我们感谢这个手稿与实验发展和批评性意见帮助阿尔贝托Severini和Vanessa Goleski。这项工作是由加拿大公共卫生署和工业研究援助计划（加拿大国家研究理事会）。从萨斯喀彻温大学出版基金获得额外的支持。

这种方法被用于Dumonceaux 等的研究。J。临床。微生物 47，4067-4077，DOI:10.1128/jcm.00112-09（2009年）。 图1描绘了整个过程的示意图。 1。珠耦合这说明了耦合聚苯乙烯Luminex公司珠的寡核苷酸探针（见表2）使用方法。卷稍微适应新的捕获探针的评价试行;括号中表示这些卷。 <ol><li>取出-20℃dessicator 1 - 乙基-3 - （3 - dimethylamiopropyl）碳二亚胺盐酸（EDC）的粉末和温暖到室温。 </li><li>重悬在20秒水浴sonicator sonciation微球，然后震荡约20秒。 </li><li> 400μL（100微升）微球转移到Eppendorf管（与此相对应5X10 6或1.2510 月 6微球） 。 Luminex公司建议使用低蛋白结合离心管（如的Eppendorf蛋白LoBind管，目录编号为0030 108.094），以防止粘管和干扰珠恢复耦合珠。我们没有发现这是一个问题，使用标准的聚丙烯离心管中，我们通常使用的（如VWR目录号87003-298）。 </li><li>佩莱在1分钟XG 14000。取下并丢弃上清液。 </li><li>重悬在50μL室温2 0.1 M的微球（12.5μL） - （N -吗啉）乙磺酸（MES），pH值4.5。 </li><li>准备在10毫克/毫升的水，在新鲜的EDC解决方案。 <ol><li>重分析规模的EDC 5-10毫克，小于1毫升这种解决方案的准备，然后加水至10毫克/毫升。 </li></ol></li><li>加入1 nmol 5&#39; -氨基C12修饰的捕获寡核苷酸（见表2）微球，并通过震荡混合。 1 nmol 5μL200微米寡核苷酸。 </li><li>振荡5秒的微球和混合添加2.5μL新鲜的EDC解决方案。 </li><li>在室温下孵育30分钟，在黑暗中。 </li><li>丢弃的EDC解决方案（步骤1.6），并准备在水上面（步骤1.6）的10毫克/毫升的EDC的新鲜样品。 </li><li>添加另一种新鲜的EDC解决方案2.5μL到5秒的微球和涡。 </li><li>在室温下孵育30分钟，在黑暗中。 </li><li>洗净加入1毫升0.02％吐温20的珠子。涡（超声以及可选20秒）重悬珠。 </li><li>离心14000 XG 1分钟。取下并丢弃上清液。 </li><li>再加入1毫升0.1％十二烷基硫酸钠（SDS），洗珠。涡（超声以及可选20秒）重悬珠。 </li><li>离心14000 XG 1分钟。取下并丢弃上清液。 </li><li>重悬在100μL（25μL）的Tris - EDTA（TE）的缓冲区[10米的珠中号的Tris - Cl的pH值8.0，1 mM的EDTA，pH值8.0。 </li><li>枚举在血球或库尔特计数器的珠子来确定股票的浓度。 </li><li>每个珠子稀释到终浓度为100珠/μLTE缓冲准备微法师组合。池加上珠相应的检测所需的复杂的（如10复杂的检测，混合在每个珠子的100/μl终浓度为10种不同的耦合珠）。 </li><li>微预混存放在4 ° C黑暗环境中。混合物可以保存几个月，如果这些条件下保存。 </li></ol> 2。分子伴侣60通用目标（cpn60 UT）的单链扩增生产和代。 <ol><li>生成聚合酶链反应每个样本（PCR）产品。包括硫代和生物素修饰的5&#39;引物（表1）。卷组合和浓度，请参阅表3。 </li><li>后立即PCR是完整的，加2μL（20个单位的T7前）onuclease到每个PCR管（PCR缓冲液，将足以为T7反应）。孵育反应在室温（22-25 ° C）为40分钟。 </li><li>在此潜伏期年底，加入12.5μL0.5 M的乙烯二胺四乙酸（EDTA）pH值8.0和组合。这给出了一个共〜64.5μl的单链PCR产物。 </li></ol> 3。 PCR产物单链寡核苷酸耦合聚苯乙烯珠杂交。 <ol><li> Prewarm仪器至60 ° C至杂交温度保持在分析过程中。