这项工作一直由美国农业部资助1999-35212-8680，2005-01378，并2010-04288-01，美国农业部方程式基金和格鲁吉亚的兽医农业研究资助国家的支持。本出版物中描述的协议，开发研究课程的一部分，基因4960H MIBO 4900L，BCMB 4960L，4960H BIOL在佐治亚大学的本科生使用几个分子流行病学研究项目的学生在毛雷尔实验室。我们要感谢支持我们的努力，将与研究本科教学，已经执行了毛雷尔和李实验室研究，许多本科生和我们的系主任，约翰Glisson。

<ol>
	<li>Dauga, C., Zabrovskaia, A., Grimont, P. A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+fragment+length+polymorphism+analysis+of+some+flagellin+genes+of+Salmonella+enterica.">Restriction fragment length polymorphism analysis of some flagellin genes of Salmonella enterica.</a> J. Clin. Microbiol. 36, 2835-2843 .</li><li>Hong, Y., Liu, T., Lee, M. D., Hofacre, C. L., Maier, M., White, D. G., Ayers, S., Wang, L., Berghaus, R., Maurer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+screen+of+broth+enrichments+for+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg,+and+Typhimurium+using+an+allelotyping+multiplex+PCR+that+targets+O+and+H+antigen+alleles.">A rapid screen of broth enrichments for Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium using an allelotyping multiplex PCR that targets O and H antigen alleles.</a> J. Food Prot. 72, 2198-2201 (2009).</li><li>Hong, Y., Liu, T., Lee, H. o. f. a. c. r. e., Maier, C. L., White, M., Ayers, D. G., Wang, S., Berghaus, L., R, M. a. u. r. e. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=J.Rapid+screening+of+Salmonella+enterica+serovars+Enteritidis,+Hadar,+Heidelberg+and+Typhimurium+using+a+serologically-correlative+allelotyping+PCR+targeting+the+O+and+H+antigen+alleles.">J.Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles.</a> BMC Microbiol. , (2008).</li><li>Liu, T., Liljebjelke, K., Bartlett, E., Hofacre, C. L., Sanchez, S., Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+nested+PCR+to+detection+of+Salmonella+in+poultry+environments.">Application of nested PCR to detection of Salmonella in poultry environments.</a> J. Food Prot. 65, 1227-1232 (2002).</li><li>Maurer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+detection+and+limitation+of+molecular+techniques.">Rapid detection and limitation of molecular techniques.</a> Ann. Rev. Food Sci. Tech. 2, 259-279 (2011).</li><li>Moscoso, H., Alvarado, I., Hofacre, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+analysis+of+infectious+bursal+disease+virus+from+bursal+tissues+collected+on+FTA+filter+paper.">Molecular analysis of infectious bursal disease virus from bursal tissues collected on FTA filter paper.</a> Avian Dis. 50, 391-396 (2006).</li><li>Sambrook, J., Fritsch, E. F., Maniatis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: a laboratory. , (1989).</li><li>Wittwer, C. T., Fillmore, G. C., Hillyard, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+polymerase+chain+reaction+capillary+tubes+with+hot+air.">Automated polymerase chain reaction capillary tubes with hot air.</a> Nucleic Acids Res. 17, 4353-4357 (1989).</li></ol>

