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>

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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><tbody><tr><td>&lt;strong&gt;Generic name for kits&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Macrofoam swab&lt;/strong&gt;</td><td>Premoistened EnviroMax Swab kit&amp;nbsp;</td><td>Puritan</td><td>2588060PFUW</td></tr><tr><td>&lt;strong&gt;RNA Lysis buffer&amp;nbsp;&lt;/strong&gt;</td><td>CDC UNEX buffer</td><td>Microbiologics</td><td>Cat No MR0501</td></tr><tr><td>&lt;strong&gt;RNA extraction spin column&lt;/strong&gt;</td><td>Midi column</td><td>Omega Biotek</td><td>Cat No R6664-02</td></tr><tr><td>&lt;strong&gt;RNA purification spin column&lt;/strong&gt;</td><td>Zymol RNA Clean and Concentrator kit&amp;nbsp;</td><td>Zymo Research</td><td>Cat No R1016</td></tr><tr><td>&lt;strong&gt;Real time RT-PCR kit&lt;/strong&gt;</td><td>AgPath kit One-Step RT-PCR Kit</td><td>Life Technologies</td><td>Cat No 4387391</td></tr><tr><td>&lt;strong&gt;Conventional RT-PCR kit&lt;/strong&gt;</td><td>Qiagen one step RT-PCR kit</td><td>Qiagen kit</td><td>Cat No 210212</td></tr><tr><td>&lt;strong&gt;Gel extraction kit&lt;/strong&gt;</td><td>Qiagen QIAquick gel extraction kit</td><td>Qiagen kit</td><td>Cat No 28704 or 28706</td></tr><tr><td></td><td>Coliphage MS2</td><td>ATCC</td><td>Cat No 15597-B1</td></tr><tr><td></td><td>RNA run-off transcripts</td><td></td><td></td></tr><tr><td></td><td>Realtime PCR platform</td><td>Applied Biosystems</td><td>Model ABI 7500</td></tr><tr><td></td><td>Optical 96-well reaction plate</td><td>Thermo Scientific</td><td>Cat No 4316813</td></tr><tr><td></td><td>&lt;a href=&quot;http://www.thermofisher.com/order/catalog/en/US/adirect/%20https:/www.thermofisher.com/order/catalog/en/US/adirect/lt?cmd=catProductDetail&amp;amp;showAddButton=true&amp;amp;productID=4306311&amp;amp;_bcs_=H4sIAAAAAAAAAI1RTWvDMAz9Nb4srDg2yXpNW3rZVyHbuRhHSQx2HWwnJf9%2BUkO6scMYGEmWnqX3%0A5Mec8e0p%2BGbUKWZMlFkNYTIa4h%2F5PqWByYqJI57r9bpJPQTnWxPRb7R3mB4jGrig6b0DdD40WOyT%0As9iBCUmHb1MYge78iYi06ZznPEd0exacF6LkD5h%2BNTr4yg1II0DH5C6rmh6imSA7GuuInsIq5TUy%0AjD6Yf%2FKU1Zav3NBrlZT13cr8s0ajGhNAJ4xsYvKoXcPkAYEHEwer5jrNFuWVdF1ieajNpbPwAhPY%0AvUrQ%2BTAjAt88w4zlm8Ly%2FYRha2yiyeUSUJM35ajHt%2BZiUS0KuWNityrHkLSjuyHUDbkgfuxgbfwx%0AD9Q0vydo9rrs4te675%2FTKhvpd74AhB0C9yUCAAA%3D&amp;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*&quot;&gt;MicroAmp Clear Adhesive Film&amp;nbsp;&lt;/a&gt;</td><td>Thermo Scientific</td><td>Cat No 4306311</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

Universidad de Cartagena UDEC

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

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

科研

环境科学

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 ...

免疫与感染

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

EPA方法1615年测量和肠道病毒水中诺如病毒发生由文化部和RT-qPCR的的。 II。总可培养病毒试验

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

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 ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

采用TaqMan从儿童凳诺如病毒的检测和Genogrouping一步法RT-PCR技术

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

医学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Quantifying Human Norovirus Virus-like Particles Binding to Commensal Bacteria Using Flow Cytometry

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is responsible for altering infection for many of these viruses. Thus, characterizing the nature of virus-bacteria binding is a foundational step needed for elucidating the mechanism(s) by which bacteria alter viral infection. For human norovirus, commensal bacteria enhance B cell infection. The virus directly binds to these bacteria, indicating that this direct interaction is involved in the mechanism of infection enhancement. A variety of techniques can be used to quantify interactions between bacteria and viruses including scintillation counting of radiolabeled viruses and polymerase chain reaction (PCR). Both methods require the use of live virus, which may need to be generated in the laboratory. Currently, none of the established in vitro culture systems available for human norovirus are robust enough to allow for generation of highly concentrated viral stocks. In lieu of live virus, virus-like particles (VLPs) have been used to characterize the interactions between norovirus and bacteria. Herein a flow cytometry method is described with uses virus specific antibodies to quantify VLP binding to gram-negative and gram-positive bacteria. Inclusion of both bacteria only and isotype controls allowed for optimization of the assay to reduce background antibody binding and accurate quantification of VLP attachment to the bacteria tested. High VLP:bacterium ratios result in VLPs binding to large percentages of the bacterial population. However, when VLP quantities are decreased, the percent of bacteria bound also decreases. Ultimately, this method can be employed in future experiments elucidating the specific conditions and structural components that regulate norovirus:bacterial interactions.