使用该仪器的软件平台上的加热器打开，一定要使用适合包含杂交珠混合式PCR板的黄铜加热块。 </li><li>悬浮微硕士混合用移液器（步长1.20），免除到Eppendorf管适量，管帽，和超声水浴sonicator在2分钟。另外，绝对的一致性，以确保在珠resuspe微预混nsion，可悬浮振荡和吹打，然后超声以上，然后配药聚丙烯Eppendorf管中所需的金额。 </li><li>免除低调96井套管PCR板（见表2）适当的水井，单链PCR产物17μL（2.2步）。添加33μL悬浮，sonciated珠的混合物，以每口井。盖硅胶套（见表2），和自来水轻轻。 </li><li>放入热循环的方案板块:95℃5分钟，10分钟，60℃60℃保持60 ° C为5分钟，结束。启动该程序。 </li><li>做出新的链亲和素 - 藻红蛋白（SA - PE）的解决方案，您将需要每孔25μL（额外的几口井）。稀释股票的SA - PE（1毫克/毫升）1:50至20微克/毫升1X四甲基氯化铵（TMAC）缓冲区（3中号TMAC SA - PE解决方案; 0.1％Sarkosyl; 50毫米的Tris - HCl，pH值8.0 4毫米EDTA，pH值8.0）。 </li><li>当热循环到达60 ° C的保持步，打开盖子，取出硅胶盖，直接添加SA - PE解决方案，以每口井（不利用的热循环板）。更换硅胶套，关闭热循环盖和恢复程序。 </li><li>当程序完成后，走板，并迅速转移到BioPlex机读。板必须在10分钟内读取。阅读在60 ° C;确保BioPlex已被预热到这个温度。可以肯定，已调整，以适应板使用，用户手册描述了使用的仪器探头高度。 </li><li>准确珠和信号检测，BioPlex门设置必须设置根据正在使用的微型。 BioRad公司聚苯乙烯珠需要4,335-10,000门设置，而磁性微球的需要设置的5,000-25,000。也有选项板采用高的光电倍增管的设置，增加了记者增益值运行。这我可以ncrease降低信号的强度，但它是很重要的，包括适当的阴性对照，作为背景的​​信号也将增加。 </li></ol> 4。代表性的成果: 我们在制定和实施这个实验的目标之一是使尽可能简单和简化。因此，我们优化了放大后的关键步骤，包括T7核酸外切酶治疗时间和杂交时间，以发展一个可以在合理的时间内完成检测。如图2a所示的扩增T7治疗是对信号的产生至关重要，因为大多数探头短或没有T7治疗很少或根本没有信号。信号呈线性增加，直到大约40分钟，此时的信号增幅放缓。 T7治疗时间2小时观察，即使在无信号的退化，这表明，硫代修饰引物的高度有效地防止轻盾吨钢绞线退化描述15。我们选择T7治疗时间为40分钟，以减低整体的协议时间，但它是从图2A的T7治疗可以去更长的时间。我们还确定了杂交时间对信号产生的影响（图2B），并发现，10分钟足够的最大信号，没有信号的进一步增加，即使后4个小时的杂交观察。因此，选择了一个10分钟的杂交步骤，再次降低整体检测时间。记住，这些结果如InstaGene（BIO - RAD）快速DNA提取技术，整体的检测，包括DNA提取，PCR和Luminex分析，在4-5个小时就可以完成。 Luminex公司或B​​ioPlex运行期间的聚苯乙烯微球的聚合水平的一个警示说明，可以检测效率有重大影响。 BioPlex软件将显示珠聚集的水平，即当不止一个珠是在激光的路径和结果中排除从总量上的杂交信号检测。视珠聚集也可以被任何微粒物质引起的，不是单一珠的适当大小，甚至可以造成气泡。在这些事件中的任何，从珠聚合，气泡，或粒子的信号被丢弃。在大多数情况下，我们发现，珠聚合最小（图3A）和100票对每个珠子事件的目标水平是很容易实现。然而，有时珠表明一个聚集温和（图3B）或严重（图3C）的水平。在这种情况下，因为大多数的数据将被丢弃，仪器可能无法达到100事件每珠型，结果可能有问题。超声步骤（步骤3.2），是为了尽量减少珠聚集。此外，存储为一个稀微的master mix（步长1.20）珠可能有帮助。我们注意到，排除TMAC的hybridization缓冲区 - 将它添加到的SA - PE稀释剂（步骤3.5） - 有助于减少珠聚集。此外，虽然我们没有测试这个新的Luminex公司或B​​ioRad公司提供的磁珠被认为是显示的倾向聚合。 