没有利益冲突的声明。

大多数市售的PCR试 ​​验检测沙门氏菌的目标属独特的基因（ 如入侵） 。然而，并非所有的沙门氏菌是导致人类或动物种群的流行疾病的能力是平等的。早期识别沙门氏菌脂多糖O抗原和flagellinH1和H2抗原的抗原差异导致分化成基于这些抗原的差异超过2500 血清型沙门氏菌。有46个不同的O抗原和86不同的鞭毛（H） 抗原属沙门氏菌中发现沙门氏菌可能拥有一（H1）或两个不同的（H1和H2 ）flagellins。今天的O，H1和H2抗原为2500 沙门氏菌血清型的不同组合确认。一个血清型，是基于个人的O和H抗原识别所得的抗原公式分配。抗原公式写为O：H1：H2和“ - ”，是指没有O -抗原阴性沙门氏菌鞭毛蛋白H1或H2和“NT”（非typeable）。例如，血清型鼠伤寒沙门氏菌被分配到一个S。肠道分离的抗原性配方B：我：1,2的肠炎是一个孤立的公式D1：G，M： - 。这种沙门氏菌人口的抗原变异是在核苷酸水平上，因此可以很容易地利用PCR扩增的差异的外在表现。 我们已经确定与特定的O和H等位基因的遗传差异，发展allelotyping PCR 检测 ，确定S. 肠炎血清型：肠炎，哈达尔，海德堡和鼠伤寒沙门氏菌（3）。正确分配和沙门氏菌的血清型的报告要求，测试与O相关联的所有等位基因：H1：为肠炎H2抗原公式（首长级薪级第1点：G，M： - ），哈达尔（C2：Z10：E，N，X），海德堡（二R：1,2），鼠伤寒沙门氏菌（B：我：1,2）。没有知识O等位基因或抗原，发现的H1克，M等位基因单独并不一定确定隔离其他的沙门氏菌血清型肠炎沙门氏菌：戈纳，加利福尼亚州和蒙得维的亚，也具有这种相同的等位基因。虽然allelotyping PCR技术最初是设计，检测中脱颖而出提及沙门氏菌血清型，这种测试可以找出另外19个血清型（见表3） 。最初的设计我们的allelotyping PCR检测O和H等位基因。在实践中，它已经变得更加实际和具有成本效益，为我们确定的O -抗原玻片凝集试验，使用O型特异性抗血清，而不是执行的O allelotyping PCR分离出沙门氏菌文化工作时。不过，我们建议使用O allelotyping PCR检测，如果你的实验室使用屏幕（2） 或输入沙门氏菌株FTA卡（6）提交本项PCR，并包括与样品的第一个屏幕中的沙门氏菌特异性PCR（4 ）。限制allelotyping PCR的只有那些被确定为沙门氏菌 PCR或生化/抗原测试样本。 在这篇文章中描述的协议已经使用爱达荷州技术Rapidcycler，热空气的热循环，允许一个规模低于许多加热块热循环时间的反应体积和运行反应条件进行了优化。为了适应这个协议与使用加热块热循环，可能需要优化反应条件，退火温度的降低/提高经验，调整引物浓度;或调整MgCl 2的浓度。例如，我们不得不适应的MJ研究的PTC - 200热循环如下协议。工作表1中描述的卷相同的反应，股市的工作concentrationof我引物稀释至2.5微米，退火温度降至55 ° C至45 ° C和变性的孵育时间，退火和延伸步骤I / G，M allelotyping PCR技术被延长至20秒。拥有适当的正面和负面的控制是必不可少的任何PCR最初的启动和优化，包括发布的协议。我们建议获取已知的沙门氏菌血清型，专门Anatum（E1：E，H：1,6），肠炎（首长级薪级第1点：G，M： - ），哈达尔（C2：Z10：E，N，X），海德堡（B： R ：1,2），蒙得维的亚（C1：G，M，S：1,2,7）和鼠伤寒沙门氏菌（B：我：1,2）;和实验室大肠杆菌 K12菌株（DH5α中，LE393，JM109等。 ）分别为本文中所描述的allelotyping PCR试验阳性和阴性对照。这些沙门氏菌血清型，将作为积极或消极的控制，取决于测试的等位基因。 ，同时执行这和任何其他诊断PCR需要遵循一定的预防措施和指引。首先，物理分离模板制备，PCR设置，凝胶电泳的关键是防止可能的PCR结转污染和误报的错误报告。此外，加入适当的控制是有必要在任何常规PCR，确定这些控制出现的问题，因为他们和协助解释结果（5）。最重要的，一致性是至关重要的，尤其是关于扩增（PCR产物）扩增大小的检测andestimation电泳条件。为了说明这一点，我们特意暂停电泳早在图1A打算。分子量标准（泳道1和13）和阳性对照（2巷）没有充分分开，准确估计大小或观察那里有小的差异，在尺寸（〜50个基点）的DNA片段的分离。充分分离的DNA片段，尤其是分子量标准，是必不可少的报告阳性是依赖于扩增的大小和区分“真阳性”，有时是在日常运行的任何PCR（5观察非特异性扩增产物的准确估计）。例如，在图1B，7巷的大小（约700 bp）的非特异性扩增。电泳被终止太早，当染料前端的中途点，在琼脂糖凝胶，将有500 bp和700 bp的DNA片段之间的小分化。因此，在7巷样本已被错误地报告为阳性，我和G，M等位基因。 最后，需要认识到测试与任何相关的限制。几个H1/H2 allelotyping PCR没有歧视性的权力，以辨别微妙的遗传等位基因的差异，属于H2的1,2，1,5，1,6，1,7抗原复合物; E，N，X / E ，N，Z15抗原复合物和H1摹抗原复杂，其中包括G，M等位基因。一个或两个氨基酸的变化，占目前在这些鞭毛抗原不同表位。因此，很难为我们设计引物，就可以利用这些有时在鞭毛蛋白基因序列（3）单碱基对差异。例如，都柏林沙门氏菌血清型（首长级薪级第1点：G，P： - ）和伯塔（D1：F，G，T： - ）也与我们克PCR阳性，mallelotyping引物。 H2的allelotyping引物不能区分的鼠伤寒沙门氏菌（B：1,2：我）从拉各斯（B：我：1,5），海德堡（B：R：1,2）从布拉德福德（乙：R：1,5）， Winneba（B：R：1,6），或圣雷莫（乙：R：1,7），或哈达尔（C2：E，N，X：Z10）从Glostrup（C2：Z10：Z15 E，N，）。虽然遇到一些这些血清型的可能性：拉各斯，布拉德福德，Winneba，圣雷莫或Glustrup是远程的，它是一种可能性，并要求提供的测试限制或其他确证试验免责声明。 总之，这种基于PCR的血清分型迅速识别S.哈达尔，海德堡和鼠伤寒沙门氏菌， 肠炎血清型肠炎，和O，H1，H2等位基因与增设19个血清型。这种检测是一种快速，成本效益的替代传统的微生物方法血清型沙门氏菌。

