经费是由美国国立卫生研究院/ NIAID补助RO1 - AI - 63457和复苏法案授予补充RO1 - AI - 63457一。我们承认当前和过去的实验组和合作者所有成员的贡献。

此视频文章的目的是提供数据格式的工作流程的概述和研究人员解释，可能只是开始这方面的工作类型（ 图 1 ）。它包括以前出版的著作中所描述的技术，简化的协议和实用技巧的示范。5,10 一般的工作流程和实验室设施: 应该有这种研究至少3个独立的工作领域。实验室应该有1）的PCR罩（清洁空气中或孤立的工作区）（稀释，分装准备和混合），2）底漆准备处理一个单独的生物安全柜和DNA此外前PCR区在PCR混合物，和3）后PCR的工作领域，为准备和装载凝胶和准备为FLA的样本。引物和DNA样本应保存在单独的冷冻机和冰箱。引物污染是在这种类型的实验室工作的行政和最持久的问题之一。为引物和PCR混合使用的移液管不应该被用于DNA。应单独设置移液器- PCR检测，PCR和后PCR的工作领域。一般来说，一名研究人员可以在12-24小时期间使用标准实验室设备处理12-18样本。 每个阶段的工作开始之前: <ul><li>穿白大褂和手套。应戴手套，随时生物样品中，引物，DNA和溴化乙锭正在处理。勤换手套。 </li><li>在PCR柜油烟机，生物安全柜或干净的工作区域的工作。 </li><li>使用新鲜的台纸或垫。 </li><li>设置废物插座，适用于液体和移液器。 </li><li>与PCR罩和吸液管，用70％乙醇内擦拭。 </li><li>设立专题移液器和工作区PCR扩增前15分钟的紫外线照射。 （紫外光交联表面的DNA污染物）。 </li><li> 始终使用喷雾防治枪头材料含有DNA，引物和PCR试 ​​剂。 </li><li>任何管/条/板含有液体的离心开放前处理，以防止气溶胶逃生和交叉污染。 </li></ul> 1，M.麻风 DNA的制备含M.临床标本麻风获得访问皮肤诊所的麻疯病患者。常规诊断样本可能是皮肤打孔切片，缝皮肤涂片或鼻拭子。一般来说，冲床活检或开衩皮肤涂片检查是最好的分子流行病学研究，因为他们是干净的，并含有足够数量的分枝麻风 。根据机构指引，必须经使用这些材料进行研究。 <ol><li>保存在一个螺丝帽小瓶1毫升70％的乙醇活检或缝皮肤涂抹样品。 </li><li>在变速台式离心机离心15分钟，在12000 XG每个样本。 </li><li>去除上清液，离心管。如果必要的话，它可能是有用的重新离心机这些样本和其他组织或DNA后恢复。 </li><li>加入500μL磷酸盐缓冲液（PBS）的组织样本，并浸泡1小时交流残余乙醇的防腐剂，并补充水分样品。 </li><li>在台式离心机离心20分钟，在12000 XG的样本。丢弃废物的容器消毒液部分填补了PBS液。 </li><li>提取细菌的DNA用Qiagen公司DNEasy血液和组织Kit中的下列规定的指引。 </li><li>应提取的DNA分装成4瓶。商店2分装在-80 ° C的测试结果的重复性，如果/当需要时或新技术成为未来使用。店之一等分在-20 ° C，并在4 ° C为更多的直接使用。 </li><li>谨慎的做法是准备一个"提取空白"的组织样本。空白是受到上文所述，但没有组织执行的所有相同的治疗。 PCR以及与病人的样本提取空白，以保证经营者的萃取技术是正确的，试剂的DNA污染。 </li></ol> 2。底漆准备<ol><li>引物位点，其中有最广泛的应变类型的数据列于表1。 4个或5个引物多重PCR结合起来。每个位点的引物进行5&#39;期间将毛细管电泳片段长度分析（FLA）检测的荧光化学标记。 </li><li>对于每个多重PCR组合，每个位点的标记的引物和相应的反向引物相结合，在单独的Eppendorf管一管的正向引物，另一个反向。股票引物准备为100μm的解决方案（ 图2a），其中一部分稀释10倍TE的使用浓度为10μm（1X的Tris - EDTA，pH值8.0）。 100μm的底漆解决方案的剩余等分储存在-20 ° C，以备后用。 </li><li>在2μm的最终浓度，引物各混合同等数量每10微米底漆。当添加到PCR混合物（ 见表3）引物组合，每个引物终浓度下降到0.2微米。 </li></ol>当合并后的引物加入到PCR混合物（见表 3），终浓度下降到0.2μm的每个。 3。使用多重PCR扩增细菌的DNA <ol><li> PCR检测设置<ul><li>记录将用于样品的数量和所需的试剂和其体积与准备工作表之前收集必要的塑料和其他材料。 </li><li>标签的无菌管/条/ PCR样本数板。请记住，包括积极和消极的控制。 </li></ul></li><li>擦拭与清洗液（如DECON ELIMINase）和程序按表2的热循环。更改手套，清洗后的前处理引物和其他试剂。 </li><li>根据表3和标签的PCR管/条/盘要使用的引物组合和样本数，准备PCR反应。 </li><li> Mastermix分装18μl每个标记的PCR样品管或井水。更换手套。 </li><li>用一个单独的生物安全柜和吸液管，用70％乙醇，以帮助清洁和消毒工作区和工具，然后工作区和移液器紫外灯15分钟，以交联的DNA污染物。更换手套。 </li><li>在清洁的生物安全柜，在每个PCR管/板，以及收购总量的20μL（图 2b）。使用所有的液体材料气雾剂预防枪头Mastermix加入DNA模板2μL。 </li><li>离心机PCR管/条/板简单组合内容。 </li><li>样品管/条/板放置在热循环和启动PCR程序。 </li><li>当程序完成后，删除从热循环和存储产品在4 ° C直到电泳。 </li></ol> 4。 PCR产物的凝胶电泳 *此协议已被多年的标准，是一个可选步骤，如果需要确认PCR产物为FLA发送前，可以聘请。 <ol><li>后PCR在一个单独的工作区，准备了2％琼脂糖凝胶。使用2.0克1X TBE琼脂糖powder/100ml（三/硼酸/ EDTA）缓冲液的烧瓶中的解决方案。约1.5-2分钟在微波炉中加热的混合物。涡流的内容，再次加热，如果需要解散琼脂糖。略有降温，倒入凝胶在使用梳子样品足够井的形式。 </li><li>删除PCR产物4℃，约30秒的离心机。 </li><li>混合2 -5μLPCR产物的5倍或6倍的凝胶样缓冲液（样缓冲液是从各公司提供的，或可在实验室中混合。食谱可在网上。）0.5 -1μl进</li><li>负载与6μl样品/样缓冲液混合凝胶的井。添加一个分子的阶梯一个（最好是一个20 bp的阶梯）。 </li><li>在100V运行约90分钟的凝胶。 </li><li>凝胶溴化乙锭溶液浸泡15-30分钟，然后在超纯蒸馏水等量时间。 </li><li>图像的凝胶，而紫外线是应用。应该有一个乐队组合在每个4-5 DNA片段（ 图3）。 </li><li>根据该机构的政策有害物质的处置凝胶。溴化乙锭溶液可用于妥善处置前多次。 </li></ol> 5。准备样品片段长度分析（FLA） <ol><li>在一个干净的Eppendorf管中，准备主，从一个全新的分装和的GeneScan -500 LIZ上浆标准（美国应用生物系统公司）0.3μl，为每一个被测试的样本中含有高迪甲酰胺的解决方案12μl混合物。 （高迪甲酰胺化学变性的DNA链之前，毛细管电泳，省去了取暖的需要。）使用96孔光学质量反应板，12.3μl甲酰胺LIZ混合分装到每个正在使用的样本，并设置板搁置。 </li><li>请为您的记录表明，DNA样本，在每口井（图4a）的板图。 </li><li>使用管/条或96孔板，加入PCR质量创造了PCR产物1:60稀释水59μlPCR产物加入1μl（从第3部分 ）。从FLA的数据或凝胶条带的亮度信号强度的基础上，稀释可能会有所调整。即使是低浓度的DNA可足够（1:120或1:180）。 </li><li>加入1μl加入稀释后的PCR产物中含有甲酰胺LIZ混合板的正确。 </li><li>可大大提高速度和效率，如果在96孔板的PCR。可以简单地按顺序排队等3板块:板块与PCR产物，无菌水稀释PCR产物2板，板甲酰胺和浆纱阶梯片段长度分析3 1。多道移液器允许快速的FLA的准备过程。 </li></ol>通过遗传分析仪的采样分析<ol start="6"><li>校准遗传分析仪检测应用flurophores每个制造商的指示。请务必使用染料设置，包括LIZ。 </li><li>补充用水冲洗干净容器，并添加新的运行与EDTA缓冲液（1X）（美国应用生物系统公司），以缓冲液槽。 </li><li>添加预先缝硅隔板，并将其放置在板的设计控股托盘。按"托盘"的遗传分析仪上的按钮，将自动进样器前进。将托盘上自动进样器和密切的遗传分析仪的大门。 </li><li>创建或导入电子表格分析。导入的文件扩展名为。PLT和制表符分隔的格式。在Excel（微软）​​或类似的计划的文件可以进行修改。 </li><li>申请注入1.6千伏电压为15秒，样品注入毛细管（50厘米长，POP - 7聚合物）毛细管电泳运行在60 ° C的电压在15千伏为1800秒。整个过程大约需要45分钟。 </li><li>一旦运行完成后，每个分析样品中的数据转换成文件扩展名为。FSA，放置在一个板文件的文件夹（ 图4b） 。每个数据文件的大小大约是100 KB，可以存储在闪存驱动器或压缩和电子邮件。数据文件可以被视为使用合适的软件，如ABI的GeneMapper或山顶的扫描仪。 </li></ol> 6。片段长度的结果分析<ol><li>从荧光毛细管电泳数据的分析，需要特殊的软件。如果这样的软件是不可用，请美国应用生物系统公司的网站， 下载峰值扫描仪。