在这里描述呼吸道病原体检测的工作是由美国康奈尔动物健康诊断中心内部开发的资金支持。纳米级PCR工作流程和相关的质量保证体系的发展是部分资金（FOA PA-13-244），并协同下，批准号1U18FD005144-美国食品和药物管理局的兽医实验室调查和反应网络（FDA兽医-LIRN）执行01。该刊物的费用由VWR和广达生物科学赞助。我们感谢加布里埃尔尼克森，Roopa Venugopalan，Veldina迷彩秀林张钰晋职，卫华王，和卡特里娜·沃克为他们写与和审查协议的援助。我们感谢迈克卡罗尔的拍摄笔者的采访。最后，我们感谢他们的有益的评论编辑器和三个匿名审稿。

没有人体或实验动物用于本协议的发展。通过序列证实扩增子的纯化及在 RNA靶体外转录生成控件。临床样品提交常规诊断测试康奈尔动物健康诊断中心。 1.保护板设计<ol><li>使用引物设计软件，以验证每个检测符合用60℃退火用引物探针测试工具标准的基于探针实时PCR循环条件（选择量化探头使用默认参数设置）。 注:所使用的RNA代表目标是改编自通用流感舒等人发表的矩阵分析目标7。是引物和探针如下:正向引物:GACCRATCCTGTCACCTCTGAC，反向引物:AGGGCATTYTGGACAAAKCGTCTA，探针:FAM-TGCAGTCCTCGCTCACTGGGCACG-QSY。这里使用的代表性的DNA测定来回适于米乘埃利亚等发表的马疱疹病毒1型（EHV-1） 的检测方法。8。是引物和探针如下:正向引物:GCTCTCAGGTTTTACGACATC，反向引物:CTTTACCCAGGCCCTTGAAA，探针:FAM-TCAACGTGGACAATACCGCAGTGATTAT-QSY。 </li><li>要想在需要的配置纳米级PCR扩增板。 注意:对于该研究，使用了18x3基因表达格式（ 表1）。为每个目标制造商的所有引物和探针（或盘点法ID）的序列。 </li></ol> 2.核酸提取<ol><li>用任何所需的方法提取总核酸（DNA和RNA）。 注意:一个自动化基于磁珠的提取试剂盒根据制造商的说明在这里使用的（更多细节的材料表）。 </li><li>制备的样品如下: <ol><li>清洁每个样品容器用10％漂白粉彻底干燥外。擦拭手套˚F英格技巧与样品防止交叉污染的10％漂白粉。 </li><li>地方鼻或用生理盐水几滴在任何无菌，密封小瓶深咽拭子（如红色顶部血液收集管中）加入，以防止在处理之前干燥。 注:棉花，塑料，木材，处理，涤纶等合成拭子都是可以接受的，但要避免藻酸钙拭子。 </li><li>为拭子和气管洗涤，添加的Dulbecco改良的Eagle培养基（DMEM），使得存在至少1-2的液体毫升。 VORTEX棉签和DMEM大力在管。然后，使用吸液管到约1ml的媒体转移到新管中。 </li><li>机械裂解组织（100-200毫克的1ml DMEM中）使用根据制造商的说明，随后通过离心在825×g下3分钟的组织破坏剂。转移500μl的上清液至新管。 </li><li>对于粪便，结合400毫克粪便与800微升1X磷酸盐缓冲的sa在线pH 7.4（PBS）中。涡旋混合1分钟或更长时间的悬浮液，直到所述样品是均质或完全中止。一旦均质，离心10分钟将悬浮液于18,000 xg离心，然后加入400μl转移到一个新试管中。 </li><li>对于全血未凝结，涡旋样品，并进行250微升等分。添加溶解剂和旋涡六滴混合。孵育在37℃下15分钟。 </li></ol></li><li>根据制造商的说明制备的裂解和洗涤缓冲液。添加MS2噬菌体或选择的裂解缓冲液的内部对照作为内部阳性提取控制9。 </li><li>添加裂解缓冲液的235微升和175微升样品（或PBS作为阴性对照）与每个胎圈管。制造商的协议进行。 注:纯化总核酸在这里洗脱90微升。 </li></ol> 3.逆转录/预放大（前置放大器） 注意:保持中使用的所有试剂和样品在任何时候都冰上前置反应。继前置放大器，把所有试剂室温。 <ol><li>组装洗脱的DNA和/或RNA，PCR反应混合物，无核酸酶水作为阴性对照，并且对于阳性放大控制汇集标准。 注:最多48个样品可以在放大板包括控制运行（包括每个撤出板从样品被拉至少一个负提取控制）。 </li><li>打印出样图。有第二人检查，地图和样品布局相匹配。 </li><li>在1.5 ml管中，添加以下准备前置主结构。 注意:这里使用的14微升最终体积。 <ol><li>加的从每个目标预混引物库，使得每个引物的终浓度为900纳米。 注:此处使用的池在10倍浓度制备，并且每个反应加入1.4微升。 </li><li>随机引物添加到600纳米或0.1×的最终浓度。 </l我&gt; <li> 2×RT-qPCR的组合加入到最终体积的一半（这里7微升）。 </li><li>轻轻涡旋和停转混合母液组件连接在一起。 </li></ol></li><li>使用该板只（列1-6）的左侧在标准的96孔平板进行前置放大。 </li><li>添加前置主混合物的8.5微升和DNA / RNA样品的5.5μl到每个孔中。 </li><li>结合所有试剂（样品，阳性对照，用前置放大器组合阴性对照）之后，彻底密封用透明的粘合剂密封的标准的96孔板中。用刀片，以防止循环过程形成密封间隙消除任何多余的密封。离心密封板用于使用PCR板喷丝20秒。检查，以确保所有的试剂都在板孔的底部相结合。 </li><li>在下列条件下运行的常规热循环前置放大器测定:在50℃下15分钟在95℃下1分钟;在95℃，然后用2分钟，在60℃下15秒的20个循环; 99.9＆＃176℃持续10分钟;保持在4℃。