Name: the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii
Uploaded: 2016-04-18T14:00:00
Description: Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

这项研究是由海军医学研究中心，科研单位6000.RAD1.J.A0310支持。在这篇文章中表达的观点是作者的，并不一定反映海军，国防部或美国政府部门的官方政策或立场。伟美程是美国政府雇员。这项工作准备为她的公务上。标题17 USC§105规定，“这个标题下版权保护不适用于美国政府的任何工作。”标题17 USC§101定义为军事服务成员或美国政府雇员制是那个人的官方职责的一部分工作美国政府的工作。

1.准备质粒DNA稀释为标准LAMP反应<ol><li>使用分光光度计在260nm处测量光密度（OD）来确定DNA浓度的质粒（ 贝氏柯克斯 RSA 493的含有靶基因IS1111a）。 1的OD 260相当于50微克/μL的DNA。 </li><li>转换的质粒DNA的量成拷贝数。自质粒的大小是6,330碱基对，该质粒的分子量为4.1×10 6克（6,330 bp的点¯x649克/碱基对）。质粒的DNA浓度为34.2纳克/微升（如来自步骤1.1确定）。 1微升该DNA?...

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以前，我们开发了一个敏感和特异LAMP检测针对插入元素IS1111a 14。被选择的IS1111元件，因为它是高度C的不同菌株中保守burnetii和细菌高份数（7〜110）（2006年克利）。我们的结果表明，灯可以检测约25 IS1111元件，其可以关联到贝氏的DNA少则一染色体拷贝的拷贝。在这项研究中， 的Bst DNA聚合酶，LAMP引物和dNTP冻干并包装在压缩铝箔袋。这些冻干...

小革兰氏阴性细菌贝氏柯克斯体是Q热，这是一个世界性的动物传染病的病原体。由于Q热的全球分销和C.高传染性burnetii，海外部署的美国军事人员和文职人员都在被感染的流行地区的风险。 Q热表现为两种形式：急性和慢性感染。急性C-发热呈现本身具有类似流感的症状，肝炎，或肺炎，通常是一种自限性疾病与低死亡率。慢性C-发热，而不太普遍，通常导致心内膜炎，如果不治疗的1,2-其具有高得多的死亡率。因此，早期诊断，指导适当的治疗为病人护理的关键。聚合酶链反应（PCR）和定量实时PCR（qPCR的）测定法已经开发了用于检测C.在细胞培养和临床标本3-5 &lt;burnetii DNA/ SUP&gt;。 PCR和定量PCR是昂贵的，往往不是在日常工作资源受限的地区一应俱全。 最初由Notomi 6中所述，环介导等温扩增（LAMP）提供了一种替代DNA扩增方法。灯使用为链置换DNA合成的Bst DNA聚合酶与识别靶基因的至少六个独立区域专门设计的引物组沿。灯的最显著优点是，在等温条件下发生扩增。因此，只需要一个水浴，加热块或培养箱。此方法已被用于检测多种立克次氏体的病原体7-9。通过凝胶电泳的扩增DNA产物的可视化是可区分假阳性真阳性由于非特异性扩增的最精确的方法。然而涉及凝胶电泳的程序不用于资源有限的芳实际EAS。被开发几种可供选择的方法来检测反应产物。这些替代方案中，如从焦磷酸镁的形成10或使用荧光嵌入染料衍生的浊度在紫外灯下11,12被可视化比运行凝胶更为有利。当储存在25℃和37℃，这是在热带和亚热带国家的环境温度，其中Q热流行13上的LAMP试剂为一个月稳定。 一盏灯试验在我们的实验室开发的检测C.存在burnetii血浆样品14英寸在这里，我们描述了从冻干试剂的制备上的LAMP反应混合物的一个简单的协议。当储存在室温冻干试剂是一个月稳定。当与方便的可视化相结合的方法，这就是使用检测C.一种理想的方法burnetii在资源有限的设置。