软件是免费的，效果相当不错。 <a href="https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&amp;catID=603624">https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&amp;catID=603624</a> </li><li>打开山顶扫描仪和"启动新项目"，"添加文件" 。装入选定的。FSA的数据文件，包括阳性和阴性对照到程序。 </li><li>选择（高亮）和"分析"所有已加载的文件。要确保每个样品设置为"尺寸标准:GS500（-250）和分析方法:浆纱默认的PP按"分析"。 </li><li>检查在每个样本中的DNA片段产生彩色高峰的碱基对的大小。 </li><li>记录中的每个碱基对的峰面积值，并把它比作阳性对照高峰。 </li><li>阳性对照样品的峰值应与表 4-7中列出的串联重复序列的碱基对和相应的数字的数字相媲美。 </li><li>确定有多少个短串联重复片段与阳性对照比较，在每个样本等位基因中。我们使用NHDP63宗正每个座位已测序的VNTR副本。 </li><li>输入到一个比较和/或数学分析的电子表格记录的数据。 VNTR"指纹"或单体型，字符串定义的位点的等位基因， 一个 M.特点麻风菌株。 </li></ol> 7。代表性的成果凝胶电泳PCR产物，将有望生产乐队为每个引物组合的轨迹（ 图 3 ）。在图3中，有2节凝胶:顶端部分组合1 PCR样品和下部组合 ​​的2个样品。每一节都包含一个20碱基对的分子梯，从8个患者的样本中获得的PCR产物。组合1也有消极的的控制和最后一个阳性对照（NHDP63株）。 （组合2的控制，在不同的凝胶。）请注意，大多数样品清楚地显示每个座位的组合5波段，1。在某些情况下，乐队可能是靠得太近，出现在只有4条带的外观单独的基因位点。 预期扩增片段大小列于表1我们的实验室使用NHDP63 M的应变 。 麻风作为阳性对照。两种类型的重复段进行了研究:微卫星位点（1-5基地重复）和小卫星位点（超过5个碱基对重复多次与段）。 <sup&gt; 5 毛细管电泳数据文件的解释依赖于2个标准:内部的DNA片段大小的标准要求的GeneScan 500LIZ（ABI）和外部细菌的DNA扩增阳性对照样品。 的FLA色谱， 山顶扫描仪软件，请使用，可以看到在图5A，5B及6。 山顶扫描仪提供了扩增片段大小的数据（碱基对X轴）和信号强度（Y轴）。 （ 图5b）对峰面积等的其他数据也可以，虽然规模和峰高值是最重要的。山峰高度超过100个单位的通常被认为是可靠的信号太弱。 图6比较，阳性对照（NHDP63）和两个病人样本，呈现出一个变化中的串联重复序列位点（GTA）9。阳性对照，序列（GTA）的重复10次。 NHDP63的VNTR和扩增大小，通过基因测序验证。患者4的PCR扩增3 BP比正面有只有9重复单位，而病人6扩增，比NHDP63揭示的11次重复（GTA）的大3 BP控制小。患者2低细菌指数（BI）与很少或根本没有DNA PCR复制，因此没有FLA信号。 在FLA数据的解释的困难，有时会出现"口吃"。 DNA聚合酶在PCR反应中，可能会产生1个或多个重复或长或短比源基因的片段。这些通常是公认的降低周围主峰高度的山峰。他们将"阶梯"，这是正确的碱基对的重复段大于或小于的主要峰值较小。其结果是相同颜色的峰的家庭。图 7显示了轨迹（TA）10 。 有时遇到的FLA的另一个困难涉及&#39;+ A&#39;或&#39;A -拖尾"，其中的DNA聚合酶[通常腺嘌呤（A）]来复制DNA片段的3&#39;端增加了一个单独的基。这表明在FLA作为一个主或口吃的高峰期是1个碱基对，比图8所示的相邻峰的权利的一个高峰。它不应该混同于一个主或口吃的高峰。被认为是一个主要的高峰期，其A尾，品种单一。 A -尾不会改变的VNTR重复数量。 （某些DNA聚合酶试剂盒的设计，专门促进拖尾，以减少这个混杂的效果，促进完成一个拖尾往往会产生一个单峰，而不是一对峰。） DNA提取和PCR产物同时包含M。麻风和人类的DNA，但与异常（TA）18，引物不够具体，他们只放大细菌的DNA，并有很少或根本没有人类的DNA扩增。 （TA）18，经常会产生一个峰值在242个碱基对，是从人类的DNA，而不是传犰狳的DNA样本（ 图 9 ）。 引物的保质期总体上是好的，他们在-20 ° C只有立即删除，保存，然后保存在4 ° C直到消耗。底漆应稳定在4 ° C的1-2周。 PCR引物组合即使是有点费时，这是建议的准备，只有足够的引物组合可立即使用。引物组合似乎长时间存放有所降低。 TE应分装，从库存量较大，可存储等分冻结或在室温。较大的分装的200 -400μl稀释干燥或浓缩底漆股（ 图2a）。 