Name: rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field
Uploaded: 2011-08-02T12:00:00
Description: Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

我们要感谢的技术支持和USGS西部生态研究中心提供资金（S. Schwarzbach）和援助（K. Spragens T.格雷厄姆）M. Scullion爱达荷州技术和R.酥。主持的创新技术中心 - 国防和国土安全部（www.idhs.org），国防部和美国空军研究实验室部支持下进行这项研究。动物处理动物护理和使用委员会的美国地质调查局西部生态研究中心和美国地质调查局捆扎实验室鸟儿许可证批准的协议。任何使用本出版物中的贸易，产品或公司名称仅是描述目的，并不意味着美国政府的认可。

1。野生鸟类捕捉，使用水雾网<ol><li>滨鸟捕获，设立一个活跃的，如沼泽，海岸线，滩涂觅食网站的薄雾网。 </li><li>幻灯片特拉梅尔线循环极周围的雾网的一端垂直插入泥极。 </li><li>伸出净，特拉梅尔循环通过插入第二个极点雾网的另一端，垂直插入泥杆，确保特拉梅尔线教。 </li><li>一旦鸟被捕获，提取净的鸟类和返回绑扎站。 </li></ol> 2。泄殖腔拭子采样<ol><li>在拍摄后?...

<ol>
	<li>Spackman, E. <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+a+real-time+reverse+transcriptase+PCR+assay+for+type+A+influenza+virus+and+the+avian+H5+and+H7+hemagglutinin+subtypes.">Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.</a> J Clin Microbiol. 40, 3256-3260 (2002).</li><li>Takekawa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+detection+of+avian+influenza+virus+in+wild+birds:+evaluation+of+a+portable+rRT-PCR+system+and+freeze-dried+reagents.">Field detection of avian influenza virus in wild birds: evaluation of a portable rRT-PCR system and freeze-dried reagents.</a> J Virol Methods. 166, 92-97 (2010).</li><li>Das, A., Spackman, E., Senne, D., Pedersen, J., Suarez, D. 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+an+internal+positive+control+for+rapid+diagnosis+of+avian+influenza+virus+infections+by+real-time+reverse+transcription-PCR+with+lyophilized+reagents.">Development of an internal positive control for rapid diagnosis of avian influenza virus infections by real-time reverse transcription-PCR with lyophilized reagents.</a> J Clin Microbiol. 44, 3065-3073 (2006).</li><li>Spackman, E., Suarez, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avian+influenza+virus+RNA+extraction+from+tissue+and+swab+material.">Avian influenza virus RNA extraction from tissue and swab material.</a> Methods Mol Biol. 436, 13-18 (2008).</li><li>Chen, R., Holmes, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+inter-species+transmission+and+geographic+subdivision+in+avian+influenza+viruses+from+wild+birds.+Virology.">Frequent inter-species transmission and geographic subdivision in avian influenza viruses from wild birds. Virology.</a> , 383-3156 (2009).</li><li>Macken, C. A., Webby, R. J., Bruno, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+turnover+by+reassortment+of+replication+complex+genes+from+avian+influenza+A+virus.">Genotype turnover by reassortment of replication complex genes from avian influenza A virus.</a> J Gen Virol. 87, 2803-2815 (2006).</li><li>Dugan, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolutionary+genetics+and+emergence+of+avian+influenza+viruses+in+wild+birds.">The evolutionary genetics and emergence of avian influenza viruses in wild birds.</a> PLoS Pathog.. 4, e1000076-e1000076 (2008).</li><li>Spackman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phylogenetic+analyses+of+type+A+influenza+genes+in+natural+reservoir+species+in+North+America+reveals+genetic+variation.">Phylogenetic analyses of type A influenza genes in natural reservoir species in North America reveals genetic variation.</a> Virus Res. 114, 89-100 (2005).</li><li>Pasick, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+molecular+based+techniques+for+the+diagnosis+and+characterization+of+avian+influenza+virus+infections..">Advances in the molecular based techniques for the diagnosis and characterization of avian influenza virus infections..</a> Transbound Emerg Dis. 55, 329-338 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OIE+Manual+of+diagnostics+tests+and+vaccines+for+terrestrial+animals+(mammals,+birds+and+bees).">OIE Manual of diagnostics tests and vaccines for terrestrial animals (mammals, birds and bees).</a> 1, 258-269 (2004).</li><li>Weber, T. P., Stilianakis, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecologic+immunology+of+avian+influenza+(H5N1)+in+migratory+birds.">Ecologic immunology of avian influenza (H5N1) in migratory birds.</a> Emerg. Infect. Dis. 13, 1139-1143 (2007).</li><li>Kim, J. -. K., Negovetich, N. J., Forrest, H. L., Webster, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ducks:+the+"Trojan+Horses"+of+H5N1+influenza.">Ducks: the "Trojan Horses" of H5N1 influenza.</a> Influenza and Other Respiratory Viruses. , 121-128 (2009).</li><li>Gilbert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flying+over+an+infected+landscape:+distribution+of+highly+pathogenic+avian+influenza+H5N1+risk+in+South+Asia+and+satellite+tracking+of+wild+waterfowl.">Flying over an infected landscape: distribution of highly pathogenic avian influenza H5N1 risk in South Asia and satellite tracking of wild waterfowl.</a> Ecohealth. , (2010).</li></ol>

