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样品中包含34.2纳克质粒DNA，等同的DNA的5×10 9份（34.2×10 -9克/ 4.1×10 6的gx 6.02×10 23）。 </li><li>添加10微升质粒DNA（5×10 9个拷贝/微升）的90微升的水用于稀释10倍，使5×10 8拷贝的DNA样品/微升。 </li><li>重复步骤1.3至制备的5×10 7的DNA样本，5×10 6，5×10 5，5×10 4，5×10 3，5×10 2，5×10 1，和5×10 0个拷贝/微升。 </li></ol> 2.样品为LAMP反应制备DNA模板<ol><li>执行从使用根据制造商的协议市售的DNA mini试剂盒的人血浆样品的DNA提取。总共使用提取200微升样品，并在20微升体积洗脱提取的DNA。 </li></ol> 3.准备2个LAMP反应缓冲液<ol><li>混合200μl的10倍聚合酶缓冲液，320微升的5M的三甲基甘氨酸，12微升的1M 硫酸镁 ，280微升dNTP混合液（各10毫摩尔）和188微升的水至1ml 2×LAMP缓冲液中。通过脉冲涡旋10秒混合。 </li></ol> 4.执行标准LAMP反应<ol><li>混合12.5微升2倍洛杉矶MP缓冲液（如在步骤3制备的40mM的Tris-HCl（pH值8.8），20mM的氯化钾，16毫硫酸镁 ，20mM的（NH 4）2 SO 4，0.2％的Triton X-100，1.6M的三甲基甘氨酸，1.4 10mM的dNTP混合液），1.2微升引物混合物，1μl的的Bst DNA聚合酶（8U /微升），以及5.3微升的水，使在0.2mL的PCR管反应混合物（20微升）。 </li><li>添加（步骤1或2）5微升DNA到20微升反应混合物中拌匀。 </li><li>靠近管和孵育在60℃的水浴或加热块60分钟。这是预先确定的最佳反应温度。 </li><li>加入5微升10×凝胶上样缓冲液以终止反应。 </li><li>负载5微升反应产物与嵌入核酸染色剂染色的2％琼脂糖凝胶。 </li><li>对于在1×TBE缓冲液35分钟运行在100伏的凝胶。 </li><li>可视化紫外光的结果。 </li></ol> 5.执行与复溶LAMP反应试剂<ol><li>混合125微升10X聚合酶缓冲液，200微升的5M的三甲基甘氨酸，7.5微升的1M 硫酸镁的，667.5微升的水至1ml重建缓冲液中。通过脉冲涡旋10秒混合。 </li><li>删除包含从密封铝箔袋的冻干试剂管。不使用如铝箔袋未被密封的管子，或者如果它被损坏。 </li><li>加入20μl重建缓冲液到每个管重新悬浮冻干试剂。 </li><li>吹打缓冲上下5次混匀。看看，以确保冻干试剂完全重悬。白色粉末，应在管消失。 </li><li>加入5微升DNA模板（从步骤1或2）的重组试剂拌匀。 </li><li>靠近管和孵育在60℃的水浴或加热块60分钟。 </li><li>加入5微升10×凝胶上样缓冲液以终止反应。 </li><li>负载5微升反应产品与嵌入核酸染色剂染色的2％琼脂糖凝胶。 </li><li>对于在1×TBE缓冲液35分钟运行在100伏的凝胶。 </li><li>可视化紫外光的结果。 </li></ol> 6.重建缓冲液含HNB或荧光嵌入染料进行LAMP反应<ol><li>混合125微升10X波尔缓冲液，200微升5米的三甲基甘氨酸，7.5微升1M的硫酸镁 ，7.5微升20毫HNB（12.5微升100倍的荧光嵌入染料）的水和660微升（或655微升）至使1毫升重建缓冲液中。通过脉冲涡旋10秒混合。 </li><li>使用在第6准备重复步骤5.2重组缓冲区 - 5.6。 </li><li>可视化的结果用肉眼（含有HNB反应）或UV光（含有荧光嵌入染料反应）。 </li></ol> 7.执行与管扫描仪实时LAMP反应<ol><li>微升的DNA加5至reconstitut含有荧光嵌入染料，靠近管，将其插入到管扫描版试剂。 </li><li>设定培养温度在60℃。测量在520nm处的荧光60分钟。 </li></ol>

