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。

<ol>
	<li>Hale, L. P., Swidsinski, A., Mendling, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+associated+with+bacterial+vaginosis.">Bacteria associated with bacterial vaginosis.</a> N. Engl. J. Med. 354, 202-203 (2006).</li><li>Hay, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+in+the+littoral+zone:+lactobacilli+losing+the+plot.">Life in the littoral zone: lactobacilli losing the plot.</a> Sex. Transm. Infect. 81, 100-102 (2005).</li><li>Morris, M., Nicoll, A., Simms, I., Wilson, J., Catchpole, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+vaginosis:+a+public+health+review.">Bacterial vaginosis: a public health review.</a> Brit. J. Obstet. Gynecol. 108, 439-450 (2001).</li><li>Myer, L., Kuhn, L., Stein, Z. A., Wright, T. C., Denny, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravaginal+practices,+bacterial+vaginosis,+and+women's+susceptibility+to+HIV+infection:+epidemiological+evidence+and+biological+mechanisms.">Intravaginal practices, bacterial vaginosis, and women's susceptibility to HIV infection: epidemiological evidence and biological mechanisms.</a> Lancet. Infect. Dis. 5, 786-794 (2005).</li><li>Wilson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+recurrent+bacterial+vaginosis.">Managing recurrent bacterial vaginosis.</a> Sex. Transm. Infect. 80, 8-11 (2004).</li><li>Nugent, R. P., Krohn, M. A., Hillier, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+diagnosing+bacterial+vaginosis+is+improved+by+a+standardized+method+of+gram+stain+interpretation.">Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation.</a> J. Clin. Microbiol. 29, 297-301 (1991).</li><li>Ison, C. A., Hay, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+a+simplified+grading+of+Gram+stained+vaginal+smears+for+use+in+genitourinary+medicine+clinics.">Validation of a simplified grading of Gram stained vaginal smears for use in genitourinary medicine clinics.</a> Sex. Transm. Infect. 78, 413-415 (2002).</li><li>Money, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+diagnosis+of+bacterial+vaginosis.">The laboratory diagnosis of bacterial vaginosis.</a> Can. J. Infect. Dis. Med. Microbiol. 16, 77-79 (2005).</li><li>Schellenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrosequencing+of+the+chaperonin-60+universal+target+as+a+tool+for+determining+microbial+community+composition.">Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition.</a> Appl. Environ. Microbiol. 75, 2889-2898 (2009).</li><li>Spear, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+diversity+of+the+vaginal+microbiota+in+HIV-infected+and+HIV-uninfected+women+with+or+without+bacterial+vaginosis.">Comparison of the diversity of the vaginal microbiota in HIV-infected and HIV-uninfected women with or without bacterial vaginosis.</a> J. Infect. Dis. 198, 1131-1140 (2008).</li><li>Brotman, R. M., Ravel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ready+or+not:+the+molecular+diagnosis+of+bacterial+vaginosis.">Ready or not: the molecular diagnosis of bacterial vaginosis.</a> Clin. Infect. Dis. 47, 44-46 (2008).</li><li>Fredricks, D. N., Fiedler, T. L., Marrazzo, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+identification+of+bacteria+associated+with+bacterial+vaginosis.">Molecular identification of bacteria associated with bacterial vaginosis.</a> N. Engl. J. Med. 353, 1899-1911 (2005).</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=Multiplex+detection+of+bacteria+associated+with+normal+microbiota+and+with+bacterial+vaginosis+in+vaginal+swabs+by+use+of+oligonucleotide-coupled+fluorescent+microspheres.">Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres.</a> J. Clin. Microbiol. 47, 4067-4077 (2009).</li><li>Molenkamp, R., van der Ham, A., Schinkel, J., Beld, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+of+five+different+DNA+targets+by+real-time+Taqman+PCR+using+the+Roche+LightCycler480:+Application+in+viral+molecular+diagnostics.">Simultaneous detection of five different DNA targets by real-time Taqman PCR using the Roche LightCycler480: Application in viral molecular diagnostics.</a> J. Virol. Meth. 141, 205-211 (2007).</li><li>Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., Rogers, Y. 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cpnDB，cpn60 UT斯达康序列数据库16的序列;匹配多个生物体的探头，要避免可能的话。此外，当确定潜在的特异性探针，我们采取的办法来探测验证一个复杂的，人工的TEmplate准备相应的克隆cpn60利息13菌群生物UT。扩增完整的"混合面板"，包括目标模板，然后生成和信号相比，从第二个模板组成的混合面板不包含目标生物体的cpn60 UT斯达康生成。产生高信号的信噪比（信号产生完整的面板和产生的噪音是没有目标模板的面板）的探针，对列入最后的检测选择。我们通常有4-5每前对列入最终的Luminex公司数组选择之一的目标生物可能探针分析。 定义一个积极的结果，是一个要考虑的问题。我们发现，不同的探头有本质的不同的信号信噪比，所以我们决定办法，即一个积极的结果是defin通过一个非常简单的统计检验:学生的t检验（单尾）。定义一个积极的结果是通过比较样品的MFI"无模板"的控制，以及时的MFI明显比控制在P &lt;0.05，结果被认为是该生物体的积极。通常情况下，两个独立产生的扩增子是重复测试。虽然PCR和T7核酸外切酶的步骤创建单链PCR产物约64.5μL（步骤2.3），只有17μL杂交（步骤3.2），足以满足3个杂交检测，我们通常从两个扩增分析重复不使用水井的数量过多，给固有的技术复制。 这个我们已经调查检测的一个方面是其半定量性质。从一个给定的探头的MFI信号与一个给定的有机体的qPCR确定的丰度（Spearman等级相关系数＆R何= 0.4686，P &lt;0.0001）13，但动态范围是有限的;信号趋于饱和，在更高的目标生物水平，符合市场预期，因为分析终点后40个循环的PCR扩增。因为它是半定量，可监测和生物体繁多的趋势观察模式（保持心中的警告信号的饱和度较高的丰度）。例如，图4显示了个人的阴道菌群，从BV的正常时间的配置文件。发生这种情况，相应的信号至 G 阴道毛滴虫 （一个BV相关的有机体17）增加，然后阴晴圆缺，而在监视时间的当然L.的信号iners增加。通讯克渍，目前BV的诊断"金标准"，即革兰氏阳性杆菌最初缺席，但在稍后的时间点检测显示模式。 Luminex公司的方法，既识别生物样品中，给小号OME信息的相对丰度。这种类型的信息可能被用于监控任何类型特别是生物体的丰度的不良趋势的样品，可以提供大量的生物体同时数据。 更重要的是，这种方法是不局限于阴道菌群分析，可以合理地应用到任何环境中是可取的一系列目标生物体的检测和部分定量。当应用到其他环境的方法，它是重要的考虑因素发生PCR抑制的可能性，共同净化通常与来自许多环境中 18的DNA的PCR抑制剂。 PCR的抑制是一个问题，可以稀释后的样品或进一步纯化，以产生足够的信号这个实验。