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">LAMP primers</td>
			<td style="width:188px;">Eurofins MWG operon</td>
			<td style="width:131px;">10 nmol, salt free</td>
			<td style="width:240px;">Sequence designed by customer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Bst DNA polymerase</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">M0275L</td>
			<td style="width:240px;">8 units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Thermo pol buffer</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">B9004S</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">dNTP mixture</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">N0447L</td>
			<td style="width:240px;">10 mM each</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Betaine</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">B0300</td>
			<td style="width:240px;">5 M, Trimethylglycine</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Magnesium Sulfate</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">M3409-1ML</td>
			<td style="width:240px;">1 M</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Bluejuice</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">10816-015</td>
			<td style="width:240px;">gel loading buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">SYBR green</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">S7585</td>
			<td style="width:240px;">10,000X, visualize products in tubes</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">GelRed</td>
			<td style="width:188px;">Phenix Research Products</td>
			<td style="width:131px;">RGB-4103</td>
			<td style="width:240px;">10,000X, visualize products in gels</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Lyophilized reagents</td>
			<td style="width:188px;">Gene Reach</td>
			<td style="width:131px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Hydroxynaphthol blue</td>
			<td style="width:188px;">Fluka</td>
			<td style="width:131px;">33936-10G</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">ESEQuant tube scanner</td>
			<td style="width:188px;">Qiagen</td>
			<td style="width:131px;"></td>
			<td style="width:240px;">real-time detection</td>
		</tr>
	</tbody>
</table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

贝氏柯克斯体 ，引起Q热剂，是一种专性细胞内细菌。基于PCR的诊断测定法已经开发了用于检测C.在细胞培养物和临床样品burnetii的DNA。 PCR需要专门的设备和大量的最终用户培训，因此，它不适合用于特别是在资源有限的地区的日常工作​​。我们已经开发出一种环介导等温扩增（LAMP）法检测C的存在burnetii患者样品英寸该方法是在在水浴或加热块约60℃的单一温度下进行。此LAMP检测的灵敏度非常相似的PCR用的每个反应约2​​5拷贝的检测限。本报告描述了使用冷冻干燥的试剂和使用hydroxynaphthol蓝色（HNB）或在反应的荧光嵌入染料UV灯结果的可视化的反应的制备方法。该LAMP试剂均为lyophili捷思并储存于室温（RT）一个月没有检测灵敏度的损失。因为反应混合配制不涉及复杂的步骤，这LAMP检测尤为强劲。这种方法非常适合在资源有限的环境中使用，其中Q热流行。

0.2毫升管内的冷冻干燥的LAMP试剂包含的Bst DNA聚合酶，引物，和dNTP。 20微升重组缓冲液用于重新暂停冻干试剂。 图1示出了与新鲜制备的试剂和冻干试剂琼脂糖凝胶上的LAMP反应的结果。冻干的试剂不降低其活性。由两种试剂制备可检测DNA模板。 图2的25份的LAMP反应示出了反应产物的HNB或存在于反应荧光嵌入染料的检测。 HNB的除了反应产生?...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