这里介绍的快速诊断方法，有利于高效，准确的监测禽流感病毒的野鸟样本进行测试。要少得多严格的便携式RRT - PCR标本的存储需求，适合于远程的情况下，维持冷链可能是不切实际的的，如果不可用液氮货主或干冰。此外，我们发现，随着冻干试剂的样品分析，足够直接与实验室为基础的分子分析的最小的知识领域的生物学家。这项技术是时间效率和成本效益。一个经营者，这是可行的运行三?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>的RNeasy微型离心柱</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>收集管（1.5 2毫升） </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区的RLT </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区RW1 </td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>缓冲区视网膜色素上皮</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td>无RNase水</td><td> Qiagen公司</td><td> 74106 </td><td>包含的RNeasy试剂​​盒</td></tr><tr><td> 14.3中号β-巯基乙醇的解决方案</td><td> Fisher Scientific则</td><td> BP176100 </td><td></td></tr><tr><td> 100％的乙醇</td><td> Fisher Scientific则</td><td> NC9602322 </td><td></td></tr><tr><td>涡精灵2，120V </td><td>科学工业</td><td> SI - 0236 </td><td></td></tr><tr><td> TaqMan探针流感A目标1（水解探针） </td><td>爱达荷州技术</td><td> Asay的- ASY - 0109 </td><td></td></tr><tr><td> LightCycler配套20毫升毛细管</td><td>罗氏应用科学部</td><td> 04929292001 </td><td></td></tr><tr><td> 2毫升管肩微量离心</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td>坚固耐用的高级病原鉴定设备（快速）7200 </td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td>基于Pentium的笔记本电脑与Windows XP专业版</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr><tr><td> LightCycler配套数据分析软件</td><td>爱达荷州技术</td><td></td><td>包括快速套件</td></tr></tbody></table>

rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field

野生鸟类有牵连的H5N1亚型高致病性禽流感（HPAI）的蔓延，促使监控候鸟迁徙的飞行路线。野生鸟类禽流感病毒（AIV）的采样往往是在偏远地区进行，但结果往往是因为需要运输的样本，以分子检测实验室配备的延误。实时逆转录聚合酶链反应（RRT - PCR）是一种分子的技术，提供了最准确和最敏感的禽流感病毒的诊断方法之一。 RRT - PCR所需的协议以前严格的实验室目前正在适应的领域。冷冻干燥（冻干）的试剂，不需要冷链，湿试剂水平的灵敏度的发展带来了现场的远程测试，以一个实际的目标。 在这里，我们目前在使用单位RRT - PCR（加固高级病原鉴定设备或快速，爱达荷州技术，盐湖城，UT），采用冻干试剂（A型流感的目标1的TaqMan的野生鸟类禽流感病毒快速诊断方法; Asay的ASY - 0109，爱达荷州技术）。该试剂含有适当浓度的单管所有必要的组件进行测试：引物，探针，酶，缓冲液和内部阳性对照，消除与保管不当或处理湿试剂相关的错误。便携式装置执行针对矩阵基因产生的结果，并在2-3小时的屏幕为A型流感。遗传亚型H5和H7型引物也可能设置为目标的血凝素基因。 该系统是适合收集的野生鸟类，迁徙滨鸟种在这里展示了使用泄殖腔和口咽的样品，在北加州拍摄西部鹬（Calidrus马利） 。动物处理动物护理和使用委员会的美国地质调查局西部生态研究中心和美国地质调查局捆扎实验室鸟儿许可证批准的协议。这项技术的主要优势是加快野生鸟类的诊断，增加包含在偏远地区爆发的机会。现场诊断也将证明有用的鉴定和研究野生种群的感染者。收集主机生物学（免疫和生理反应，感染）和空间生态（受感染的鸟类的迁徙表现）信息的机会将提供野生鸟类在何种程度上可以作为禽流感病毒的载体，在长距离的见解。

Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

这项研究描述了在使用一台便携式的RRT - PCR系统的野生鸟类禽流感的诊断。该方法采用冻干试剂的屏幕野生鸟类在非实验室环境中，典型的爆发情况下的优势。利用分子工具提供了快速诊断的准确和灵敏的替代品。

在野生鸟类禽流感病毒快速诊断：使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

Research

JoVE Journal

Immunology and Infection

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

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

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 ...

科研

免疫与感染

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

禽流感监测与FTA卡：实地方法，生物安全，和运输问题解决了

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Influenza Virus Propagation in Embryonated Chicken Eggs

在鸡胚的流感病毒传播

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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 ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Influenza A Virus Studies in a Mouse Model of Infection

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...