10-50μL较小的分装，用于补充引物组合3和第4卷。 TE是价格低廉，使用后应丢弃等分。 多重酶试剂盒是相当昂贵的，并应保持冻结，直到使用（-20℃）。 PCR准备，任何未使用的多重解决方案应立即返回到4 ° C。小Qiagen公司的多重PCR试剂盒配备了3管多重组合，每片含0.85毫升（850μl）的解决方案;足够的约65-70 PCR产物。 一般来说，它是最好的，以避免重复，在这种类型的实验室工作，包括DNA样本，引物和多重套件解决方案所用的材料主要的温度变化。所有材料应储存在-20 ° C，直到需要，并保持在4℃直至消耗。长的DNA长期储存应在-80 ° C 所有材料中含有花的组织，引物和/或DNA应蒸压和处置时不再使用任何。所有样品处理用溴化乙锭或甲酰胺应视为危险废物，并根据该机构的政策有害物质的处置。 <img src="/files/ftp_upload/3104/3104table1.jpg" alt="表1" /> 表1:扩增大小应变NHDP63 <img src="/files/ftp_upload/3104/3104table2.jpg" alt="表2" /> 表2:骑自行车的VNTR PCR技术的参数<img src="/files/ftp_upload/3104/3104table3.jpg" alt="表3" /> 表3:PCR的制备<img src="/files/ftp_upload/3104/3104table4.jpg" alt="表4" /> 表4:组合1的等位基因调用<img src="/files/ftp_upload/3104/3104table5.jpg" alt="表5" /> 表5:组合2的等位基因调用<img src="/files/ftp_upload/3104/3104table6.jpg" alt="表6" /> 表6:组合3的等位基因调用<img src="/files/ftp_upload/3104/3104table7.jpg" alt="表7" /> 表7:组合4等位基因电话<img src="/files/ftp_upload/3104/3104fig1.jpg" alt="图1"/> 图1:VNTR FLA工艺流程图<img src="/files/ftp_upload/3104/3104fig2.jpg" alt="图2"/> 图2。 （一）编制上的引物组合1（Eppendorf管中的图片礼貌<a href="http://www.clker.com">www.clker.com</a> ）。 （二）PCR的设置（8 PCR的）（Eppendorf管www.clker.com图片礼貌） <img src="/files/ftp_upload/3104/3104fig3.jpg" alt="图3"/> 图3。组合1和2的VNTR PCR产物DNA琼脂糖凝胶<img src="/files/ftp_upload/3104/3104fig4a.jpg" alt="图4a"/> <img src="/files/ftp_upload/3104/3104fig4b.jpg" alt="图4b"/> 图4。 （一）的FLA板地图（二）FLA的数据文件:*. FSA <img src="/files/ftp_upload/3104/3104fig5a.jpg" alt="图5a"/> <img src="/files/ftp_upload/3104/3104fig5b.jpg" alt="图5b"/> 图5。 （一）阳性对照的FLA色谱（NHDP63）组合1的VNTR位点（B） 峰扩增的规模和丰富的扫描数据（GTA）的9 <img src="/files/ftp_upload/3104/3104fig6.jpg" alt="图6"/> 图6。PCR检测样品比较轨迹（GTA）9 PC（NHDP63） <img src="/files/ftp_upload/3104/3104fig7.jpg" alt="图7"/> 图7。主要口吃峰（TA）10 <img src="/files/ftp_upload/3104/3104fig8.jpg" alt="图8"/> 图8:+ A（A尾）毗邻主或口吃峰的峰。 <img src="/files/ftp_upload/3104/3104fig9.jpg" alt="图9"/> 图9:（电讯局长）18个主峰，口吃峰和人类的DNA峰<img src="/files/ftp_upload/3104/3104fig10a.jpg" alt="图10a"/> <img src="/files/ftp_upload/3104/3104fig10b.jpg" alt="图10b"/> 图10:（一）M的应变分化麻风 VNTR数据，（二 ）M.应变分化麻风基于MLVA DNA指纹