项目启动前，这些条件。 </li><li>打开传统的热循环仪，选择前置循环程序和启动程序。放置96孔板中的热循环，只有当热循环仪（未盖）的底部到达通过监测温度读数在屏幕上用于cDNA生产（50℃）所需的温度。在此之前，保持板冷以减少产生假阳性结果的风险。 </li><li>一旦前置放大器运行完成，验证板一直保持密封。 </li><li>立即进行步骤4.1。过夜存储该板，如在步骤4.2至4.5中所述，除了代替松散覆盖板进行稀释，密封用透明的粘合剂密封，并代替拉链锁内袋，保存在4℃下的板。 </li></ol> 4.稀释前置放大器板<ol><li>除去扩增板的适当数量的往复m为冷冻（最多4可以一次运行）。不要打开包装。允许板预热在室温下至少15分钟。记录盘的序列号和批号。 注:未开封板可在室温下保持长达24小时，但对于最佳操作，从不超过30分钟，需要之前冷冻除去这些。 </li><li>离心完成前置放大器板使用PCR板微调20秒。打开放大机及电脑上，并启动相应的程序。 </li><li>删除从PCR设置区域中的所有不必要的物品。戴上双层手套和套管盖。 </li><li>从前置放大器板取下密封，小心地取出并丢弃外手套。通过上下吹打加入56微升TE缓冲液中，以列96-前置放大器板1-6和混合的各孔5:稀释前置产品1。只去吸管的第一站，从底部吸液和分配上涨，但没有创造气泡。加入TE缓冲所有6列，无论使用的水井。 </li><li>重新密封带任何明确的粘合剂或铝箔密封板，离心它采用PCR板微调20秒。抛开这个板块（松散覆盖），直到6.1步完成。丢弃剩余的手套，套盖。 </li></ol> 5.准备384孔板转移到放大板<ol><li>建立覆盖和密封板放大所需材料:盖子，塞，浸渍液，板压，乙醇喷瓶，不起毛的实验室湿巾注射器。取下注射器盖并锁定一个小塑料尖到注射器（需要力）。喷射浸没流体的少量到纸巾以除去空气积聚（这是确定是否一些小气泡残留在尖端）。除去从真空密封的铝袋在注射器在60分钟内使用浸没流体。一旦注射器被打开，不重新安装供以后使用的瓶盖。 </li><li>检查该印版装载液体处理器的废物箱是空的，打开的提示框。写在当一个新盒子被打开的提示框盖提示的到期日期，并确保该提示是没有过期。打开液体处理器和计算机并打开液体处理器软件，这将执行自检。 </li><li>点击"设置和装载"。点击"浏览"选择从样品追踪软件创建的CSV文件。如果没有显示文件，进入"仪器/编辑首选项"，然后单击"更改"的"样品盘文件夹"。选择其中的CSV文件保存的文件夹。然后，单击"设置和加载"，然后选择"浏览"，选择该文件。 </li><li>接下来，单击"浏览"旁边的"板夹位置1"，并选择具有相同的序列号板的TPF文件（由制造商提供）。 </li></ol> 6.液体处理器转移注意:不要启动网络灌装在384孔板，直到所有的上述步骤完成。蒸发量小卷的关注 - 不要让384孔板坐在里面发现了液体。 <ol><li>一旦所有的上述步骤完成后换上新的，很好地符合双手套，套盖。然后，添加2.5微升的扩增主混合物至384孔板中。 </li><li>根据地图转移2.5微升稀释前置产物到384孔板在表2中，并立即用箔密封紧紧盖住板。丢弃外手套，套盖。 </li><li>离心机在PCR板喷丝20秒的384孔板中。 </li><li>不要取下铝箔封口，但放置在384孔板的液体处理器。 </li><li>打开包装含包裹放大板并小心地将它与右边的序列号的第一个插槽的液体处理器 - 持有的边缘的情况下不接触面ÔF中的板。开口的一小时内使用展开板材。 注意:这样的目的，是为了保护板从被触摸，因为这可能会扰乱通孔内的少量的引物和探针。 </li><li>从液体处理器内的384孔板取下铝箔封口一旦一切就绪。点击下一步，然后选中所有的复选框，因为每个步骤完成。点击OK开始运行。 </li><li>关闭的大门液体处理器，并立即开始灌装过程。留旁边的液体处理器和立即进行下一步骤，一旦板被充满。 </li><li>而液体处理程序正在运行时，从壳体盖的底部除去的保护膜。 注意:在壳体盖的底部的粘接剂是由一个红胶带和保护膜覆盖。这部电影需要先访问繁文缛节被删除。 <ol><li>留在原地顶部薄膜直到步骤6.15。总理繁文缛节通过稍微拉到releas从胶电子吧。 </li></ol></li><li>从液体处理器取出装入（饱满）扩增板和轻轻地用在右边的序列号将其放置在板压机。 </li><li>将盖子上与在底部（朝向板指向）右侧和粘合剂的切口端板的顶部。 </li><li>拉下板压杠杆小心关闭它;灯将闪烁20秒。在此期间，请勿触摸平板机。一旦指示灯变为稳定的绿色，仔细地抬起杠杆，并小心地抱着它的边缘取下密封板。 </li><li>而垂直地保持密封放大板由壳体的边缘，立即插入注射器尖端到在壳体的端部的装载端口，则分配所述浸没流体慢慢在一个轻柔的连续运动以填充板和之间的空间盖。留一个小气泡在角落里，一旦板浸入。 </li><li>同时继续为h老板垂直的边缘，通过插头插入端口，顺时针旋转插头扭动，施加足够的压力，直到手柄脱落密封装货港。把手的情况下的边缘牢固地使手柄断裂的力不会导致的情况下下降。如果板丢弃，丢弃它。 </li><li>用抹的无绒布已用乙醇彻底清洁喷的情况。干燥的情况下，向下擦拭外壳用干净​​的擦拭。轻轻地处理;一定不能在井上面的玻璃施加压力。 </li><li>使密封板的放大机。在"仪器"选项卡，选择"仪表控制台"。选择放大机，然后点击"开门"按钮。 </li><li>放置在板适配器的第一槽的放大板，检查在左上角的适配器正确排队与A 1。