<table border="1"><tbody><tr><th>材料名称</th><th>类型</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td>卢里亚Bertani </td><td></td><td>费舍尔</td><td></td><td> （或任何类似的源） </td></tr><tr><td>离心管</td><td> 1.5 ml容量</td><td>费舍尔</td><td></td><td> （或任何类似的源） </td></tr><tr><td>乙醇</td><td> 200证明</td><td>费舍尔</td><td></td><td> （或任何类似的源） </td></tr><tr><td> DH 2 O </td><td>分子生物学级</td><td>西格玛</td><td></td><td> （或任何类似的来源; PCR技术只） </td></tr><tr><td> 10 × PCR缓冲液： </td><td> 20毫米氯化镁2 </td><td>爱达荷州技术</td><td></td><td> （缓冲与牛血清白蛋白，聚蔗糖和柠檬黄） </td></tr><tr><td></td><td> 30毫米氯化镁2 </td><td></td><td></td><td></td></tr><tr><td></td><td> 40毫米氯化镁2 </td><td></td><td></td><td></td></tr><tr><td> PCR引物</td><td></td><td></td><td></td><td></td></tr><tr><td>二甲基亚砜</td><td></td><td></td><td></td><td></td></tr><tr><td>的dNTP </td><td></td><td>罗氏公司</td><td></td><td> （或任何类似的源） </td></tr><tr><td> Taq DNA聚合酶</td><td></td><td>丹维尔</td><td></td><td> （或任何类似的源） </td></tr><tr><td>毛细管移液器</td><td> 10μL体积</td><td>爱达荷州技术</td><td></td><td></td></tr><tr><td>快速扩增仪</td><td></td><td>爱达荷州技术</td><td></td><td></td></tr><tr><td>琼脂糖</td><td>低的平等就业机会;分子生物学级</td><td> BioRad公司</td><td></td><td> （或任何类似的源） </td></tr><tr><td> 50 × TAE缓冲液中或5倍TBE缓冲液</td><td></td><td>费舍尔</td><td></td><td> （或任何类似的源） </td></tr><tr><td>溴化乙锭</td><td></td><td> BioRad公司</td><td></td><td> （或任何类似的源） </td></tr><tr><td>水平，潜水凝胶电泳凝胶模具商会</td><td></td><td> BioRad公司</td><td></td><td> （或任何类似的源） </td></tr><tr><td>电源</td><td></td><td> BioRad公司</td><td></td><td> （或任何类似的源） </td></tr><tr><td>分子成像凝胶文档系统</td><td></td><td> BioRad公司</td><td></td><td> （或任何类似的源） </td></tr></tbody></table> 表4。材料

an allelotyping pcr for identifying salmonella enterica serovars enteritidis, hadar, heidelberg, and typhimurium