针对G. 5 plex的Luminex公司阵列的应用结果阴道毛滴虫，A。阴道，L。 iners，L. crispatus，和L。 gasseri连同相应的革兰氏染色阴道拭子涂片如图4所示。这些样本来自在多个时间点的单个人。时间为0，个人与BV诊断的基础上革兰氏染色（图4A），并从同一样品的Luminex公司的结果（图4B）显示阵列，G.代表的生物阴道毛滴虫是最普遍的，而A。阴道和L. iners也是积极的。 9天之后，个人仍然是BV的阳性和Signal为G。阴道有大幅增加，而A。阴道和L. iners仍为阳性。更重要的是，经过这段时间的个人开始过渡到一个正常的菌群，革兰氏阳性杆菌涂片检测（图4A）， 而 G的信号阴道减弱和信号为L。 iners增加（图4B）。我们用这种方法（G.阴道和/或A.阴道样本呈阳性反应，被认为是BV 的积极）13 BV的原始定义，这个人是积极BV在所有时间点， 虽然未能检测G.革兰氏染色阴道在后两个时间点。随着Luminex公司这里介绍的方法，趋 ​​势可以很容易地相比，基于测序的方法，和Luminex公司检测结果证实革渍 13，同时提供对生物体的身份和丰富的附加 ​​信息。 <img src="/ files/ftp_upload/3344/3344fig1.jpg"ALT ="图1"/&gt; 图1。Luminex公司的协议确定一个复杂的临床或环境样品的菌群轮廓示意图。该协议开始已经从样本中提取的利息的DNA模板。 （1）生成模板DNA的PCR产物链特定的生物素化， 硫代修饰cpn60 UT斯达康PCR引物， 再加上未修改cpn60 UT PCR引物（表1），（2），这将产生一个PCR产物池中的微生物与生物素硫代一个链上的修改;（3）精华的T7核酸外切酶，它可以不降低硫代修饰的分支和因此产生的单链DNA的，是在5&#39;端用生物素修饰的双链PCR技术产品;（4）夫妇聚苯乙烯珠特定物种cpn60 UT斯达康探头-每个珠子具有独特光谱地址（由珠表示），我小号有迹可寻Luminex公司或生物复杂的仪器;（5）杂交的单链PCR产物产生利益的样本种特异性寡核苷酸，加上珠套件;（6）添加绑定到的链亲和素 - 藻红蛋白共轭生物素标记的单链PCR产物和杂交指标的行为;（7）确定光谱地址（珠身份）和杂交信号的强度，使用Luminex公司或生物复杂的仪器。至少有100珠计算每个珠子的身份和中位数的荧光强度（MFI）的藻红蛋白信号输出报告。多达100个不同的珠类型可以同时进行评估，但检测显示三珠类型的歧视说明。 （8）当在PCR的小额信贷机构重复相同的样本产生是明显大于在一个给定珠负面控制（之一，尾学生的吨的测试，p &lt;0.05），样本是认为，有机体阳性。<html><img src="/files/ftp_upload/3344/3344fig2.jpg" alt="图2" /> 图2。Luminex公司检测参数优化。针对链球菌anaerobius五种不同的探针，用混合模板产生的扩增子包括质粒的克隆 cpn60 20已知与阴道的细菌，UT 斯达康 ，包括体育anaerobius。 （一）。 T7核酸外切酶治疗时间的影响。其次上面所描述的协议，但不同杂交和中位数的荧光强度（MFI）的所有探测器测定前用T7核酸外切酶的治疗时间。 （二）。杂交时间的影响。上面所描述的协议之后，各种杂交倍。同一组的探针被用于从同一模板生成A和中位数的荧光强度（MFI）在确定所有探头的扩增。 "&gt; <img src="/files/ftp_upload/3344/3344fig3.jpg" alt="图3" /> 图3。测定BioPlex软件珠聚集。三个例子BioPlex运行在珠聚集水平（一）接受（5％）;（二）边缘（50％）;及（C）不可接受的（80％）。 <img src="/files/ftp_upload/3344/3344fig4.jpg" alt="图4" /> 图4 5 plex的Luminex公司数组BV采取连续样品来自同一个体诊断中的应用。 （一）。革兰氏染色的幻灯片显示了传统的诊断用于评估BV每个样品。 （二）。 5 plex的Luminex公司数组的应用在本协议中所述相同的四个样品（a）所示的准备和执行。细菌的MFI信号的目标是通过我们的定义（单尾学生 t检验，P &lt;0.05）是由星号（*）表示显著积极。

