Name: multiplex detection of bacteria in complex clinical and environmental samples using oligonucleotide-coupled fluorescent microspheres
Uploaded: 2011-10-23T14:00:00
Description: Watch this Scientific Journal Video about Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres at JoVE.com

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 - dimethylamio...

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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>

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 ...

Research

JoVE Journal

Immunology and Infection

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

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 ...