定向与条形码的板朝上和朝仪器的前部。点击"关闭门"按钮。注意使用适配器托盘 - 有10个用途的限制。 </li><li>在屏幕底部的选项卡，Run菜单下，选择OpenArray。点击"获取板的ID。"空盒将与有关铭牌信息自动填充。要开始运行，请单击"开始→运行"。 </li><li>关闭液体处理器软件和关闭液体处理器。将在尖机架顶端盖。丢弃从垃圾箱的提示和清洁。清洁和消毒的所有项目，包括在工作面上，吸管，垃圾斗，和提示框。 </li><li>重新封装用透明胶密封并储存在-20°C的前置放大器板。 </li></ol> 7.结果分析<ol><li>一旦运行完成，保存文件，然后点击与运行名称选项卡上的"X"在屏幕的底部，关闭文件。 </li><li>点击"打开门"在屏幕上打开门的顶部，拆下放大板，检查没有浸液泄露出去了。点击"关闭门"，在屏幕的顶部关门。 </li><li>点击"打开"，并选择运行文件，将其打开。按绿色屏幕右上方分析按钮。点击屏幕的左侧，然后在"开始导出""导出"导出结果（如.txt文件）。它打开后最小化文件。然后，单击"导出QC形象。" </li><li>根据以下标准的图像分析程序审查QC图片: <ol><li>使用预READ_CHANNEL_4.tiff和POST-READ_CHANNEL_4.tiff，通过检查，有没有暗斑或污迹评估装载质量。 注:此信道检测到的染料是由制造商到引物和探针，该反应被加载时被释放到溶液中。在这个通道中的读数表明，反应是正确的加载并与染料混合引物和探针出席了会议。 </li><li>检查使用S02_C001_t03_p001_m1_x2_e1_cp＃_spotfind.tiff加载后的泄漏或搅拌到板上。如果图像看起来黑暗，增加亮度（按Ctrl + Shift + C打开控制），直到整个板块是可见的。检查图像看起来均匀，无阴影，气泡，或流离失所样本。 </li><li>评估使用STAGE2_CYCLE1_CHANNEL_1.tiff和STAGE2_CYCLE40_CHANNEL_1.tiff下盖（亮点）盖放置和碎片。 </li></ol></li><li>可选:打开包含宏的结果（补充代码文件）的电子表格。在导出数据文件，右键单击该选项卡上的在屏幕的底部，并选择"移动或复制"。在"预定"栏，选择"结果Macro.xlsm"。点击"移动结束"，并勾选"创建副本"。然后，点击确定。 <ol><li>如果结果微距选项不会出现在"预订"领域，SIM卡层复制导出的数据文件工作表的内容（点击左上角的单元格以突出显示所有细胞和Ctrl-C）并粘贴到一个新的标签。 </li></ol></li><li>删除旧的"导出"选项卡，然后重命名新添加的选项卡中的"导出"。 </li><li>点击"ALT-F8"（或单击视图，然后宏），然后选择"宏"，然后单击"运行"。然后点击"ALT-F8"然后选择"Macro2"，然后点击"运行"重复此为Macro2。 </li><li>重命名使用的相关信息（日期和序列号）的结果宏文件，把表以上这些信息，并保存为导出的结果文件夹相同的文件名。最后，打印出宏观生成的结果表。 </li><li>在放大机软件，由在屏幕的左侧选择分析/扩增曲线检查每个样品和控制针对宏表的曲线。然后，选择样品标签，然后点击每个样品确认有2或3的每个表中的阳性结果的阳性扩增曲线。 </li><li>关闭放大机。 </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+mRNA+using+real-time+reverse+transcription+PCR+(RT-PCR):+trends+and+problems.">Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems.</a> J Mol Endocrinol. 29 (1), 23-39 (2002).</li><li>McCall, M. N., 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+benchmark+for+microRNA+quantification+algorithms+using+the+OpenArray+platform.">A benchmark for microRNA quantification algorithms using the OpenArray platform.</a> BMC bioinformatics. 17 (1), 138 (2016).</li><li>Pozzi, A., Previtali, C., Cenadelli, S., Gandini, L., Galli, A., Bongioni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+traceability+of+cattle+using+an+OpenArray+genotyping+platform.">Genetic traceability of cattle using an OpenArray genotyping platform.</a> Anim Genet. 47 (1), 133-134 (2016).