<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

Nanyang Technological University

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

Multiplexed Fluorescent Microarray for Human Salivary Protein Analysis Using Polymer Microspheres and Fiber-optic Bundles

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array technology employed combines the advantages of microsphere-based suspension array fabrication with the use of fluorescence microscopy. As described in the video protocol, commercially available 4.5 &#956;m polymer microspheres were encoded into seven different types, differentiated by the concentration of two fluorescent dyes physically trapped inside the microspheres. The encoded microspheres containing surface carboxyl groups were modified with monoclonal capture antibodies through EDC/NHS coupling chemistry. To assemble the protein microarray, the different types of encoded and functionalized microspheres were mixed and randomly deposited in 4.5 &#956;m microwells, which were chemically etched at the proximal end of a fiber-optic bundle. The fiber-optic bundle was used as both a carrier and for imaging the microspheres. Once assembled, the microarray was used to capture proteins in the saliva supernatant collected from the clinic. The detection was based on a sandwich immunoassay using a mixture of biotinylated detection antibodies for different analytes with a streptavidin-conjugated fluorescent probe, R-phycoerythrin. The microarray was imaged by fluorescence microscopy in three different channels, two for microsphere registration and one for the assay signal. The fluorescence micrographs were then decoded and analyzed using a homemade algorithm in MATLAB.