当前商业PCR的测试， 确定沙门氏菌靶基因，该属植物所特有的。但是，有两个品种，6个亚种，超过2500 种沙门氏菌血清型，而不是所有的都在他们的意义，等于对公众健康。例如，发现S。肠道亚种 IIIA亚利桑那州的一个表上鸡蛋层农场相比是微不足道的孤立的S.肠道亚种我沙门肠炎，沙门氏菌病的挂表鸡蛋消费的首要原因。血清型抗原的差异，在脂多糖（LPS）（O抗原）和鞭毛（H1和H2抗原）的基础上确定。这些抗原的差异与此表型的基因及相关基因的等位基因多样性的外观。 我们已经开发出一种allelotyping，多重PCR键上的四个主要南之间的遗传差异在家禽肠道亚种我血清型的发现和与重大人类疾病在美国相关。 PCR引物对目标的关键基因，或到一个特定的沙门氏菌血清型的独特序列，旨在产生一个大小为特定的基因或等位基因的扩增 。，沙门氏菌血清型分配到隔离PCR检测结果，结合具体脂多糖鞭毛蛋白基因的等位基因。本文中描述的多重PCR的具体检测的S.肠道亚种我血清型肠炎，哈达尔，海德堡，和鼠伤寒沙门氏菌。 在这里，我们将演示如何使用多重PCR反应，以确定为沙门氏菌的血清型隔离。

Watch this Scientific Journal Video about An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium at JoVE.com

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

我们描述了一个多重PCR快速检测沙门氏菌血清型肠炎，哈达尔，海德堡和鼠伤寒沙门氏菌。具体的沙门氏菌血清型，可确定的目标是多重PCR技术，以独特的O -抗原生物合成集群和一个给定的血清型的鞭毛的基因和序列。血清型，然后分配到一个隔离基础上的具体化，规模扩增产物（PCR产物）对应的目标等位基因外观的沙门氏菌。

一个用于标识Allelotyping PCR扩增沙门氏菌肠炎血清型，哈达尔，海德堡和鼠伤寒沙门氏菌

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus.  However, there are two species, six subspecies, and over ...

an-allelotyping-pcr-for-identifying-salmonella-enterica-serovars

Research

JoVE Journal

Immunology and Infection

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

21.6K 次观看.  University of Georgia. 我们描述了一个多重PCR快速检测沙门氏菌血清型肠炎，哈达尔，海德堡和鼠伤寒沙门氏菌。具体的沙门氏菌血清型，可确定的目标是多重PCR技术，以独特的O -抗原生物合成集群和一个给定的血清型的鞭毛的基因和序列。血清型，然后分配到一个隔离基础上的具体化，规模扩增产物（PCR产物）对应的目标等位基因外观的沙门氏菌。

视频: An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative tumor of vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the form of pleural effusion lymphomas (PEL) and some forms of multicentric Castleman's disease.3-5 Only 1-5% of cells in KS lesions actively support lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV), the etiological agent associated with KS, and it is clear that cellular factors must interact with viral factors in the process of oncogenesis and tumor progression.6,7 Identifying novel host-factor determinants which contribute to KS pathology is essential for developing prognostic markers for tumor progression and metastasis as well as for developing novel therapeutics for the treatment of KS.8 The accompanying video details the methods we use to identify host cell gene expression programs altered in dermal microvascular endothelial cells (DMVEC) after KSHV infection and in KS tumor tissue.9 Once dysregulated genes are identified by microarray analysis, changes in protein expression are confirmed by immunoblot and dual labeled immunofluorescence. Changes in transcriptional expression of dysregulated genes are confirmed in vitro by quantitative real-time polymerase chain reaction (qRT-PCR). Validation of in vitro findings using archival KS tumor tissue is also performed by dual labeled immunochemistry and tissue microarrays.8,10 Our approach to identifying dysregulated genes in the KS tumor tissue microenvironment will allow the development of in vitro and subsequently in vivo model systems for discovery and evaluation of potential novel therapeutic for the treatment of KS.

Identifying Dysregulated Genes Induced by Kaposi&#039;s Sarcoma-associated Herpesvirus KSHV

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

确定失调卡波西氏肉瘤相关疱疹病毒（KSHV）诱导的基因

Currently KS is the most predominant HIV/AIDS related malignancy in Southern Africa and hence the world.1,2 It is characterized as an angioproliferative ...