</li><li>Grigorenko, 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=Multiplex+screening+for+blood-borne+viral,+bacterial,+and+protozoan+parasites+using+an+OpenArray+platform.">Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform.</a> J Mol Diagn. 16 (1), 136-144 (2014).</li><li>Shu, 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=Design+and+performance+of+the+CDC+real-time+reverse+transcriptase+PCR+swine+flu+panel+for+detection+of+2009+A+(H1N1)+pandemic+influenza+virus.">Design and performance of the CDC real-time reverse transcriptase PCR swine flu panel for detection of 2009 A (H1N1) pandemic influenza virus.</a> J Clin Microbiol. 49 (7), 2614-2619 (2011).</li><li>Elia, G., 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=Detection+of+equine+herpesvirus+type+1+by+real+time+PCR.">Detection of equine herpesvirus type 1 by real time PCR.</a> J Virol Methods. 133 (1), 70-75 (2006).</li><li>Dreier, J., St&#246;rmer, M., Kleesiek, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Bacteriophage+MS2+as+an+Internal+Control+in+Viral+Reverse+Transcription-PCR+Assays.">Use of Bacteriophage MS2 as an Internal Control in Viral Reverse Transcription-PCR Assays.</a> J Clin Microbiol. 43 (9), 4551-4557 (2005).</li><li>Pusterla, N., Hussey, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equine+Herpesvirus+1+Myeloencephalopathy.">Equine Herpesvirus 1 Myeloencephalopathy.</a> Vet Clin North Am Equine Pract. 30 (3), 489-506 (2014).</li><li>Firth, C., Lipkin, W. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomics+of+emerging+pathogens.">The genomics of emerging pathogens.</a> Annu Rev Genomics Hum Genet. 14, 281-300 (2013).</li><li>Lecuit, M., Eloit, M. <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+of+whole+genome+NGS+for+infectious+disease+diagnosis.">The potential of whole genome NGS for infectious disease diagnosis.</a> Expert Rev Mol Diagn. 15 (12), 1517-1519 (2015).</li><li>Dunne, G., Gurfield, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+Veterinary+Diagnostic+Laboratory,+a+Model+for+the+One+Health+Initiative.">Local Veterinary Diagnostic Laboratory, a Model for the One Health Initiative.</a> Vet Clin North Am Small Anim Pract. 39 (2), 373-384 (2009).</li><li>Moutailler, S., 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=Co-infection+of+Ticks:+The+Rule+Rather+Than+the+Exception.">Co-infection of Ticks: The Rule Rather Than the Exception.</a> PLOS Negl Trop Dis. 10 (3), e0004539 (2016).</li></ol>

