The authors have no acknowledgements.

1.棉签取样的场<ol><li>穿一双干净的手套。 </li><li>测量取样区域的大小，而无需使用卷尺或直尺接触到表面。尝试尽可能准确地估计该地区并填写报告表（补充表1）。 </li><li>检查可能的泄漏和标签样本运输袋，包棉签拭子套件。 </li><li>移动拭子穿过取样面积如下:在水平方向的行程，在垂直方向的一个行程，并在对角方向的一个笔划。不要擦拭表面面积超过700 平方厘米大。 </li><li>将每个拭子插入管并拧紧瓶盖。 </li></ol> 2.储存和运输拭子的实验室<ol><li>保持在4℃拭子长达48小时。如果需要较长时间的存储，存储所述拭子在-20℃（或-70℃）。 </li><li>保持管在0-4°C（ 即。在隔热容器运送至实验室和船舶中收集的24-48小时内使用冷包）。 </li></ol> 3.病毒浓度，病毒RNA提取纯化注意:所有的离心步骤使用台式离心机以5,000×g离心5分钟，在室温下，除非另有说明。与通用核酸提取（UNEX）缓冲区时要格外小心。戴上护目镜或面罩。 <ol><li>标签1个15毫升管，并为每个样品一种RNA的中型柱。包括在每个实验中的负提取控制。 </li><li>要为10拭子准备裂解液，将25ml UNEX缓冲与25毫升的PBST混合（PBS pH值7.2含0.02％吐温80）。 </li><li>添加噬菌体MS2悬浮液（10 6 PFU /微升）50微升到50毫升的裂解溶液。 </li><li>将棉签在15毫升管加入5 ml裂解液。混合并孵育在室温下10分钟。 </li><li>5毫升100％乙醇添加至每个管并涡旋10秒。 </li><li>小心取出15毫升管拭子（它包含UNEX缓冲）轻轻压在管的一侧以除去多余的液体，然后丢弃棉签。其余各卷应该是9-10毫升之间。 </li></ol> 4.中型柱病毒核酸提取<ol><li>小心地从步3.6 4.5毫升UNEX /乙醇混合物转移到一个中型柱，离心，弃滤液。 </li><li>装载从同一混合物另4.5毫升到同一列中，离心，并弃去滤液。 </li><li>对于第一次洗涤，添加3.5毫升70％乙醇到中型柱，离心并弃去滤液。 </li><li>对于第二次洗涤，加入另一3.5毫升70％乙醇到中型柱。离心并弃去滤液。 </li><li>对于干式旋，离心迷笛列，除去乙醇所有痕迹（可产生负面影响您的病毒RNA恢复）。 </li><li>放置南部列在一个新的15ml离心管中。加入250微升洗脱缓冲到中型柱;离心分离得到最高的病毒RNA恢复之前等待1分钟。 </li><li>存储所提取的核酸在-70℃或立即进行到步骤5。 </li></ol> 5.浓缩病毒核酸RNA使用清洁和集中套件<ol><li>添加的RNA结合缓冲液在上述步骤4.7和涡洗脱10秒RNA的250μL500μL。 </li><li>加入750μL100％乙醇，涡旋10秒。 </li><li>标记每个样品离心柱。加载750微升样品到离心柱。 </li><li>离心旋转柱以12,000 xg离心1分钟。丢弃的流量通过。 </li><li>加载剩余的750微升到旋转柱和离心机以12,000 xg离心1分钟。 </li><li>为预洗，以12,000 xg离心400μL的RNA的准备缓冲器添加到每个旋转柱和离心1分钟。丢弃的流量通过。 </li><li>对于第一次洗涤，以12,000 xg离心添加800μL的RNA洗涤缓冲液到每个旋转柱和离心1分钟。丢弃的流量通过。 </li><li>对于第二次洗涤，加入400μL的RNA的洗涤缓冲液以12,000 xg离心旋转柱和离心1分钟。丢弃的流量通过。 </li><li>对于干式纺丝，离心旋转柱以12,000 xg离心以除去洗涤缓冲液的所有痕迹2分钟。 </li><li>仔细离心柱转移到一个干净的1.5 ml离心管。 </li><li>添加25微升洗脱缓冲液到旋转柱并孵育在室温下1分钟。这会增加从塔病毒RNA的回收。 </li><li>通过以10,000 xg离心离心旋转柱1分钟收集RNA。 </li><li>无论是在-70℃直接进行RT-qPCR的（步骤6）或商店的RNA。 </li></ol> 6.基因型组I的Mutiplex RT-qPCR的检测，和II诺如病毒，和大肠杆菌噬菌体的MS 2（补充表2） <ol><li>清洁的工作表面，pipettES，并与核糖核酸酶净化解决方案的离心机，以减少可能的污染。 </li><li>在冰上解冻RT-qPCR的试剂。在冰上解冻RNA（在一个单独的区域/间）。 </li><li>确定反应的数量和它们添加更多的至少10％（ 例如 ，如果需要10反应，使11个主混合物）。 </li><li>涡单个主结构组件，除了25X RT-qPCR的酶，5秒。小心通过用手指轻弹管混合酶。 </li><li>短暂离心母液成分为5秒。 </li><li>根据试剂盒说明书准备诺如病毒GI和GII的实时荧光定量RT-PCR检测预混和添加特定的诺如病毒的寡核苷酸引物和探针在1.5 ml离心管（补充表3）。 </li><li>吹打5-10倍上下混合母液（不推荐涡旋）。等分试样22微升在实时PCR的96孔板的每个孔中的主混合物。 </li><li>涡样品RNA5秒，并进行了简短（5秒）离心收集。 </li><li>加入样品RNA和GI和GII阳性对照3μL到RT-qPCR的板（遵循从表2模板）。添加核酸酶Freewater的3微升在无模板对照（NTC）的孔中。 </li><li>密封用光学粘合剂膜实时板。 </li><li>在1300 xg离心仔细离心实时板1分钟，以除去任何气泡或液滴可能存在于孔中。 </li><li>设置了实时PCR仪和设置下列热循环条件:1）10分钟的RT步骤。在45℃（2）激活Taq聚合酶，10分钟。在95℃，（3）在95℃下45个循环的15秒，60秒，在60℃。 </li></ol> 7.诺如病毒的定量的拭子样品<ol><li>检查的阳性和阴性对照的结果来验证，RT-qPCR的结果（ 补充表3）。 </li><li>确定每个标准曲线是否满足可接受值在补充表2中给出，并计算使用公式1的标准曲线的总体标准偏差。 （公式1）％的效率= 100×10（平均Ct值截距）/斜率 。 </li><li>如果在PCR仪软件不计算斜率为每个标准曲线，确定斜率和R使用统计软件2值回归（ 例如 Excel，SPSS，SAS或）。此外，计算公式如下I标准曲线％的效率</li><li>记录由热循环仪软件基于满足补充表4规定的标准，标准曲线所有测试样本计算出来的RNA拷贝数。重新运行与标准曲线牛逼帽子的样品不符合标准或有假阳性对照。检查大肠杆菌噬菌体MS2，其中加入作为内部对照来监测PCR抑制的Ct值。 </li><li>确定RNA拷贝p的总数由总RNA洗脱剂的体积（25至50μl）的该用于RT-qPCR的反应中的RNA（3-5微升）的比例，在步骤7.2计算出的RNA拷贝数乘法器样品。可替代地，通过用分析的对象的表面积（厘米2）除以总RNA拷贝数计算出的RNA浓度。 </li></ol> 8.基因分型实时定量RT-PCR阳性样品由赫米嵌套常规PCR扩增<ol><li>制备第一轮主混合物的每个引物组的RT-PCR测定的（ 补充表1,2和5）。 </li><li>诺如病毒添加RNA阳性的5微升至预混20μL。 </li><li>在下列条件下运行的RT-PCR:在42℃（2）激活Taq聚合酶，15分钟30分钟（1）RT步骤在95℃，和（3）在95℃下的30秒40个循环，30秒，在50℃，并在72℃下60秒。后40个循环，孵育在72℃另外10分钟。 </li><li>准备为每个引物组的RT-PCR测定的（ 补充表2和5）在第二轮脑膜混合物。 </li><li>加入23μl反应混合液，并添加第一轮RT-PCR产物（从第一轮）2微升，每管（最好是第一轮产品应在无RNase水稀释1/10）。 </li><li>重复步骤8.3。 </li><li>制备在100ml 1×Tris-乙酸EDTA的2％琼脂糖凝胶（40毫摩尔Tris，20mM的乙酸，和1mM EDTA，pH为8.3）。通过在微波炉中加热该混合物琼脂糖溶解后，将混合物冷却到60-70℃后加入10微升每100mL制得的凝胶的核酸染色，倒入该凝胶并插入梳子。让凝胶沉降至少30分钟。 </li><li>混合15微升各RT-PCR产物与6×上样染料的3微升。负荷各样品的15微升和electrophorese在100V琼脂糖凝胶1小时。 </li><li>从凝胶合适的大小（330个基点GI和341个基点GII）的消费目标RT-PCR片段d使用商业凝胶提取试剂盒纯化RNA。纯化的PCR产物现在可用于Sanger测序。 </li></ol>