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+phosphorothioate+primers+and+exonuclease+hydrolysis+for+the+preparation+of+single-stranded+PCR+products+and+their+detection+by+solid-phase+hybridization.">The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.</a> PCR. Methods. Appl. 3, 285-291 (1994).</li><li>Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H., Hemmingsen, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cpnDB:+A+Chaperonin+Sequence+Database.">cpnDB: A Chaperonin Sequence Database.</a> Genome. Res. 14, 1669-1675 (2004).</li><li>Bradshaw, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+association+of+Atopobium+vaginae+and+Gardnerella+vaginalis+with+bacterial+vaginosis+and+recurrence+after+oral+metronidazole+therapy.">The association of Atopobium vaginae and Gardnerella vaginalis with bacterial vaginosis and recurrence after oral metronidazole therapy.</a> J. Infect. Dis. 194, 828-836 (2006).</li><li>Dumonceaux, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enumeration+of+specific+bacterial+populations+in+complex+intestinal+communities+using+quantitative+PCR+based+on+the+chaperonin-60+target.">Enumeration of specific bacterial populations in complex intestinal communities using quantitative PCR based on the chaperonin-60 target.</a> J. Microbiol. Meth. 64, 46-62 (2006).</li></ol>

没有利益冲突的声明。

特异性的信号的产生是至关重要的，你必须有信心，信号观察真实地反映了从该生物体产生的扩增检测。如PrimerPlex（总理Biosoft）的软件可以帮助设计探针，将有效地杂交，但他们可能会或可能不会跨非目标物种杂交。如在本议定书中所述，当使用通用引物进行PCR，重要的是要牢记，代表所有的生物样品中扩增产生。它是重要的，因此，分析潜在的探针交叉杂交的可能性比较探针设计软件建议c...

<table border="1"><tbody><tr><td> 寡核苷酸名称 </td><td> 公司 </td><td> 序列1 </td></tr><tr><td> H279BP </td><td rowspan="3"> Invitrogen公司，内幕交易审裁处，或其他</td><td> 生物素OEFO GAIIIIGCIGGIGAYGGIACIACIAC </td></tr><tr><td> H280 </td><td> YKIYKITCICCRAAICCIGGIGCYTT </td></tr><tr><td> H1612BP </td><td> 生物素OEFO GAIIIIGCIGGYGACGGYACSACSAC </td></tr><tr><td> H1613 </td><td></td><td> CGRCGRTCRCCGAAGCCSGGIGCCTT </td></tr><tr><td colspan="3"> 1O，硫代- C，E，硫代- G;楼硫代一个;我，肌苷，Y，C或T，R，A或G，K，T或G，S，C或G 。 </td></tr></tbody></table> 见表1。修改后的通用cpn60 PCR寡核苷酸序列。 <table border="1"><tbody><tr><td> 试剂名称</td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 1 - 乙基-3 - （3 - dimethylamiopropyl）碳二亚胺盐酸（EDC） </td><td>刺穿</td><td> 22980 </td><td></td></tr><tr><td>荧光聚苯乙烯珠:MicroPlex微球（Luminex公司），或生物复杂的羧基珠（BIO - RAD） </td><td> Luminex公司或B​​io - Rad公司</td><td> Bio - Rad公司:171 - 506xxx其中xxx对应珠标识符</td><td>磁珠成为寡核苷酸耦合，并可能提供一定的优势。没有尝试过这些作者。 </td></tr><tr><td>捕获寡核苷酸（5&#39;氨基C12修饰） </td><td> Invitrogen公司，内幕交易审裁处，或其他</td><td>各种</td><td>脱盐的纯度水平是可以接受的的。用来描述阴道拭子的捕获探针的序列是在这协议是基于 13的手稿。 </td></tr><tr><td> T7核酸外切酶</td><td> New England Biolabs公司</td><td> M0263S </td><td></td></tr><tr><td>链霉亲和R -藻红蛋白（SA - PE） </td><td> Invitrogen公司</td><td> S - 866 </td><td>要小心，以获得高纯度的SA - PE;这个目录建议</td></tr><tr><td>套管96孔PCR板</td><td>费舍尔</td><td> CS006509 </td><td>融入96孔的热循环和BioPlex机</td></tr><tr><td>套管密封垫</td><td>费舍尔</td><td> CS006555 </td><td>可重新使用，用肥皂水洗，冲洗干净，干燥</td></tr><tr><td> 5M TMAC </td><td>西格玛</td><td> T3411 </td><td></td></tr><tr><td>生物复杂或Luminex公司仪器</td><td> Bio - Rad公司或Luminex公司</td><td> Bio - Rad公司:171-000201 </td><td></td></tr><tr><td> PrimerPlex探针设计软件</td><td>总理Biosoft </td><td> www.premierbiosoft.com </td><td>建议对Luminex公司探头DE的迹象，虽然可以使用其他软件平台</td></tr></tbody></table> 表2特定的试剂和设备。 <table border="1"><tbody><tr><td> 组件 </td><td> μL/检测 </td><td> μl/100分析 </td><td> 终浓度 </td></tr><tr><td> 10x PCR缓冲液（Invitrogen公司） </td><td> 5 </td><td> 500 </td><td> 1X </td></tr><tr><td> 50毫米MgCl 2的 （Invitrogen公司） </td><td> 2.5 </td><td> 250 </td><td> 2.5毫米</td></tr><tr><td> 10 mM的dNTP的</td><td> 1 </td><td> 100 </td><td>每次0.2毫米</td></tr><tr><td> H279BP，25μM </td><td> 0.25 </td><td> 25 </td><td> 375牛米</td></tr><tr><td> H1612BP，25μM </td><td> 0.75 </td><td> 75 </td><td> 125纳米</td></tr><tr><td> H280，25微米</td><td> 0.25 </td><td> 25 </td><td> 375牛米</td></tr><tr><td> H1613，25微米</td><td> 0.75 </td><td> 75 </td><td> 125纳米</td></tr><tr><td>水</td><td> 34 </td><td> 3400 </td><td> --- </td></tr><tr><td>总计</td><td> 44.5 </td><td> 4450 </td><td></td></tr></tbody></table> 表3。推荐的PCR引物（表1）与修改cpn60 UT混合物。该试剂盒被设置为5μL模板DNA和0.5μLTaq DNA聚合酶（Invitrogen公司）（2.5U）。通常情况下，大量的准备（如1​​00检测足够），并储存在-20 ° C

multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres

细菌性阴道病（BV）是一个经常性的多种微生物综合征的特点是在"正常" 菌群改变乳酸菌占主导地位， 其中包括阴道加德纳菌，Atopobium阴道的细菌种类占主导地位的菌群， 及其他1-3。这种情况是关联与对健康的负面结果，包括艾滋病毒收购4范围内，它可能难以管理临床 5 。此外，BV的诊断依赖于使用的各6,7数值标准取得的阴道拭子涂片革兰氏污渍。虽然这个诊断是简单，价格低廉，适合资源有限的环境，它可以受到相关的主观解释，并没有给出一个详细的个人资料的阴道菌群8的组成问题。最近的深度测序工作已经透露了​​一个丰富，形式多样的阴道菌群明确诊断相比，那些被认为正常9,10个人，这导致在11,12 BV的分子诊断的潜在目标识别与BV的个人采取的样本之间的差异。这些研究提供了丰富的有用信息，但深度测序尚未实际在临床上的诊断方法。最近，我们描述的方法，迅速 ​​分析多重格式的阴道菌群，使用寡核苷酸耦合的荧光珠检测在 Luminex公司平台13。像目前的革兰氏染色的方法，这种方法是快速和简单，但增加了额外的优势，利用分子测序探针设计研究产生的知识。因此，这种方法提供了一种方法来配置文件的阴道拭子，可用于诊断BV具有较高的特异性和灵敏度比赛中的主要微生物ared革兰氏染色同时提供更多的信息物种的存在和丰富的半定量和快速的方式。这种复用的方法是，远远超出了目前定量PCR分析，特别是生物，目前仅限于5或6个不同的检测单个样品14范围扩展。更重要的是，该方法不仅限于细菌在阴道拭子检测，可以很容易地适应快速分析几乎所有的微生物群落的兴趣。例如，我们最近开始申请污水处理厂使用这种方法的诊断工具的发展。

Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

我们描述了一个复用的微生物样本内使用寡核苷酸耦合的荧光珠的检测方法。从样本内的所有生物体的扩增杂交的探针耦合珠小组。一个Luminex公司或生物复杂的仪器是用于查询每个珠珠型和杂交信号。

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

multiplex-detection-bacteria-complex-clinical-environmental-samples

Research

JoVE Journal

Immunology and Infection

16.1K 次观看.  Agriculture and Agri-Food Canada. 我们描述了一个复用的微生物样本内使用寡核苷酸耦合的荧光珠的检测方法。从样本内的所有生物体的扩增杂交的探针耦合珠小组。一个Luminex公司或生物复杂的仪器是用于查询每个珠珠型和杂交信号。