玛西娅·斯莱特和埃伦奥滕贝格是产生在这篇文章中使用的试剂和仪器的Thermo Fisher Scientific公司的员工。

这个程序已被用于在我们的实验室超过六个月（每周2-3次）的过程中的呼吸道病原体例行测试。我们使用相同的过程粪便样本和细菌分离株在肠道病原体分析上单独定制的板块也取得了成功。一旦板生产的，经验丰富的员工可以完成一个标准工作日之内2-7步骤。最重要的步骤是前置放大器板的适当的密封，前置放大样品板之间的传输（其必须迅速完成，以避免蒸发），和放大板的最终覆盖物。宏电子表格作为结果分析（检查样品和对照曲线）指导的使用也是很关键的，因为它大大减少了所需的这一过程的时间和文书工作的数量。所提供的电子表格宏是一个例子;这将需要修改或重新创建为不同配置平板。这很容易PE有人用电子表格的基本知识rformed。 由于加载到放大板（33 NL）样品的非常少量的，需要预扩增。在这个协议中（未示出）的优化，我们比较了一些预扩增参数，包括主混合物，加入随机引物，周期数，退火时间，和稀释扩增前。每个目标都有自己的最佳条件，而这里描述的那些代表那些得到全面检测的最佳极限为我们的面板。此面板涵盖了广泛的病原性的目标，并且在常规兽医诊断测试中遇到的样品类型，但修改到预扩增程序可以针对不同的面板是必要的。生产商优化扩增条件是基于一个标准的基于探针的实时PCR用10分钟在95℃下酶激活协议接着变性在95℃和安伊令/延伸60℃。引物和探针是预先印刷在平板上也使用制造商优化条件，因此不需要滴定。结合逆转录和预扩增步骤对于所有样品（无论目标的类型的），以便在工作流以保持效率是必要的。拥有所有样品反转录也是最大限度地提高灵敏度有利。此外，使用预混因为这是最小化抑制剂优化前置放大器的多功能性可以在同一个板结合不同类型的样品。 该疾病预防中心（CDC）舒等 7纳升规模的反应说明，并适应这里流感矩阵法是一种通用流感检测适合于从人类和伴侣动物测试样本。它是用微升规模REA设计的所有甲型流感病毒的M基因的通用检测ctions。它已在世界各地用作CDC人流感病毒面板和CDC猪流感面板的一部分。超高压-1试验8在这里适应检测的马，可能会导致流产和神经系统疾病（由Pusterla和赫西10综述）的重要病原菌呼吸。适应这些测试的高通量平台与物种无关内部控制的意义在于它们可以掺入一个OneHealth监视的方法。拥有对高通量检测平台这些分析将有助于临床设施，候车亭和性能事件应急准备。 上述结果代表支原体cynos例外，这表明在分析性能相当多的变异板所有目标。这可能是由于引物的亚最佳熔融温度（T M），其理想情况下应58-60＆＃176; C（理想的探头Tm为68-70°C）。这个平台的一个限制是，它需要的时间比用于订购个别探针，这限制了快速修改序列的能力的设计和制造的板。在一般的实时PCR的另一个限制是无法检测新颖的或意外的病原体。这可以通过设计在多个物种匹配这样的序列测定法可以克服在一定程度上，但偏全基因组测序基础的方法更适合于新的药剂的发现。11,12 纳米实时PCR允许一个新的范例为综合征基于而非基于物种测试，这是用于减少在高通量分子诊断试剂和劳动力成本是有用的。通过这种方法大规模面板检测可以促进OneHealth监视努力，例如那些由邓恩和Gurfield 13和Moutailler 等人 14所述。拭子样本收集年初，一般在临床发作的3天，提供了识别的呼吸道病原体的存在的最佳机会。传染病的出现通常是不可预测的，并且可以在不同的物种，而不需要试剂的修改来进行的试验是理想的防备。这种技术的未来应用很可能是在pathotyping，耐药性分析，并进一步基于综合症临床诊断面板。纳米级的实时PCR是用于多个样品和病原体的类型的快速，高通量筛选最有用的，并且将通过无偏见地或部分地偏置的基于测序的方法，用于识别新和紧急病原体来补充。

