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>
	<li>Anderson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Q+fever+United+States,+2013:+recommendations+from+CDC+and+the+Q+Fever+Working+Group.">Diagnosis and management of Q fever United States, 2013: recommendations from CDC and the Q Fever Working Group.</a> MMWR Recomm. Rep. 62 (3), 1-30 (2013).</li><li>Rolain, J. M., Boulos, A., Mallet, M. N., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+ratio+of+serum+doxycycline+concentration+to+MIC+and+rapid+decline+of+antibody+levels+during+treatment+of+Q+fever+endocarditis.">Correlation between ratio of serum doxycycline concentration to MIC and rapid decline of antibody levels during treatment of Q fever endocarditis.</a> Antimicrob. Agents and Chemother. 49 (7), 2673-2676 (2005).</li><li>Fournier, P. E., Raoult, D. <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+PCR+and+serology+assays+for+early+diagnosis+of+acute+Q+fever.">Comparison of PCR and serology assays for early diagnosis of acute Q fever.</a> J. Clin. Microbiol. 41 (11), 5094-5098 (2003).</li><li>Schneeberger, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+with+serum+samples+is+indispensable+for+early+diagnosis+of+acute+Q+fever.">Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever.</a> Clin. Vaccine Immunol. 17 (2), 286-290 (2010).</li><li>Turra, M., Chang, G., Whybrow, D., Higgins, G., Qiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+acute+Q+fever+by+PCR+on+sera+during+a+recent+outbreak+in+rural+South+Australia.">Diagnosis of acute Q fever by PCR on sera during a recent outbreak in rural South Australia.</a> Ann. N Y Acad. Sci. 1078, 566-569 (2006).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Paris, D. H., Blacksell, S. D., Newton, P. N., Day, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+rapid+and+sensitive+detection+of+Orientia+tsutsugamushi.+by+loo-isothermal+DNA+amplification.">Simple, rapid and sensitive detection of Orientia tsutsugamushi. by loo-isothermal DNA amplification.</a> Trans. R. Soc. Trop. Med. Hyg. 102 (12), 1239-1246 (2008).</li><li>Huber, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+assay+targeting+the+47-kDa+gene+of+Orientia+tsutsugamushi.:+A+rapid+and+sensitive+alternative+to+real-time+PCR.">Loop-mediated isothermal amplification assay targeting the 47-kDa gene of Orientia tsutsugamushi.: A rapid and sensitive alternative to real-time PCR.</a> J. Med. Microb. Diagn. 1 (112), (2012).</li><li>Pan, L., Zhang, L., Wang, G., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+sensitive+detection+of+the+ompB+gene+of+spotted+fever+group+rickettsiae+by+loop-mediated+isothermal+amplification.">Rapid, sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.</a> BMC Infect. Dis. 12 (254), (2012).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem. Biophys. Res. Commun. 289 (1), 150-154 (2001).</li><li>Qiao, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+rapid+detection+of+Bacillus+anthracis.spores.">Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis.spores.</a> Biotechnol. Lett. 29 (12), 1939-1946 (2007).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat. Protoc. 3 (5), 877-882 (2008).</li><li>Thekisoe, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Loop-Mediated+Isothermal+Amplification+(LAMP)+reagents+and+its+amplification+efficiency+on+crude+trypanosome+DNA+templates.">Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates.</a> J. Vet. Med. Sci. 71 (4), 471-475 (2009).</li><li>Chen, H. W., Ching, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+loop-mediated+isothermal+amplification+assays+for+rapid+and+easy+detection+of+Coxiella+Burnetii.">Development of loop-mediated isothermal amplification assays for rapid and easy detection of Coxiella Burnetii.</a> J. Microbiol Methods. 107, 176-181 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+In-House+Real-Time+LAMP+Assays+with+a+Commercial+Assay+for+the+Detection+of+Pathogenic+Bacteria.">A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.</a> Molecules. 20 (6), 9487-9495 (2015).</li></ol>

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...

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><tbody><tr><td>LAMP primers</td><td>Eurofins MWG operon</td><td>10 nmol, salt free</td><td>Sequence designed by customer</td></tr><tr><td>Bst DNA polymerase</td><td>New England Biolabs</td><td>M0275L</td><td>8 units/ml</td></tr><tr><td>10x Thermo pol buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>dNTP mixture</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each</td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B0300</td><td>5 M, Trimethylglycine</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M3409-1ML</td><td>1 M</td></tr><tr><td>10x Bluejuice</td><td>Invitrogen</td><td>10816-015</td><td>gel loading buffer</td></tr><tr><td>SYBR green</td><td>Invitrogen</td><td>S7585</td><td>10,000x, visualize products in tubes</td></tr><tr><td>GelRed</td><td>Phenix Research Products</td><td>RGB-4103</td><td>10,000x, visualize products in gels</td></tr><tr><td>Lyophilized reagents</td><td>Gene Reach</td><td></td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Fluka</td><td>33936-10G</td><td></td></tr><tr><td>ESEQuant tube scanner</td><td>Qiagen</td><td></td><td>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-development-lyophilized-loop-mediated-isothermal-amplification

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

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

循环介导等温扩增 (灯管) 法快速识别烟粉虱

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

科研

环境科学

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

抗体分析的荧光素酶免疫沉淀系统（LIPS）

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

生物学

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 ...

免疫与感染

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 ...

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 ...

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 ...

生物工程

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

优化的基于PCR的检测支原体

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

使用等温扩增进行人类呼吸道细菌感染诊断

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

作者聚焦:使用微流控芯片推进呼吸道病原体的快速检测 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

冻干环介导等温扩增试剂的检测中的发展

摘要

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此视频中的章节

循环介导等温扩增 (灯管) 法快速识别烟粉虱

使用循环介质等温扩增检测灌溉水中的植物浮肿

抗体分析的荧光素酶免疫沉淀系统（LIPS）

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

应用荧光共振能量转移（FRET）审查效应转运效率由

毛细管阵列中多核酸的视觉检测

优化的基于PCR的检测支原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

冻干环介导等温扩增试剂的检测中的发展

摘要

探索更多视频

此视频中的章节

循环介导等温扩增 (灯管) 法快速识别烟粉虱

使用 循环介质等温扩 增检测灌溉水中的植物浮肿

抗体分析的荧光素酶免疫沉淀系统（LIPS）

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

应用荧光共振能量转移（FRET）审查效应转运效率由

毛细管阵列中多核酸的视觉检测

优化的基于PCR的检测支原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

通过基于微流控芯片的环介导等温扩增快速检测引起下呼吸道感染的细菌病原体

使用循环介质等温扩增检测灌溉水中的植物浮肿