<ol>
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作者什么都没有透露。

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。这些冻干试剂的灵敏度保持在25份;类似于至少一个月的新鲜制备的试剂当储存在室温。以确保铝箔袋打开之前正确密封是非常重要的。不使用如铝箔袋未被密封的管子，或者如果它被损坏。这可能会导致补液Ø˚F这导致其反应性的损失冻干试剂。另一关键步骤是与重建缓冲液冻干试剂的再悬浮液。在LAMP反应不会与未充分再悬浮的试剂正常工作。因为反应缓冲液和三甲基甘氨酸溶液的高粘度的高盐浓度的，它们不能被成功地与的Bst DNA聚合酶，LAMP引物和dNTP冻干。特步要准备与这些组件的重建缓冲液。潜在的LAMP反应可以在具有低或没有三甲基甘氨酸低盐浓度进行测试。如果存在具有相同的检测灵敏度，但具有低的盐和低三甲基甘氨酸浓度的反应条件下，冻干的试剂只需要用水进行复原。这将进一步简化反应步骤。 近日，王某等 15相比十二个与商品化等温预混套件-house LAMP检测，发现室内试验有假阳性结果。在我们的实验室，我们已开发了几个灯泡测定用于检测不同的细菌和从来没有发现在这些内部检测假阳性结果。此外，在LAMP法的发展的开始阶段，所述测定法与两个内部的制备主混合物和商品化主混合物进行测试，并在检测"的敏感性和特异性没有观察到任何差别。 我们的研究也提出了几种方法，不仅可以检测LAMP产物无需打开反应管以降低实验室交叉污染的可能性，但也有一个短的处理时间来读取结果进行比较，以凝胶电泳法。使用这些检测方法的优势将大大提高我们迅速地进行分析和诊断与C.的个人能力burnetii </eM&gt;感染在资源有限的地区。这将允许急性Q热的快速和适当的治疗，并减少未来慢性感染的潜力。 不同于基于PCR的方法，其中需要一个热循环，加热块，水浴或培养箱维持60℃左右的恒定温度是所有必需的LAMP检测。这使得该测定特别适用于资源有限的设置。不像原来LAMP检测， 的Bst DNA聚合酶，LAMP引物和dNTP需要被储存在-20℃。这些冻干试剂可在室温下保存，这使得它更加有用，其中Q热流行一个资源有限的设置。在4℃，25℃，37℃的冻干试剂的长期稳定性研究目前正在进行中。今后，我们愿在使用冻干试剂这种LAMP检测可以检测C.证明burnetii与灵敏度相似PCR技术在远程设置。

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的除了反应产生从紫色到蓝色的可视颜色变化与存在于反应DNA模板25,50和100个拷贝。荧光嵌入染料的反应中的夹杂物显示用UV光强荧光信号时的DNA模板25，50，和100个拷贝存在。使用了管扫描器在这项研究中进行实时检测。本机可同时执行8的反应。使用管扫描器监控实时荧光嵌入染料存在的荧光信号图3所示。的荧光信号是高于基线后14，18，21和23分钟，10 6，10 4，10 3，和在反应中的DNA模板的100个拷贝。 <img alt="图1" src="/files/ftp_upload/53839/53839fig1.jpg" /> 琼脂糖电泳 图1. LAMP结果与新鲜配制的试剂（A）和冻干试剂（B）。反应混合物在60℃保温60分钟。 （A）1巷100 bp的阶梯;泳道2 - 3,4 - 5,6 - 7,8 - 9是具有2.5×10 3的反应，2.5×10 2，2.5×10，且无质粒DNA的拷贝。 （B）10巷- 11，12 - 13，14 - 15，16 - 17 REA用2.5×10 3 ctions，2.5×10 2，2.5×10和质粒DNA的拷贝0。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53839/53839fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53839/53839fig2.jpg" /> 图2. 方法来检测LAMP产品。HNB（A）或荧光嵌入染料（B）被用来检测LAMP产品。反应在60℃下60分钟进行。道1到5，反应0，10，质粒25，50，和100份。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53839/53839fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53839/53839fig3.jpg" /> 图3。 </LAMP产品 STRONG&gt; 实时检测。反应在60℃下60分钟进行。反应用10 6（黑），10 4（红色），10 3（绿），100（黄色），10（蓝），1（橙色）和0（棕浅棕色）质粒的拷贝。三是检测独立进行。他们高度重复性。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53839/53839fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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-development-lyophilized-loop-mediated-isothermal-amplification