随着对快速全面检测在临床诊断为人类和动物多个代理的需求，除非用于大量样品被测试为单一疾病的病原体检测单生物体分子诊断方法是沉重的负担。在兽医方面，高通量的诊断方法是特别重要的，由于用于从各种各样的物种的覆盖病原体的额外需要。 OneHealth方法对食源性疾病的管理和新兴病原体监测的测试需求，包括一些细菌的例子，病毒（DNA或RNA为基础），寄生虫和真菌。以，为了提高测试效率结合为多种分析物一起（复用）的测试的挑战是灵敏的一个可能发生的损失，并用于优化和测定法的验证一个很大的负担。 纳升规模的实时聚合酶链反应（PCR）是一个ALTER原产于多路复用的实践中，它允许许多不同的反应，在同一样品1同时运行。该OpenArray平台是这一原则2的申请;它结合芯片技术与实时PCR。基于空间复用的概念，每个样本为通孔中分离大量的目标测试。这个平台最初用于基于花青双链DNA结合化学2和使用暗猝灭探针3现在可用于基于探测的化学。这个平台已主要被用于遗传分析人类4和动物5。它最近被适于通过Grigorenko 等检测血源性DNA和RNA 的病原体。6使用两步逆转录/扩增前（前级）的程序。 我们在这里描述的一步基于前置过程，用于在呼吸秒检测两种类型的病原体retions以及各种能够完全在标准工作日执行的其他类型的样品。在一步法前置放大器完成后，样品被转移到384孔平板，它是通过附带此纳米的PCR平台的自动化液体处理系统所接受的格式。系统绘制在多达4板（192样品和对照）的表面的主混合物和样品一次。在此之后加载过程中，我们描述的样本是如何放大和使用总结了适合一张打印页面上的表3技术复制每个样品/目标组合的平均宏电子表格进行分析。 建立了分析使用该平台的样本中提取的DNA和/或RNA的统一方法。多种样品中提取，逆转录，和预扩增在96孔格式中，最大限度地减少发生错误的可能性。测试的呼吸道样本包括鼻拭子，深咽拭子，反式气管洗涤，支气管肺泡灌洗，和肺组织。由于一些测试还可以存在于末梢血或粪便的试剂中，我们引入这些类型的样品的入过程。这种简化的工作流程有助于节省相比，通过实现通过基于面板病原体和毒素基因检测效率检测到位单独测试运行在多个板块的实验时间和资源。这里展示的呼吸面板有18测定每个印在通孔一式三份，可容纳高达每板48样本。检测出的马病原体包括马腺病毒1和2，马动脉炎病毒，马鼻炎病毒A和B，马疱疹病毒（EHV）类型1和4，和马链球菌 。犬病原体包括犬呼吸道冠状病毒（betacoronavirus），犬瘟热病毒，犬腺病毒，犬副流感病毒，犬肺病毒， 支气管炎博德特和 支原体cynos。一个通用流感也包括一个分析和内部控制（MS2噬菌体RNA）。

<table border="0" cellpadding="0" cellspacing="0" width="751">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:227px;">Zap-OGlobin II lytic reagent</td>
			<td style="width:239px;">Beckman Coulter</td>
			<td style="width:119px;">7546138</td>
			<td style="width:167px;">Optional reagent for preparing blood samples.</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:45px;width:227px;">Extraction kit (MagMAX Total Nucleic Acid Isolation Kit)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">AM1840</td>
			<td>Other extraction kits appropriate for the desired sample types may be substituted. This kit comes with PBS, lysis buffer, and wash buffer.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:227px;">2X One-Step RT-qPCR mix (qScript XLT One-Step RT-qPCR ToughMix )</td>
			<td style="width:239px;">Quanta Biosciences</td>
			<td style="width:119px;">95134-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Gene-specific primer pool</td>
			<td style="width:239px;">IDT</td>
			<td style="width:119px;">custom order</td>
			<td>Can be generated by the user or purchased with the amplification plates.</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:227px;">Random primer mix</td>
			<td style="width:239px;">New England Biolabs</td>
			<td style="width:119px;">S1330S</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:227px;">Standard 96-well plate</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">N8010560</td>
			<td>This can be any plate that fits into the conventional PCR machine used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:41px;width:227px;">Amplification master mix (OpenArray master mix)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4462164</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Clear adhesive seal</td>
			<td style="width:239px;">Thermo-Fisher</td>
			<td style="width:119px;">430611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Foil seal</td>
			<td style="width:239px;">Excel Scientific</td>
			<td style="width:119px;">AF-100</td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:227px;">384-well plate</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4406947</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:227px;">Amplification plate (QuantStudio 12K Flex OpenArray plate)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4470813</td>
			<td>Can be customized with any assays conforming to standard TaqMan conditions.&#160;</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:30px;width:227px;">Accessories kit</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4469576</td>
			<td>Contains the case lids, plugs, and immersion fluid.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:227px;">AccuFill tips</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4457246</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Amplification machine (QuantStudio 12K Flex Real-Time PCR System)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4471090</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:227px;">Liquid handler (OpenArray AccuFill System)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4457243</td>
			<td>This is purchased with the amplification machine.</td>
		</tr>
		<tr height="36">
			<td height="36" style="height:37px;width:227px;">Primer design software (Primer Express)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4363991</td>
			<td>See manufacturer&#39;s instructions for detailed primer and probe specifications.</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:29px;width:227px;">Image analysis program (ImageJ)</td>
			<td style="width:239px;">NIH</td>
			<td style="width:119px;"></td>
			<td>Available at http://imagej.nih.gov/ij/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spreadsheet program (Excel)</td>
			<td style="width:239px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td>Available at https://products.office.com/en-us/excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">PCR plate spinner</td>
			<td style="width:239px;">VWR</td>
			<td style="width:119px;">89184-608</td>
			<td>This device has only one speed (2500 rpm); the rotor starts when the lid is closed and stops when the button is pushed.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">TE buffer</td>
			<td style="width:239px;">Millipore</td>
			<td style="width:119px;">8890-100ML</td>
			<td>10mM Tris(hydroxymethyl)aminomethane hydrochloride, 1mM Ethylenediaminetetraacetic acid, pH 8.0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">DMEM</td>
			<td style="width:239px;">Thermo-Fisher/Gibco</td>
			<td style="width:119px;">11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Tissue disruptor (TissueLyser II)</td>
			<td style="width:239px;">Qiagen</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:227px;">Conventional thermal cycler (Veriti)</td>
			<td style="width:239px;">Thermo-Fisher/Applied Biosystems</td>
			<td style="width:119px;">4375786</td>
			<td>Any conventional thermal cycler with a heated lid can be used.</td>
		</tr>
	</tbody>
</table>