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	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+of+leprosy+for+control+programmes.">Chemotherapy of leprosy for control programmes.</a> Technical Report Series. 675, 18-22 (1982).</li><li>Cole, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Massive+gene+decay+in+the+leprosy+bacillus.">Massive gene decay in the leprosy bacillus.</a> Nature. 409, 1007-1011 (2001).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leproma+Web+Server.">Leproma Web Server.</a> Leproma Web Server [Internet]. , (2004).</li><li>Groathouse, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Polymorphic+Loci+for+Molecular+Typing+of+Strains+of+Mycobacterium+leprae.">Multiple Polymorphic Loci for Molecular Typing of Strains of Mycobacterium leprae.</a> J. of Clinical Microbiology. 42, 1666-1672 (2004).</li><li>Young, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+Short+Tandem+Repeat+Sequences+to+Study+Mycobacterium+leprae+in+Leprosy+Patients+in+Malawi+and+India.">Use of Short Tandem Repeat Sequences to Study Mycobacterium leprae in Leprosy Patients in Malawi and India.</a> Public Library of Science: Neglected Tropical Diseases. 2, E214-E214 (2008).</li><li>Monot, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+Variable-Number+Tandem+Repeats+Appropriate+for+Genotyping+Mycobacterium+leprae.">Are Variable-Number Tandem Repeats Appropriate for Genotyping Mycobacterium leprae.</a> J. of Clinical Microbiology. 46, 2291-2297 (2008).</li><li>Kimura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Variable+Number+Tandem-Repeat+Genotyping+for+Mycobacterium+leprae+Clinical+Specimens.">Rapid Variable Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens.</a> J. of Clinical Microbiology. 47, 1757-1766 (2009).</li><li>Weng, X. i. a. o. m. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Distribution+of+Mycobacterium+leprae+Genotypes+in+a+Region+of+High+Leprosy+Prevalence+in+China:+a+3-Year+Molecular+Epidemiological+Study.">Identification and Distribution of Mycobacterium leprae Genotypes in a Region of High Leprosy Prevalence in China: a 3-Year Molecular Epidemiological Study.