<ol>
	<li>Isakbaeva, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+transmission+on+cruise+ship.">Norovirus transmission on cruise ship.</a> Emerg. Infect. Dis. 11, 154-158 (2005).</li><li>Lopman, B. A., Gasta&#241;aduy, P., Park, G. W., Hall, A. J., Parashar, U. D., Vinj&#233;, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+transmission+of+norovirus+gastroenteritis.">Environmental transmission of norovirus gastroenteritis.</a> Curr. Opin. Virol. 2 (1), 1-7 (2011).</li><li>Malek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreak+of+norovirus+infection+among+river+rafters+associated+with+packaged+delicatessen+meat,+Grand+Canyon,+2005.">Outbreak of norovirus infection among river rafters associated with packaged delicatessen meat, Grand Canyon, 2005.</a> Clin Infect Dis. 48 (1), 31-37 (2009).</li><li>Atmar, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norwalk+virus+shedding+after+experimental+human+infection.">Norwalk virus shedding after experimental human infection.</a> Emerg. Infect. Dis. 14 (10), 1553-1557 (2008).</li><li>Glass, R. I., Parashar, U. D., Estes, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+gastroenteritis.">Norovirus gastroenteritis.</a> N. Engl. J. Med. 361 (18), 1776-1785 (2009).</li><li>Park, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+New+Environmental+Sampling+Protocol+for+Detection+of+Human+Norovirus+on+Inanimate+Surfaces.">Evaluation of a New Environmental Sampling Protocol for Detection of Human Norovirus on Inanimate Surfaces.</a> Appl. Environ. Microbiol. 81 (17), 5987-5992 (2015).</li><li>Barker, J., Jones, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+spread+of+infection+caused+by+aerosol+contamination+of+surfaces+after+flushing+a+domestic+toilet.">The potential spread of infection caused by aerosol contamination of surfaces after flushing a domestic toilet.</a> J. Appl. Microbiol. 99, 339-347 (2005).</li><li>Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosolization+of+a+Human+Norovirus+Surrogate,+Bacteriophage+MS2,+during+Simulated+Vomiting.">Aerosolization of a Human Norovirus Surrogate, Bacteriophage MS2, during Simulated Vomiting.</a> PloS one. 10, 0134277 (2015).</li><li>Atmar, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+50%+human+infectious+dose+for+Norwalk+virus.">Determination of the 50% human infectious dose for Norwalk virus.</a> J. Infect. Dis. 209 (7), 1016-1022 (2014).</li><li>Petrignani, M., van Beek, J., Borsboom, G., Richardus, J. H., Koopmans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+introduction+routes+into+nursing+homes+and+risk+factors+for+spread:+a+systematic+review+and+meta-analysis+of+observational+studies.">Norovirus introduction routes into nursing homes and risk factors for spread: a systematic review and meta-analysis of observational studies.</a> J. Hosp. Infect. 89 (3), 163-178 (2015).</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=Centers+for+Disease+Control+Prevention.+Norovirus+outbreak+in+an+elementary+school--District+of+Columbia,+February+2007.">Centers for Disease Control Prevention. Norovirus outbreak in an elementary school--District of Columbia, February 2007.</a> MMWR. Morb. Mortal. Wkly. Rep. 56 (51-52), 1340-1343 (2008).</li><li>Cheesbrough, J. S., Barkess-Jones, L., Brown, 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=Possible+prolonged+environmental+survival+of+small+round+structured+viruses.">Possible prolonged environmental survival of small round structured viruses.</a> J. Hosp. Infect. 35, 325-326 (1997).</li><li>Julian, T. R., Tamayo, F. J., Leckie, J. O., Boehm, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+surface+sampling+methods+for+virus+recovery+from+fomites.">Comparison of surface sampling methods for virus recovery from fomites.</a> Appl. Environ. Microbiol. 77, 6918-6925 (2011).</li><li>Taku, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+detection+of+caliciviruses+from+food+contact+surfaces.">Concentration and detection of caliciviruses from food contact surfaces.</a> J. Food. Prot. 65, 999-1004 (2002).</li><li>Scherer, K., Ellerbroek, L., Schulenburg, J., Johne, R., Klein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+a+swab+sampling+method+for+the+detection+of+norovirus+and+rotavirus+on+artifically+contaminated+food+and+environmental+surfaces.">Application of a swab sampling method for the detection of norovirus and rotavirus on artifically contaminated food and environmental surfaces.