The overall goal of this experiment is to develop a simple LAMP method using lyophilized reagents for the detection of C.burnetii in patient samples. The main advantage of this protocol is that no coaching is required for the storage of the reagents. This method is ideal for use in resource-limited settings where Q fever is endemic.Demonstrating the procedure will be Tatiana, a technician from our laboratory. Beginning with DNA for the target gene IS1111a from C.burnetii RSA493, determine the plasmid concentration with a spectrophotometer at 260 nanometers. Convert the plasmid DNA concentration into copy number using the following calculations, where 6330 equals the length and base pairs of the plasmid, and 34.2 nanograms per microliter is the concentration of the plasmid, as just determined using the spectrophotometer.Then prepare 8-to 10-fold serial dilutions of the plasmid to arrive at the following number of copies per microliter. To prepare the DNA template for the LAMP reaction, begin with 200 microliters of human plasma samples, and with the DNA miniprep kit, extract the DNA according to the manufacturer's protocol. Dilute the final sample into a 20-microliter volume.Prepare 1 mililiter of 2x LAMP reaction buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds. To perform the standard LAMP reaction, in a 0.2 milliliter PCR tube, mix 12.5 microliters of 2x LAMP buffer, 1.2 microliters of primer mix, 1 microliter of Bst DNA polymerase, and 5.3 microliters of water.Next, add 5 microliters of DNA to the 20 microliter reaction and mix well, then incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. After the incubation, terminate the reaction by adding 5 microliters of 10x gel loading buffer to the tube. Then load 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain.Run the gel in 1x TBE at 100 volts for 35 minutes, then use UV light to visualize the results. To perform the LAMP reaction with reconstituted reagents, prepare 1 milliliter of reconstitution buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds.Next, remove the tubes that contained the lyophilized reagents from the sealed aluminium foil bag. For Step 4.2, it is very important to make sure the aluminium foil bag is properly sealed before opening it. Do not use the tubes if the aluminum bag is not sealed or is damaged.Add 20 microliters of reconstitution buffer to each tube of reagents and mix by pipetting up and down five times. Ensure that the lyophilized reagents are completely re-suspended. Then, add 5 microliters of DNA template to the reconstituted reagents and mix well.Incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. To terminate the reaction after the incubation, add 5 microliters of 10x gel loading buffer, then add 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain. Run the gel in 1x TBE buffer at 100 volts for 35 minutes, then visualize the results by UV light.To perform the LAMP reaction with hydroxynaphthol blue, or HNB, or intercalating dye, prepare 1 milliliter of reconstitution buffer by combining the following reagents that include HNB or a fluorescent intercalating dye. Mix by pulse-vortexing for 10 seconds. Carry out the LAMP reaction as just described and use UV light or the naked eye to visualize the results.To perform the LAMP reaction with a tube scanner, add 5 microliters of DNA to the reconstituted reagents containing the fluorescent intercalating dye, then insert the closed tube into the tube scanner. Set the incubation temperature to 60 degrees Celsius and measure the fluorescence at 520 nanometers for 60 minutes. This figure shows the LAMP reaction results on agarose gels with freshly-prepared reagents and lyophilized reagents.Observe the lyophilized reagent maintains its activity. LAMP reactions prepared by both reagents detected 25 copies of DNA template. In this figure, the detection of the reaction's products with HNB or fluorescent intercalating dye in the reaction is shown.The addition of HNB to the reaction produces a visual color change, from purple to blue, with 25, 50, and 100 copies of DNA template present in the reactions. In addition, the inclusion of fluorescent intercalating dye in the reaction shows a strong fluorescent signal with UV light when similar DNA copy numbers are present. Shown here are the results from using the tube scanner to monitor the fluorescent signals in real-time with fluorescent intercalating dye.The fluorescent signals are above baseline after 14, 18, 21, and 23 minutes, with 10^6, 10^4, 10^3, and 100 copies of DNA template in the reactions. Once mastered, this procedure can be done in 90 minutes. After its development, this assay paved the way for quick diagnosis of Coxiella burnetii infection in resource-limited areas.After watching this video, you should have a good understanding of how to reconstitute lyophilized LAMP reagents. Don't forget that patient samples can be infectious and safety precautions should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Generation of Recombinant Arenavirus for Vaccine Development in FDA-Approved Vero Cells

在FDA批准的Vero细胞疫苗开发产生重组沙粒

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field  4. The use of cell-based ...

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Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

定性和定量测定为检测和蛋白质抗菌药物的表征

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

应用荧光共振能量转移（FRET）审查效应转运效率由<em&gt;贝氏柯克斯体</em&gt; siRNA沉默中

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

乙型肝炎病毒记者系统的开发监控复制周期的早期阶段

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...