科研

免疫与感染

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

一个<em在体外</em&gt;模型研究人类巨噬细胞分化和极化的异质性

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. 
We generated model xenograft tumors in mice by subcutaneous injection of a human ovarian cancer cell line, OVCAR-8 (NCI DCTD Tumor Repository, Frederick, MD). 
We transformed attenuated strains of S. typhimurium bacteria (ELH430:SL1344 phoPQ- 1) with a constitutively expressed luciferase (luxCDABE) plasmid for visualization2. These strains specifically colonize tumors while remaining essentially non-virulent to the mouse1. 
Once measurable tumors were established, bacteria were injected intravenously via the tail vein with varying dosage. Tumor-localized, bacterial gene expression was monitored in real time over the course of 60 hours using an in vivo imaging system (IVIS). At each time point, tumors were excised, homogenized, and plated to quantitate bacterial colonies for correlation with gene expression data. 
Together, this data yields a quantitative measure of the in vivo growth and gene expression dynamics of bacteria growing inside tumors.

Measuring Growth and Gene Expression Dynamics of Tumor-Targeted S. Typhimurium Bacteria

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium bacterial colonies growing inside tumors. This video covers tumor cell preparation and implantation, bacteria preparation and injection, whole-animal luminescence imaging, tumor excision, and bacterial colony counting.

肿瘤靶向性生长和基因表达的动力学测量<em&gt; S。鼠伤寒沙门氏菌</em&gt;细菌

The goal of these experiments is to generate quantitative time-course data on the growth and gene expression dynamics of attenuated S. typhimurium ...

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission of TSE diseases, such as chronic wasting disease (CWD), is presumed to be from animal to animal2,3 as well as from environmental sources4-6. Scavengers and carnivores have potential to translocate PrPRes material through consumption and excretion of CWD-contaminated carrion. Recent work has documented passage of PrPRes material through the digestive system of American crows (Corvus brachyrhynchos), a common North American scavenger7.
We describe procedures used to document passage of PrPRes material through American crows. Crows were gavaged with RML-strain mouse-adapted scrapie and their feces were collected 4 hr post gavage. Crow feces were then pooled and injected intraperitoneally into C57BL/6 mice. Mice were monitored daily until they expressed clinical signs of mouse scrapie and were thereafter euthanized. Asymptomatic mice were monitored until 365 days post inoculation. Western blot analysis was conducted to confirm disease status. Results revealed that prions remain infectious after traveling through the digestive system of crows and are present in the feces, causing disease in test mice.

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas. We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

用于识别传染性朊病毒通过后通过一个鸟类的消化系统程序

Infectious prion (PrPRes) material is likely the cause of fatal, neurodegenerative transmissible spongiform encephalopathy (TSE) diseases1. Transmission ...

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense against gram-negative bacterium Salmonella typhimurium. Salmonella establishes persistent infection in the intestine of C. elegans and results in early death of infected animals. A number of immunity mechanisms have been identified in C. elegans to defend against Salmonella infections. Autophagy, an evolutionarily conserved lysosomal degradation pathway, has been shown to limit the Salmonella replication in C. elegans and in mammals. Here, a protocol is described to infect C. elegans with Salmonella typhimurium, in which the worms are exposed to Salmonella for a limited time, similar to Salmonella infection in humans. Salmonella infection significantly shortens the lifespan of C. elegans. Using the essential autophagy gene bec-1 as an example, we combined this infection method with C. elegans RNAi feeding approach and showed this protocol can be used to examine the function of C. elegans host genes in defense against Salmonella infection. Since C. elegans whole genome RNAi libraries are available, this protocol makes it possible to comprehensively screen for C. elegans genes that protect against Salmonella and other intestinal pathogens using genome-wide RNAi libraries.

A Protocol to Infect Caenorhabditis elegans with Salmonella typhimurium

C. elegans has emerged as a new genetic model to study host-pathogen interactions. Here we describe a protocol to infect C. elegans with Salmonella typhimurium coupled with the double-strand RNAi interference technique to examine the role of host genes in defense against Salmonella infection.

一个协议感染<em&gt;秀丽隐杆线虫</em&gt;与<em&gt;鼠伤寒沙门氏菌</em

In the last decade, C. elegans has emerged as an invertebrate organism to study interactions between hosts and pathogens, including the host defense ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

高通量检测到的表型<em&gt;沙门氏菌</em&gt;伤寒协会，侵袭和复制的巨噬细胞

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

高通量设置的建立筛选小分子能调节C-二GMP信令<em&gt;绿脓杆菌</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR RS-PCR

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

基因分型<em&gt;金黄色葡萄球菌</em&gt;由核糖体垫片的PCR（RS-PCR）

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

巨噬细胞中含有吞噬的鼠伤寒沙门氏菌的分离

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

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

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

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

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

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 ...