</a> Food. Environ. Virol. 1 (42), 42-49 (2009).</li><li>Herzog, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+sample+recovery+efficiency+for+bacteriophage+P22+on+fomites.">Evaluation of sample recovery efficiency for bacteriophage P22 on fomites.</a> Appl. Environ. Microbiol. 78, 7915-7922 (2012).</li><li>Vega, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CaliciNet:+A+Novel+Surveillance+Network+for+Norovirus+Gastroenteritis+Outbreaks+in+the+United+States.">CaliciNet: A Novel Surveillance Network for Norovirus Gastroenteritis Outbreaks in the United States.</a> Emerging Infectious Diseases. 17 (8), 1389-1395 (2011).</li><li>Rolfe, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+internally+controlled,+one-step,+real-time+RT-PCR+assay+for+norovirus+detection+and+genogrouping.">An internally controlled, one-step, real-time RT-PCR assay for norovirus detection and genogrouping.</a> J Clin Virol. 39 (4), 318-321 (2007).</li><li>Kittigul, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+GII-4+2006b+variant+circulating+in+patients+with+acute+Thailand+during+a+2006-2007+study.">Norovirus GII-4 2006b variant circulating in patients with acute Thailand during a 2006-2007 study.</a> J. Med. Virol. 82 (5), 854-860 (2010).</li><li>Teunis, P. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norwalk+virus:+how+infectious+is+it.">Norwalk virus: how infectious is it.</a> J. Med. Virol. 80 (8), 1468-1476 (2008).</li><li>Wollants, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+norovirus+sampling+method+using+sodium+dodecyl+sulfate/EDTA-pretreated+chromatography+paper+strips.">Evaluation of a norovirus sampling method using sodium dodecyl sulfate/EDTA-pretreated chromatography paper strips.</a> J. Virol. Methods. 122, 45-48 (2004).</li><li>Weir, M. H., Shibata, T., Masago, Y., Cologgi, D., Rose, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effect+of+Surface+Sampling+and+Recovery+of+Viruses+and+Non-Spore+Forming+Bacteria+on+a+QMRA+Model+for+Fomites.">The Effect of Surface Sampling and Recovery of Viruses and Non-Spore Forming Bacteria on a QMRA Model for Fomites.</a> Environ. Sci. Technol. 50 (11), 5945-5952 (2016).</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=Microbiology+of+food+and+animal+feed-Horizontal+method+for+determination+of+hepatitis+A+virus+and+norovirus+in+food+using+real-time+RT-PCR.">Microbiology of food and animal feed-Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR.</a> International Organization for Standardization (ISO). , (2013).</li><li>Huslage, K., Rutala, W. A., Sickbert-Bennett, E., Weber, D. 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+quantitative+approach+to+defining+"high-touch"+surfaces+in+hospitals.">A quantitative approach to defining "high-touch" surfaces in hospitals.</a> Infect. Control. Hosp. Epidemiol. 31 (8), 850-853 (2010).</li><li>Wu, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+norovirus+outbreak+at+a+long-term-care+facility:+the+role+of+environmental+surface+contamination.">A norovirus outbreak at a long-term-care facility: the role of environmental surface contamination.</a> Infect. Control. Hosp. Epidemiol. 26 (10), 802-810 (2005).</li><li>Ikner, L. A., Gerba, C. P., Bright, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+recovery+of+viruses+from+water:+a+comprehensive+review.">Concentration and recovery of viruses from water: a comprehensive review.</a> Food Environ. Virol. 4 (2), 41-67 (2012).</li><li>Gallimore, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+monitoring+for+gastroenteric+viruses+in+a+pediatric+primary+immunodeficiency+unit.">Environmental monitoring for gastroenteric viruses in a pediatric primary immunodeficiency unit.</a> J. Clin. Microbiol. 44 (2), 395-399 (2006).</li><li>Ganime, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+of+human+adenoviruses+and+rotavirus+species+A+on+fomites+of+hospital+pediatric+units.">Dissemination of human adenoviruses and rotavirus species A on fomites of hospital pediatric units.</a> Am J Infect Control. , (2016).</li><li>Verani, M., Bigazzi, R., Carducci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+contamination+of+aerosol+and+surfaces+through+toilet+use+in+health+care+and+other+settings.">Viral contamination of aerosol and surfaces through toilet use in health care and other settings.</a> Am J Infect Control. 42 (7), 758-762 (2014).</li></ol>