high-throughput detection of respiratory pathogens in animal specimens by nanoscale pcr

纳升规模实时PCR使用空间复用，以允许多个测定以平行于单个板而不结合反应在一起的典型缺点运行。我们设计和评估基于这个原理，以迅速识别常见病剂在狗和马的急性呼吸系统疾病的存在的面板。这份手稿描述了样品制备，放大和目标病原体序列分析纳米级PCR诊断工作流程，重点放在从微升规模的反应不同的程序。在介绍了呼吸面板，18测定分别设立了一式三份，可容纳高达每板48样本。通用提取和前置放大工作流程用于高通量样品制备优化，以适应多个矩阵和DNA和RNA基于病原体。代表性的数据提出了一种RNA的目标（甲型矩阵）和一个DNA目标（马疱疹病毒1）。对病原体的全面，基于校正子组，以快速，准确地进行测试的能力，为提高效率和诊断测试的人体工程学和急性呼吸道疾病的诊断和管理的重要工具。

结果示于图1和2为代表的RNA测定（流感基质）和DNA测定（EHV-1）与阳性对照并提交用于常规诊断测试临床样品的组合。贯穿本典型反应发出的荧光信号示于图1，其中每个周期图表原始荧光值。所有相对循环阈值（CT）的值是自动由放大机软件产生的。背景荧光用作装载质量进行评估的独立指标，而不是将其纳入前计算Ct值的荧光读数。 检测每个靶分析限（LOD）的计算基于在95％的检测电平的Ct值加上2个标准差的总体平均中的控制为一池所有目标。的最高稀释度，其中重复的至少95％呈阳性反应的具有代表性的分析为50份;的5份检测重复的50％是成功的。无论是检测下面一个副本进行检测。对于RNA和DNA测定检测限分别以21.92和20.05的Ct值来计算。这些值被认为是对为阳性与嫌疑人（可能不重复）的报告值的截止值。 的目标的分析性能的其它方面，使用在三个不同的日子（ 图2）运行合并阳性对照的系列稀释液进行评估。代表RNA和DNA测定的平均效率为101.1％和106.6％;对所有目标的整体平均效率为101.3％。该试验也有良好的线性（R 2&gt; 0.98）。复制内的变化的通孔通常是两个临床样品的0.2标准偏差之内的ð控制。观察到任何目标之间没有交叉反应性。 在补充文件的最终结果工作表显示为10个临床诊断样本和4控制代表性的定量结果。临床样品是类型的样品的子集定期地测试包括鼻/口咽部拭子，肺组织，和粪便的样品。在这个组中的一个（马）粪便样品示出一个失败的MS2的内部控制，这表明抑制剂的样品中的存在。这通常是通过在洗脱样品和/或重新提取的稀释管理。作为这里的情况下，负放大控制应该是负的所有目标，并且所述阴性萃取控制应该只包含内部控制。在临床组，样品制作的Ct值betacoronavirus， 支气管败血波氏杆菌 ，犬瘟热病毒A，犬副流感病毒，犬肺病毒和流感一个。 <img alt="图1" src="/files/ftp_upload/54781/54781fig1.jpg" /> 图1.扩增曲线。荧光读数作图的RNA的测定（A）和DNA分析（B）的扩增循环。三角形被示表示Ct值。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54781/54781fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54781/54781figure2.jpg" /> 图2.标准曲线。从在三个不同的日子进行测试的RNA和DNA分析的标准曲线绘制在为了证明使用阳性对照的扩增的线性度和范围。循环阈值（CT）的值作图的RNA（A）或DNA的拷贝数的对数（10）（ <strong&gt; B）标准。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54781/54781fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表格1" src="/files/ftp_upload/54781/54781table1.jpg" /> 表1.示意图扩增板和目标位置的。用于纳米实时PCR检测在此平台上的板是显微镜载玻片大小和布置在通孔48的子阵列64，与通孔共3,072个别反应。一个子阵列被这里所示，一式三份18的目标。每个样品由液体处理器添加到一个子阵列。板涂覆有亲水性和疏水性化合物通过表面张力保留在通孔的试剂。不锈钢芯片"光刻图案和湿蚀刻，以形成3,072微机械加工，320微米diamete的一个直线阵列r为对目标的33 NL每一个"2缩写孔如下:BCOR，犬呼吸道冠状病毒（betacoronavirus）; BORD， 支气管败血波氏 CAV，犬腺病毒; CPIV，犬副流感病毒; CPNV，犬肺病毒; DISTA / B，犬。犬瘟热病毒A和B; EADV1 / 2，马腺病毒1/2; EAV，马动脉炎病毒; EHV1 / 4，马疱疹病毒类型1/4; ERVA / B，马鼻炎病毒A / B; IVM，甲型矩阵; MCYNOS， 支原体cynos; MS2，内部控制（MS2噬菌体RNA）;色曲， 马链球菌 。 <img alt="表2" src="/files/ftp_upload/54781/54781table2.jpg" /> 表2盘传送的地图。用于使用固定的8通道移液器从96孔前置放大器板到384孔板转移的彩色编码的板转移图被示出。交替，也可以使用一个可调节的吸移管。相同的过程，每次执行h的无论<html>嗷嗷许多样品都在板。 <img alt="补充文件" src="/files/ftp_upload/54781/54781sup.jpg" /> 补充文件。包含两个宏进行格式化结果到一个汇总表启用了宏的电子表格文件（如在协议中所述）提供。由宏生成的典型结果表是包含在文件中（在最终结果）。的两个宏将填充在样品名称中的第一列（最多48个样品），并为每个目标对于那些有阳性结果三个Ct值的平均值;注意，未扩增的目标出现在该表为空白。此可容易地印刷在一张纸上，并用作用于有效地检查原始数据的参考。在最终结果选项卡中，10个临床样本和4控制代表性的结果显示。用于分析的名称缩写列于表1中的传奇。www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54781/supplemental_code_file_R1.xlsm"目标="_空白"&gt;请点击这里下载该文件。