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

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

10.4K 次观看.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

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

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

从现场收集到的蚊子传染病毒Vero细胞培养法检测

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

科研

免疫与感染

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

The development and implementation of arenavirus reverse genetics represents a significant breakthrough in the arenavirus field 4. The use of cell-based arenavirus minigenome systems together with the ability to generate recombinant infectious arenaviruses with predetermined mutations in their genomes has facilitated the investigation of the contribution of viral determinants to the different steps of the arenavirus life cycle, as well as virus-host interactions and mechanisms of arenavirus pathogenesis 1, 3, 11 . In addition, the development of trisegmented arenaviruses has permitted the use of the arenavirus genome to express additional foreign genes of interest, thus opening the possibility of arenavirus-based vaccine vector applications 5 . Likewise, the development of single-cycle infectious arenaviruses capable of expressing reporter genes provides a new experimental tool to improve the safety of research involving highly pathogenic human arenaviruses 16 . The generation of recombinant arenaviruses using plasmid-based reverse genetics techniques has so far relied on the use of rodent cell lines 7,19 , which poses some barriers for the development of Food and Drug Administration (FDA)-licensed vaccine or vaccine vectors. To overcome this obstacle, we describe here the efficient generation of recombinant arenaviruses in FDA-approved Vero cells.

Rescue of recombinant arenaviruses from cloned cDNAs, an approach referred to as reverse genetics, allows researchers to investigate the role of specific viral gene products, as well as the contribution of their different specific domains and residues, to many different aspects of the biology of arenavirus. Likewise, reverse genetics techniques in FDA-approved cell lines (Vero) for vaccine development provides novel possibilities for the generation of effective and safe vaccines to combat human pathogenic arenaviruses.

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 ...

Assessing the Development of Murine Plasmacytoid Dendritic Cells in Peyer's Patches Using Adoptive Transfer of Hematopoietic Progenitors

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal Peyer&#39;s patch (PP). Common dendritic cell progenitors (CDPs, lin- c-kitlo CD115+ Flt3+) were purified from the bone marrow of C57BL6 mice by FACS and transferred to recipient mice that lack a significant pDC population in PP; in this case, Ifnar-/- mice were used as the transfer recipients. In some mice, overexpression of the dendritic cell growth factor Flt3 ligand (Flt3L) was enforced prior to adoptive transfer of CDPs, using hydrodynamic gene transfer (HGT) of Flt3L-encoding plasmid. Flt3L overexpression expands DC populations originating from transferred (or endogenous) hematopoietic progenitors. At 7-10 days after progenitor transfer, pDCs that arise from the adoptively transferred progenitors were distinguished from recipient cells on the basis of CD45 marker expression, with pDCs from transferred CDPs being CD45.1+ and recipients being CD45.2+. The ability of transferred CDPs to contribute to the pDC population in PP and to respond to Flt3L was evaluated by flow cytometry of PP single cell suspensions from recipient mice. This method may be used to test whether other progenitor populations are capable of generating PP pDCs. In addition, this approach could be used to examine the role of factors that are predicted to affect pDC development in PP, by transferring progenitor subsets with an appropriate knockdown, knockout or overexpression of the putative developmental factor and/or by manipulating circulating cytokines via HGT. This method may also allow analysis of how PP pDCs affect the frequency or function of other immune subsets in PPs. A unique feature of this method is the use of Ifnar-/- mice, which show severely depleted PP pDCs relative to wild type animals, thus allowing reconstitution of PP pDCs in the absence of confounding effects from lethal irradiation.