Authors have no conflicting interest. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

诺如病毒有18和10 3病毒颗粒20之间的50％的人感染剂量。因此，即使表面的低水平的污染可能对公共健康危险。拭子采样协议的若干方面进行了评价，包括:1）不同拭子的材料，2）在运输过程中贮存条件拭子，3）病毒RNA的浓度，和4）噬菌体MS2作为内提取的控制。 直到最近，才由棉，涤纶，锦纶和抗静电擦拭）制成拭子的性能进行了评估，并提出适合...

<html>诺如病毒的人是流行性和散发性急性胃肠炎全世界1，2，3的主要原因。该病毒是极具传染性并通过直接的人发生互动的人或间接通过受污染的食物，水或环境表面接触传播。诺如病毒可棚长时间和长期的环境表面上的病毒的生存已记载1，2，3。在高峰脱落，十亿病毒颗粒被每克释放粪便和呕吐物还含有足够数量的病毒颗粒中，以引起感染4，5，6，7，8，EF"&gt; 9,10，另外，可以很容易地发生无生命的表面和人的皮肤之间的病毒传输2，11，12，因此，环境污染的监测可有助于爆发调查和评估的清理的有效性和消毒程序。 几个环境取样协议已经用于检测轮状病毒了描述，大肠杆菌噬菌体MS2，猫杯状病毒（FCV），和噬菌体P22 13，14，15，16。然而，在这些研究中，包括快速干燥（&lt;1小时）和小的表面区域中所述的验证条件（25×100厘米2）时，可能不能充分代表字段设置。此外，预期ENVIRO的低的污染水平nmental表面需要是能够检测很少的病毒颗粒的协议。 我们开发了诺如病毒的检测和分型基于大泡，表面取样方法。这种方法已经在几次爆发诺沃克病毒被证实。该协议包括1）如何收集环境表面拭子样本（2）如何收集和运输到实验室过程中最好保持样本的完整性，以及3）诺如病毒的实验室测试和打字。