The goal of this protocol is to quantify binding of the eukaryotic pathogen human norovirus to bacteria. After performing an initial virus-bacterium attachment assay, flow cytometry is used to detect virally-bound bacteria within the population.

使用流细胞测定量化人类诺如病毒样颗粒与准细菌结合

Commensal bacteria are well established to impact infection of eukaryotic viruses. Direct binding between the pathogen and the host microbiome is ...

Swabbing the Urban Environment - A Pipeline for Sampling and Detection of SARS-CoV-2 From Environmental Reservoirs

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries implemented strategies based on direct human testing, face covering, and surface disinfection. Under the assumption that the main route of transmission includes aerosols and respiratory droplets, efforts to detect SARS-CoV-2 in fomites have focused on locations suspected of high prevalence (e.g., hospital wards, cruise ships, and mass transportation systems). To investigate the presence of SARS-CoV-2 on surfaces in the urban environment that are rarely cleaned and&#160;seldomly disinfected, 350 citizens were enlisted from the greater San Diego County. In total, these citizen scientists collected 4,080 samples. An online platform was developed to monitor sampling kit delivery and pickup, as well as to collect sample data. The sampling kits were mostly built from supplies available in pandemic-stressed stores. Samples were processed using reagents that were easy to access despite the recurrent supply shortage. The methods used were highly sensitive and resistant to inhibitors that are commonly present in environmental samples. The proposed experimental design and processing methods were successful at engaging numerous citizen scientists who effectively gathered samples from diverse surface areas. The workflow and methods described here are relevant to survey the urban environment for other viruses, which are of public health concern and pose a threat for&#160;future pandemics.

A citizen science project was designed to recruit San Diego residents to collect environmental samples for SARS-CoV-2. A multilingual web-based platform was created for data submission using a user-friendly mobile device interface. A laboratory information management system facilitated the collection of thousands of geographically diverse samples with real-time outcome tracking.

城市环境 -环境水库SARS-CoV-2采样和检测管道

To control community transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during the 2020 global pandemic, most countries ...

生物学

An Assay to Quantify the Binding of Human Norovirus VLPs to Intestinal Bacteria

After preparing the reagents, antibodies, and bacteria as described in the manuscript, assemble all the materials in a BSL II biosafety cabinet.
Virus-like particles for human norovirus are listed as BSL II pathogens, and all work performed using VLPs should be conducted in a certified biosafety cabinet.
Add 10 micrograms of human norovirus VLPs to each tube of bacteria, and mix thoroughly by pipetting. Incubate the tubes for 1 hour at 37 degrees Celsius with constant rotation. After incubation, centrifuge the tubes at 10,000 times g for 5 minutes.
Aspirate and discard the supernatant and resuspend the bacterial pellet in 1 milliliter of PBS. After repeating the wash steps once, centrifuge the tubes again at 10,000 times g for 5 minutes. Discard the supernatant and resuspend the VLP bacteria pellet in 150 microliters of 5% blocking buffer.
A common error that occurs is that tubes are swapped and the wrong treatment is added. To prevent this, arrange all tubes in lines that correspond to the correct treatment, and add one treatment all at once to the tubes.
For each bacterial sample, prepare 50 microliters of diluted human norovirus G2 antibody. Using 5% blocking buffer, dilute the antibody 1 to 125 for E. coli case samples. Prepare 50 microliters of diluted isotype antibody for each bacterial sample using the same dilution ratios. Divide each attachment assay sample into 350-microliter aliquots by transferring them into clean 1.5-milliliter centrifuge tubes.
To create unstained controls, add 50 microliters of blocking buffer to the first aliquot from each bacterial sample. For the stained samples, add 50 microliters of the G2 antibody dilution to the second set of aliquots. Create the isotype controls by adding 50 microliters of the diluted isotype antibody to the third set of aliquots.
Incubate all the samples on ice and in the dark for 30 minutes. Then, centrifuge all the samples at 10,000 times g for 5 minutes. Discard the supernatants and resuspend each sample in 150 microliters of FCSB. Again, centrifuge all the samples at 10,000 times g for 5 minutes. Again, discard the supernatants and resuspend the samples in 100 microliters of FCSB.
After centrifuging the samples one final time and discarding the supernatant, resuspend the samples in 150 microliters of FCSB. Transfer each sample to a tube containing 400 microliters of FCSB for a total volume of 550 microliters. Store the samples at 4 degrees Celsius until they are analyzed by flow cytometry.