We describe a procedure for profiling salivary proteins using multiplexed microsphere-based antibody arrays. Monoclonal antibodies were covalently linked to fluorescent dye-encoded 4.5 &#x03BC;m polymer microspheres using carbodiimide chemistry. The modified microspheres were deposited in fiber-optic microwells to measure protein levels in saliva using fluorescence sandwich immunoassays.

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

Herein, we describe a protocol for simultaneously measuring six proteins in saliva using a fiber-optic microsphere-based antibody array. The immuno-array ...

科研

化学

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

使用多色荧光检测液滴微流控光纤

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

生物工程

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

毛细管阵列中多核酸的视觉检测

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

免疫与感染

A Rapid Method for Multispectral Fluorescence Imaging of Frozen Tissue Sections

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample that can provide information about antigen coexpression and spatial distribution of the markers. However, a lack of suitable antibodies for formalin-fixed tissues may restrict the nature of markers that can be detected. In addition, the staining method is time-consuming. Here we describe a rapid method to perform multispectral fluorescence imaging on frozen tissues. The method includes the fluorophore combinations used, detailed steps for the staining of mouse and human frozen tissues, and the scanning, acquisition, and analysis procedures. For staining analysis, a commercially available semiautomated multispectral fluorescence imaging system is used. Through this method, up to six different markers were stained and detected in a single frozen tissue section. The machine learning analysis software can phenotype cells that can be used for quantitative analysis. The method described here for frozen tissues is useful for the detection of markers that cannot be detected in FFPE tissues or for which antibodies are not available for FFPE tissues.

We describe a rapid staining method to perform multispectral imaging on frozen tissues.

冷冻组织部分多光谱荧光成像的快速方法

Multispectral fluorescence imaging on formalin-fixed paraffin-embedded (FFPE) tissues enables the detection of multiple markers in a single tissue sample ...

血清中 疏螺旋体特异性 IgG 和 IgM 的多重免疫检测

Simultaneous Detection of Different Antibody Classes in a Multiplexed Serological Test

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different antibody isotypes because they appear at different stages of the host immune response. With Lyme borreliosis, the pathogenic agent can be one of the multiple members of the Borrelia species. Therefore, correct sample classification requires evaluating the immunoreactivity against different antigens of different Borrelia species. Additionally, anti-pathogen IgG and IgM responses can have different elicitation time courses during disease progression. Here we demonstrate the development of a two-reporter multiplex immunoassay that has utility in identifying Borrelia-specific immune response in human serum samples by simultaneously evaluating both IgG and IgM immunoreactivity against different bacterial antigens in the same reaction well. This dual-reporter approach retains the analytical performance of single-reporter methods while conserving time and resources and reducing sample size requirements. This assay allows essentially double the serological information to be generated from a blood sample in half the time.

作者聚焦:扩大用于莱姆疏螺旋体病诊断和病原体研究的多重免疫检测的范围 

A three-channel dual-reporter fluorescence flow analysis system was used to develop a bead-based multiplex immunoassay that simultaneously evaluates serum samples for IgG and IgM elicited against multiple antigens of different Borrelia species that cause Lyme borreliosis in Europe and North America.

在多重血清学检测中同时检测不同抗体类别

To monitor the progression of infectious diseases, it is useful to assess immunoreactivity against various antigenic determinants, and measure different ...

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

摘要

探索更多视频

此视频中的章节

复用荧光微阵列人类唾液蛋白质分析用高分子微球和光纤束

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

使用多色荧光检测液滴微流控光纤

与复用电子检测的粒子的空间跟踪微流体平台

毛细管阵列中多核酸的视觉检测

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

冷冻组织部分多光谱荧光成像的快速方法

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别

在多重血清学检测中同时检测不同抗体类别