Name: detection of bacteria using fluorogenic dnazymes
Uploaded: 2012-05-28T13:00:00
Duration: 13 min 20 s
Description: Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

自然科学和工程研究理事会（NSERC）的加拿大与Sentinel生物活性纸网络，为这项工作提供了资金。

<ol>
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没有利益冲突的声明。

今天最常见的细菌检测方法是缓慢的（经典的微生物）或（抗体，PCR）技术要求高。因此，我们认为，应配合向速度和简单的检测工具的下一代。为此，我们已经建立了一个RNA裂解和荧光信号核酶可用于开发简单的检测报告通过荧光信号的产生细菌的存在。功能核酶探针，RFD的EC1的，被激活的大肠杆菌的生长过程中产生的CEM在培养基中的大肠杆菌 。由于我们的方法使用细菌的原油外混合物作为检测目标和绕过艰苦的目标提取和扩增步骤，它可以用来设置很简单，“混合和阅读”类型的细菌检测的检测。使用我们的核酶不仅限于基于荧光检测方法。例如，比色法检测使用相同的核酶系统检测Ç设计使用以前报道的方法，利用有机染料结合滚环扩增32，我们预计使用细菌检测脱氧核酶作为一个有吸引力的途径，产生新的细菌生物传感器具有更大的操作简便。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号或型号 </td></tr><tr><td>洋菜</td><td> BioShop加拿大</td><td> AGR003 </td></tr><tr><td>过硫酸铵（APS） </td><td> BioShop加拿大</td><td> AMP001 </td></tr><tr><td>丙烯酰胺/双丙烯酰胺（40％，29:1） </td><td> BioShop加拿大</td><td> ACR004 </td></tr><tr><td>硼酸</td><td> BioShop加拿大</td><td> BOR001 </td></tr><tr><td>溴酚蓝</td><td> Sigma-Aldrich公司</td><td> B8026 </td></tr><tr><td> EDTA的</td><td>电磁学</td><td> EXO539-1 </td></tr><tr><td>盐酸</td><td> Sigma-Aldrich公司</td><td> 38281 </td></tr><tr><td>肝素钠</td><td> bioshop加拿大</td><td> HEP001 </td></tr><tr><td> LB培养基</td><td> Sigma-Aldrich公司</td><td> L3022 </td></tr><tr><td> MgCl 2的 </td><td>电解二氧化锰化学制品</td><td> B10149-34 </td></tr><tr><td>氯化钠</td><td> BioShop加拿大</td><td> SOD002 </td></tr><tr><td>醋酸钠</td><td>电解二氧化锰化学品</td><td> SXO255-1 </td></tr><tr><td>氢氧化钠</td><td>电解二氧化锰化学品</td><td> SXO590-1 </td></tr><tr><td> SDS </td><td> BioShop加拿大</td><td> SDS001 </td></tr><tr><td> TEMED </td><td> BioShop加拿大</td><td> TEM001 </td></tr><tr><td> Tris碱</td><td> BioShop加拿大</td><td> BST666 </td></tr><tr><td>吐温20 </td><td> Sigma-Aldrich公司</td><td> P9416 </td></tr><tr><td>尿素</td><td> BioShop加拿大</td><td> URE001 </td></tr><tr><td> xylenecyanol法郎</td><td> Sigma-Aldrich公司</td><td> X4126 </td></tr><tr><td> DNA的浓缩</td><td> Thermo Scientific的</td><td>学者的DNA Speed​​Vac离心120 </td></tr><tr><td> millex过滤器单元</td><td> Millipore公司</td><td> SLGP033RS </td></tr><tr><td>凝胶加载提示</tD&gt; <td> Diamed </td><td> TEC200EX - K </td></tr><tr><td> ImageQuant软件</td><td>分子动力学</td><td>版本5.0 </td></tr><tr><td> kimwipes </td><td>金佰利专业</td><td> 34705 </td></tr><tr><td>迷你Vortexer </td><td>厂商VWR </td><td> 58816-121 </td></tr><tr><td>封口膜</td><td>佩希内塑料包装</td><td> PM996 </td></tr><tr><td>培养皿</td><td> Fisher Scientific则</td><td> fisherbrand 08-757-12 </td></tr><tr><td>加stripettor（移液枪） </td><td>康宁</td><td> 07764714 </td></tr><tr><td>石英比色皿</td><td>瓦里安公司</td><td> 66-100216-00 </td></tr><tr><td>沙克尔/孵化器</td><td>新不伦瑞克省科技</td><td>经典系列C24 </td></tr><tr><td>台风扫描器</td><td> GE医疗集团</td><td> 9200变模式</td></tr><tr><td>离心分离</td><td>贝克曼</td><td> Allegra的X22-R的</td></tr><tr><td>紫外分光光度计</td><td> Thermo Scientific的</td><td> GenesysUV 10 </td></tr><tr><td>荧光分光光度计</td><td>瓦里安公司</td><td>卡里的Eclipse </td></tr></tbody></table>