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Generic name for kits</td>
			<td style="width:259px;"></td>
			<td style="width:153px;"></td>
			<td style="width:185px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Macrofoam swab</td>
			<td>Premoistened EnviroMax Swab kit&#160;</td>
			<td>Puritan</td>
			<td>2588060PFUW</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;RNA Lysis buffer&#160;</td>
			<td>CDC UNEX buffer</td>
			<td>Microbiologics</td>
			<td>Cat No MR0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA extraction spin column</td>
			<td>Midi column</td>
			<td>Omega Biotek</td>
			<td>Cat No R6664-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">RNA purification spin column</td>
			<td>Zymol RNA Clean and Concentrator kit&#160;</td>
			<td>Zymo Research</td>
			<td>Cat No R1016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Real time RT-PCR kit</td>
			<td>AgPath kit One-Step RT-PCR Kit</td>
			<td>Life Technologies</td>
			<td>Cat No 4387391</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional RT-PCR kit</td>
			<td>Qiagen one step RT-PCR kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 210212</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Gel extraction kit</td>
			<td>Qiagen QIAquick gel extraction kit</td>
			<td>Qiagen kit</td>
			<td>Cat No 28704 or 28706</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;"></td>
			<td>Coliphage MS2</td>
			<td>ATCC</td>
			<td>Cat No 15597-B1</td>
			<td></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;"></td>
			<td>RNA run-off transcripts</td>
			<td></td>
			<td></td>
			<td style="width:285px;">Bacteriophage MS2 (ATCC No. 15597-B1) can be cultivated using Escherichia coli (E.coli) Famp (ATCC No. 700891).&#160;</td>
		</tr>
		<tr height="148">
			<td height="148" style="height:148px;"></td>
			<td>Realtime PCR platform</td>
			<td>Applied Biosystems</td>
			<td>Model ABI 7500</td>
			<td style="width:285px;">GI and GII RNA run off transcripts were quantified spectrophotometrically at A260, diluted in diethyl pyrocarbonate-treated water to 1 &#215; 106 copies/ &#956;l, and stored at &#8722;80&#176;C with 1.0 U /&#956;l RNasin (Promega, Madison, WI).&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td>Optical 96-well reaction plate</td>
			<td>Thermo Scientific</td>
			<td>Cat No 4316813</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td><a href="http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;showAddButton=true&amp;productID=4306311&amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;returnURL=http%3A%2F%2Fwww.thermofisher.com%3A80%2Forder%2Fcatalog%2Fen%2FUS%2Fadirect%2Flt%3Fcmd%3DcatDisplayStyle%26catKey%3D101%26filterType%3D1%26OP%3Dfilter%26filter%3Dft_1101%252Ff_2005260*">MicroAmp Clear Adhesive Film&#160;</a></td>
			<td>Thermo Scientific</td>
			<td>Cat No 4306311</td>
			<td></td>
		</tr>
	</tbody>
</table>

swab sampling method for the detection of human norovirus on surfaces

诺如病毒的人是流行性和世界各地的零星胃肠炎的主要原因。因为大多数感染或者直接经由人对人的路线通过环境表面或食物传播或间接地受污染的非生物媒介和无生命的表面是用于病毒的期间诺罗病毒爆发的传播重要的车辆。 我们开发和评估使用大泡拭子用于检测和从硬表面人类诺如病毒的打字的协议。纤维拭子或防静电抹布相比，大泡拭子允许从高达700 cm 2的马桶座面恢复病毒（范围1.2-33.6％）。该协议包括用于病毒从拭子提取和使用旋转柱的病毒RNA的进一步浓缩的步骤。总共127已经从在游船和长期护理设施的表面，其中诺罗病毒性胃肠炎已经收集到217拭子样品（58.5％）报道药检呈阳性被RT-qPCR的诺如病毒GII。这些29（22.8％）的可以成功基因分型。总之，检测使用我们开发可以帮助确定环境污染的暴发期间水平以及检测病毒的当临床样品是不可用的协议环境表面上诺罗病毒的;它也可能有利于整治策略的有效性进行监测。

图1呈现拭子采样协议的流程图。这个协议包括四个主要步骤; 1）样品的采集，2）样品存储和运输，3）病毒RNA纯化和浓缩和4），RT-qPCR分析和基因分型。 <img alt="图1" src="/files/ftp_upload/55205/55205fig1.jpg" /> 图1:流量为诺如病毒的环境表面取样最终协议的图表 <a href="http://ecsource.jove.com/files/ftp_upload/55205/5...

Watch this Scientific Journal Video about Swab Sampling Method for the Detection of Human Norovirus on Surfaces at JoVE.com

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

swab-sampling-method-for-the-detection-of-human-norovirus-on-surfaces

Research

JoVE Journal

Immunology and Infection

19.5K 次观看.  Centers for Disease Control and Prevention. A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces. 

视频: Swab Sampling Method for the Detection of Human Norovirus on Surfaces

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

Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

手指棒运动诱导淋巴细胞凋亡的测定血液抽样方法

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement ...

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

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 have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

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 ...

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, ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

We describe here a combination of the glass-supported lipid bilayer technique of forming immunological synapses with the super-resolution imaging technique of stimulated emission depletion (STED) microscopy. The goal of this protocol is to provide users with the instructions necessary to successfully carry out these two techniques.

的自然杀伤细胞免疫突触上的玻璃支承平面脂双层超分辨率成像

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

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 ...

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 ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

黏膜衬里流体为体内上呼吸道免疫介质水平量化的无创性取样

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...