This protocol describes experimental procedures to assess the differentiation of plasmacytoid dendritic cells in Peyer&#8217;s patch from common dendritic cell progenitors, using techniques involving FACS-mediated cell isolation, hydrodynamic gene transfer, and flow analysis of immune subsets in Peyer&#8217;s patch.

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

This protocol details a method to analyze the ability of purified hematopoietic progenitors to generate plasmacytoid dendritic cells (pDC) in intestinal ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in mice. Recently, myeloid differentiation factor 88 (MyD88) signaling was shown to be important for initial T cell priming and memory T cell development during WNV NS4B-P38G mutant infection. In this study, two flow cytometry-based methods &#8211; an in vitro T cell priming assay and an intracellular cytokine staining (ICS) &#8211; were utilized to assess dendritic cells (DCs) and T cell functions. In the T cell priming assay, cell proliferation was analyzed by flow cytometry following co-culture of DCs from both groups of mice with carboxyfluorescein succinimidyl ester (CFSE) - labeled CD4+ T cells of OTII transgenic mice. This approach provided an accurate determination of the percentage of proliferating CD4+ T cells with significantly improved overall sensitivity than the traditional assays with radioactive reagents. A microcentrifuge tube system was used in both cell culture and cytokine staining procedures of the ICS protocol. Compared to the traditional tissue culture plate-based system, this modified procedure was easier to perform at biosafety level (BL) 3 facilities. Moreover, WNV- infected cells were treated with paraformaldehyde in both assays, which enabled further analysis outside BL3 facilities. Overall, these in vitro immunological assays can be used to efficiently assess cell-mediated immune responses during WNV infection.

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

Two flow cytometry-based methods &#8211; an in vitro T cell priming assay and intracellular cytokine staining were utilized to measure antigen presenting capacity of dendritic cells and antigen-specific T cell responses to a West Nile virus mutant infection in mice.

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor Fc&#949;RI

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and Fc&#949;RI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine Fc&#949;RI&#945; was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine &#945;-chain formed a functional receptor complex with the endogenous rat &#946;- and &#947;-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine Fc&#949;RI&#945; transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using &#946;-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% &#177; 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3.

The current study describes the development and applications of a genetically engineered assay system based on the transfection of rat basophilic leukemia cells with the equine Fc&#949;RI&#945; gene. Transfected cells express a functional receptor where the release of mediators of the allergic response can be activated by IgE and antigen.

的发展<em&gt;在体外</em&gt;模型研究的交互系统<em&gt;雅科仕</em&gt;<em&gt; caballus</em&gt;以其高亲和力受体了FcεRI的IgE

The interaction of IgE with its high-affinity Fc receptor (Fc&#949;RI) followed by an antigenic challenge is the principal pathway in IgE mediated ...

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

环介导等温扩增（LAMP）法进行的物种特异性检测<em&gt;艾美耳球虫</em&gt;可感染鸡

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

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

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

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

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

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

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

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 replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

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

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

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

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

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

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

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 ...

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冻干环介导等温扩增试剂的检测中的发展

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此视频中的章节

从现场收集到的蚊子传染病毒Vero细胞培养法检测

在FDA批准的Vero细胞疫苗开发产生重组沙粒

评估小鼠浆细胞树突状细胞在淋巴集结的开发利用造血祖细胞的过继转移

在 MyD88的介导的细胞免疫应答西尼罗河病毒变异株感染体外分析

的发展

环介导等温扩增（LAMP）法进行的物种特异性检测

生成和多表型高内涵筛选

定性和定量测定为检测和蛋白质抗菌药物的表征

应用荧光共振能量转移（FRET）审查效应转运效率由

对于检测到的敏感性和特异性显色琼脂培养基发展