记的dNTP混合液</td><td> InDevR公司</td><td> MI - 5009 </td><td></td></tr><tr><td> LAMBDA核酸外切酶</td><td>震中生物技术</td><td> LE032K </td><td> 2500 ü，10U/μl </td></tr><tr><td>流感集成电路芯片引物组合</td><td> InDevR </td><td> N / A </td><td>尚未可供出售</td></tr><tr><td>轨道摇床</td><td> Madell技术</td><td> ZD - 9556 - A </td><td></td></tr><tr><td>洗箱</td><td> InDevR公司</td><td> MI - 4002 </td><td></td></tr><tr><td>清洗架</td><td> InDevR公司</td><td> MI - 4003 </td><td></td></tr><tr><td> 2倍的杂交缓冲液</td><td> InDevR公司</td><td> MI - 5004 </td><td></td></tr><tr><td>校准芯片</td><td> InDevR公司</td><td> AP - 5006 </td><td></td></tr><tr><td>洗净缓冲器公元</td><td> InDevR公司</td><td> MI - 5005 </td><td></td></tr><tr><td> ampliRED </td><td> InDevR公司</td><td> AP - 5004 </td><td></td></tr><tr><td> ampliTAG </td><td> InDevR公司</td><td> AP - 5001 </td><td></td></tr><tr><td> 2X ampliTAG缓冲器</td><td> InDevR公司</td><td> AP - 5002 </td><td></td></tr><tr><td> ampliPHY，ampliPHY增强剂</td><td> InDevR公司</td><td> AP - 5003 </td><td></td></tr></tbody></table>

ampliphox colorimetric detection on a dna microarray for influenza

DNA芯片已成为一个病原体检测的有力工具。1-5例如，已被证明的能力类型和亚型流感病毒的例子很多。6-11识别和亚型流感DNA芯片应用在公共健康和诊所，以便及早发现，迅速介入，并最大限度地减少流感大流行的影响。传统的荧光是目前最常用的基因芯片检测方法。然而，由于基因芯片技术对临床使用过程，取代昂贵的仪器，成本低的检测技术表现出相似的性能特点，荧光将使芯片检测更具吸引力和成本效益。 ampliPHOX比色法检测技术是用于研究中的应用，和传统荧光11的幅度内之一为了一个近似的10倍仪器成本较低的优势，比焦荧光微阵列芯片扫描仪需要与主要的检测限，检测。另一个优点是体积小巧，仪器的可移植性和灵活性允许不同于传统的荧光仪器。因为聚合技术是不是天生荧光检测线性的，但是，它是最好的适合密度较低的芯片应用中存在一定的顺序是/没有答案所需，如病原体检测阵列。目前最大的现货密度与ampliPHOX检测兼容〜1800点/阵列。由于现场密度的限制，高密​​度微阵列不适合ampliPHOX检测。 在这里，我们目前作为一个信号放大方法对流感病毒的检测和表征（流感集成电路芯片）开发的低密度芯片ampliPHOX比色法检测技术。虽然这个协议使用一个特定的应用程序，任何芯片，结合生物素化的目标，可以标记，并以类似的方式检测ampliPHOX检测流感集成电路芯片（DNA芯片）。被捕获的芯片设计和生物素化的目标是用户的责任。一旦阵列上已捕获生物素化的目标，ampliPHOX检测可以进行与链霉亲和标签的共轭（ampliTAG）的数组，第一个标记。 （ampliPHY）使用ampliPHOX读者仪器，聚合单体溶液的光曝光后只发生在含有ampliTAG标记的目标地区。聚合物形成一种无毒的解决方案，以改善视觉对比度，成像和使用的一个简单的软件程序包（ampliVIEW）分析，随后可沾上。从整个联合国提取样本流感集成电路芯片检测结果可在约6个小时，和上面所述的ampliPHOX检测步骤，可以在大约30分钟完成。

Watch this Scientific Journal Video about ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza at JoVE.com

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

ampliPHOX比色法检测技术是作为一种廉价的替代芯片荧光检测。 ampliPHOX聚合的基础上，产生的固体聚合物点，在短短几分钟内肉眼可见。结果将被自动成像和解释一个简单但功能强大的软件包。

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

ampliphox-colorimetric-detection-on-a-dna-microarray-for-influenza

Research

JoVE Journal

Immunology and Infection

9.9K 次观看.  InDevR Inc.. ampliPHOX比色法检测技术是作为一种廉价的替代芯片荧光检测。 ampliPHOX聚合的基础上，产生的固体聚合物点，在短短几分钟内肉眼可见。结果将被自动成像和解释一个简单但功能强大的软件包。

视频: ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

质粒DNA重组流感病毒的一代

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

科研

免疫与感染

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 ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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

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 of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

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

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

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 ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

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 ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

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

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

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

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

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