detection of bacteria using fluorogenic dnazymes

挂钩经由食物传染的医院获得性病原体的帐户和百万人死亡，住院以及巨大的经济损失每一年的爆发。预防此类疾病暴发和持续流行的地方不断增加的需求分析方法，可以准确地找出罪魁祸首病原体在最早阶段的影响最小。虽然是大阵的病原体检测的有效方法，他们没有能满足以下五个体现为一种理想的检测方法总理要求：高特异性（检测只利益细菌），灵敏度高（能够检测低作为一个单一的活细菌细胞），时间短，结果（几分钟到几小时），伟大的操作简便（无需冗长的抽样程序和使用专门的设备），和成本效益。例如，传统的微生物方法具有高度特异性，但需要很长一段时间（DAYS到几周）获得一个明确的结果。1聚合酶链反应和抗体为基础的技术，提供更短的等待时间（数小时至数天），但它们需要使用昂贵的试剂和/或设备精良。2-4因此，仍然存在为对科研开发创新的细菌检测方法，提供一个或多个上述要求的改进特性的需求量很大。我们的实验室是研究作为一类新型生物传感应用，包括细菌检测的分子探针的脱氧核酶潜在兴趣。 脱氧核酶（也称为核酶或DNA酶）是人造的单链DNA分子具有催化化学反应的能力。6-8这些分子可以从一个巨大的随机DNA序列池（其中包含多达10个隔离16个单独序列的过程）称为“ 体外选择”OR“的SELEX”（9-16这些特殊的DNA分子系统进化指数富集的配体）。已广泛研究，近年来随着分子生物传感应用工具。6-8 我们的实验室已经建立了隔离的RNA裂解荧光脱氧核酶（RFDS;图1） 在体外选择程序和调查作为分析工具的使用RFDS 17-29 RFDS催化裂解的DNA-RNA嵌合基板在一个单核苷酸。交界处（R）的两侧，是由荧光（F）和（Q）的猝灭。接近的F和Q呈现的uncleaved的基板的最小荧光。然而，卵裂事件，导致分离的F和Q，这是伴随着荧光强度显着增加。 最近，我们开发的RFDS隔离细菌检测的方法。5这些特殊RFDS的分离“点亮“在原油外混合背后由特定类型的细菌在他们的环境，或在他们所培养的媒体（ 图1）离开（CEM）的存在。原油混合使用，绕过繁琐的净化过程，使用外的目标，确定一个合适的目标利益为生物传感器的发展微生物（这可能需要数月或数年才能完成）。意味着化验过程很简单，因为那里是没有需要的步骤，以获取细胞内的目标。 利用上述方法，我们得出一个RFD的切割底物只由大肠杆菌产生的CEM（FS1的。图2A） 大肠杆菌 （CEM - EC）。5这五大肠杆菌检测RFD的，名为RFD的EC1的（ 图2A），被认为是严格CEM - EC，但没有响应从其他细菌的主机（ 图3）商业电子讯息。 在这里，我们预先选择的窗口新台币设立大肠杆菌的关键实验程序， 大肠杆菌检测实验使用RFD的EC1的和有代表性的结果。

Watch this Scientific Journal Video about Detection of Bacteria Using Fluorogenic DNAzymes at JoVE.com

Detection of Bacteria Using Fluorogenic DNAzymes

我们最近报道一种新的方法，产生荧光脱氧核酶探针，可用于设立一个简单的“混合和阅读的”细菌检测荧光法。这些特殊的DNA探针，催化裂解1生色修饰DNA-RNA的存在原油外的一个特定的细菌，从而转化为细菌检测荧光信号的产生，产生的混合物（CEM）的嵌合基板。在这份报告中，我们将描述模型的细菌检测采用特定的核酶探针记“RFD的EC1的”为重点的实验程序，<em&gt;大肠埃希氏菌（大肠杆菌）</em&gt;。

检测使用荧光脱氧核酶的细菌

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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

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科研

免疫与感染

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

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 ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

在实时以下的肺炎球菌毒力因子在小鼠急性肺炎模型的影响采用发光细菌

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

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 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 ...

Biomimetic Materials to Characterize Bacteria-host Interactions

仿生材料表征细菌宿主相互作用

Bacterial attachment to host cells is one of the earliest events during bacterial colonization of host tissues and thus a key step during infection. The ...

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

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

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 ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

基于分子印迹的电容生物传感器超灵敏检测生物标志物

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

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

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

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 ...

Assaying for Inorganic Polyphosphate in Bacteria

细菌中无机聚磷酸盐的检测

Inorganic polyphosphate (polyP) is a biological polymer found in cells from all domains of life, and is required for virulence and stress response in many ...