Watch this Scientific Journal Video about High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR at JoVE.com

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

的DNA和RNA基于病原体由纳米级的PCR的高通量的检测是使用综合征犬和马呼吸PCR面板说明。

在动物标本呼吸道病原体的高通量检测通过纳米PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

high-throughput-detection-respiratory-pathogens-animal-specimens

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

11.9K 次观看.  Cornell University Animal Health Diagnostic Center. 的DNA和RNA基于病原体由纳米级的PCR的高通量的检测是使用综合征犬和马呼吸PCR面板说明。 

视频: High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

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

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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, ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

小胶质细胞的小分子RNA检测在正常中枢神经系统的实时PCR技术在神经发炎

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

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

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

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

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

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

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

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

沙门氏菌筛选的高通量平台.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Immunohistochemistry Test for the Lyssavirus Antigen Detection from Formalin-Fixed Tissues

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. While the direct fluorescent antibody (DFA) test or the direct rapid immunohistochemical test (DRIT) are most commonly utilized for the antigen detection, both tests require fresh and/or frozen tissues for impressions on slides prior to the antigen detection using antibodies. If samples are collected and fixed in formalin, neither test is optimal for the antigen detection, however, testing can be performed by conventional immunohistochemistry (IHC) after embedding in paraffin blocks and sectioning. With this IHC method, tissues are stained with anti-rabies antibodies, sections are deparaffinized, antigen retrieved by partial proteolysis or other methods, and incubated with primary and secondary antibodies. Antigens are stained using horseradish peroxidase / amino ethyl carbazole and counterstained with hematoxylin for the visualization using a light microscope. In addition to the specific antigen detection, formalin fixation offers other advantages like the determination of histological changes, relaxed conditions for specimen storage and transport (under ambient temperatures), ability to test retrospective cases and improved biological safety through the inactivation of infectious agents.

Here, we present an immunohistochemistry test protocol for the detection of rabies virus antigen as an alternative diagnostic test for formalin-fixed tissues.

从正式固定组织检测利沙病毒抗原的免疫化学测试

One of the primary diagnostic modalities for rabies is the detection of viral ribonucleoprotein (RNP) complex (antigen) in the infected tissue samples. ...

Detection of SARS-CoV-2 Neutralizing Antibodies using High-Throughput Fluorescent Imaging of Pseudovirus Infection

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.

The protocol described here outlines a fast and effective method for measuring neutralizing antibodies against the SARS-CoV-2 spike protein by evaluating the ability of convalescent serum samples to inhibit infection by an enhanced green fluorescent protein-labeled vesicular stomatitis virus pseudotyped with spike glycoprotein.

使用假病毒感染的高通量荧光成像检测SARS-CoV-2中和抗体

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the ...