视频: Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

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

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

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

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

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to the best of these authors' knowledge, no method has been developed to allow for the in situ visualization of the 
bacteria in histological sections.
While bacterial localization has been determined in vivo in previous studies using plate counts 3, radiometric labeled 4, 
or bioluminescent bacteria 5, most of these studies have only been performed at the gross organ level, with one exception 6. This limitation 
is of particular concern because E. ictaluri has a complex infection cycle 1,7, and it has a variety of virulence factors 8,9. 
The complex interaction of E. ictaluri with its host is similar in many respects to Salmonella typhi 10, which is in the same taxonomic family.
Here we describe a technique allowing for the detection of bacteria using indirect immuno-histochemistry using the monoclonal Ed9 antibody described by Ainsworth et al.11.
Briefly, a blocking serum is applied to paraffin embedded histological sections to prevent non-specific biding. Then, the 
sections are incubated with the primary antibody: E. ictaluri specific monoclonal antibody Ed9. Excess antibodies are rinsed away and the FitC 
labeled secondary antibodies are added. After rinsing, the sections are mounted with a fluorescent specific mounting medium. 
This allowed for the detection of E. ictaluri in situ in histological sections of channel catfish tissues.

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Here we describe a procedure allowing the labeling of Edwardsiella ictaluri in situ in histological sections from channel catfish Ictalurus punctatus using indirect immunohistochemistry with monoclonal antibodies Ed9 as a primary, and fluorescent FitC labeled antibodies as a secondary. This allowed for the detection of the bacterium using fluorescent microscopy.

使用荧光免疫化学检测爱德华ictaluri</em斑点叉尾鮰>（一毛虫）样品

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

确定临床抗体样品的吞噬活性

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

检测使用荧光脱氧核酶的细菌

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in SJL mice and cardiac myosin heavy chain-&#945; (Myhc) 334-352-induced experimental autoimmune myocarditis (EAM) in A/J mice. Two sets of cocktails of dextramer reagents were used, where dextramers+ cells were analyzed by laser scanning confocal microscope (LSCM): EAE, IAs/PLP 139-151 dextramers (specific)/anti-CD4 and IAs/Theiler&#8217;s murine encephalomyelitis virus (TMEV) 70-86 dextramers (control)/anti-CD4; and EAM, IAk/Myhc 334-352 dextramers/anti-CD4 and IAk/bovine ribonuclease (RNase) 43-56 dextramers (control)/anti-CD4. LSCM analysis of brain sections obtained from EAE mice showed the presence of cells positive for CD4 and PLP 139-151 dextramers, but not TMEV 70-86 dextramers suggesting that the staining obtained with PLP 139-151 dextramers was specific. Likewise, heart sections prepared from EAM mice also revealed the presence of Myhc 334-352, but not RNase 43-56-dextramer+ cells as expected. Further, a comprehensive method has also been devised to quantitatively analyze the frequencies of antigen-specific CD4 T cells in the &#8216;Z&#8217; serial images.

In Situ Detection of Autoreactive CD4 T Cells in Brain and Heart Using Major Histocompatibility Complex Class II Dextramers

The protocol to detect self-reactive CD4 T cells in brain and heart by direct staining with major histocompatibility complex class II dextramers has been described in this report. For comprehensive analysis, a reliable method to enumerate the frequencies of antigen-specific CD4+ T cells in situ is also devised.

原位检测自身反应性CD4 + T细胞在大脑和心脏使用主要组织相容性复合II类Dextramers的

This report demonstrates the use of major histocompatibility complex (MHC) class II dextramers for detection of autoreactive CD4 T cells in situ in myelin ...

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled 16S rRNA-targeting DNA probes has been described. This protocol can be applied to study the role of bacteria in various diseases such as periodontitis, cancers, and inflammatory immune diseases.

原位检测使用地高辛标记的DNA探针石蜡包埋组织中的细菌靶向16S rRNA基因

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been ...

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

In the current climate of scarce resources, new technologies are emerging that allow researchers to conduct studies cheaper, faster and with more precision. Here we describe the development of a bead-based salivary antibody multiplex immunoassay to measure human exposure to multiple environmental pathogens simultaneously.

制定唾液抗体多重免疫测定来测量人体暴露于环境中的病原体

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and ...

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 ...

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

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