</a> J. of Clinical Microbiology. 45, 1728-1734 (2007).</li><li>Weng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+leprosy+in+Qiubei+County,+Yunnan,+China:+Insights+from+an+eight+year+molecular+epidemiology+investigation.">Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an eight year molecular epidemiology investigation.</a> Infection, Genetics and Evolution. , (2010).</li><li>Sakamuri, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Population-Based+Molecular+Epidemiology+of+Leprosy+in+Cebu,+Philippines.">Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines.</a> J. of Clinical Microbiology. 47, 2844-2854 (2009).</li><li>Fontes, A. N. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+diversity+of+Mycobacterium+leprae+isolates+from+Brazilian+leprosy+patients.">Genetic diversity of Mycobacterium leprae isolates from Brazilian leprosy patients.</a> Leprosy Review. 80, 302-315 (2009).</li></ol>

麻风患者皮肤样本收集需要熟练的医师或在皮肤诊所工作的技术人员。处理这些样品的实验室工作人员必须非常小心，穿实验室外套，手套和防护眼镜和处理感染人类或犰狳组织样本时，在一个生物安全柜工作。表面和工具的消毒也很关键。避免污染的DNA样本，在一个干净，无菌的生物安全柜的工作是非常重要的。 提取DNA提取试剂盒从像Qiagen公司公司的发展变得相对容易的感谢。方向，必须认真遵守。所有的样品应当在不使用时保持低温。避免重复样品，极端的温度变化。 在这项工作中使用的DNA引物可下令从各公司已在引在本文末尾的参考文献列表。应采取护理工作与自由的环境中的DNA引物，以避免污染。引物来作为干粉，必须混在一起用TE稀释至100μm的浓度，然后分成小批量（ 图2a） 。引物的工作解决方案，进一步稀释到10μm的浓度。再次，不要用引物使用，并准备在地区/抽油烟机的DNA样本。 PCR产物，也应该在不使用时保持低温。 3％琼脂糖凝胶的制备将提供更好的DNA条带的分离，但需要更长的时间来运行。 2％是纯定性的目的，通常就足够了。溴化乙锭溶液染色的DNA琼脂糖凝胶是一种高活性genotoxin，是通过皮肤吸收。处理这个的解决方案，并它使用非常谨慎，总是一定要戴手套染色凝胶。处理任何DNA样本或溴化乙锭的材料后，彻底洗净双手。处置的溴化乙锭染色溶液，冲洗和凝胶，根据​​机构指引。凝胶是不是经常需要;一旦FLA的方法已经建立，这一步可以消除。它是时间和试剂消耗。 甲酰胺的解决方案，在准备为FLA的样品使用剧毒，应谨慎处理。其使用后要洗手。下列机构的指导方针处理。 读的FLA的结果， 使用峰值的扫描仪软件可以是具有挑战性的。一个等位基因调用（串联重复序列数目）的基本已经拟定了一套在CSU（表 4-7） 。 （应该指出的是， 表4-7可能不是所有的包容性。M.其他菌株麻风研究，可能会发现列出的范围以外的拷贝数的等位基因。）一个特别的挑战，涉及到"口吃"的高峰。这些家庭的高峰，尤其是只涉及2-3个碱基重复，如（TA）10，可疑交易报告，。有时选择了正确的的高峰阅读困难（ 图7） 。在这个数字中，阳性对照在190 bp的高峰是主峰。峰较低的高度，2，4，甚至6个碱基对大于或小于主峰，被称为"口吃峰。"一个拖尾，也可以引起混乱。一个拖尾前面所述的文字和图8。最后，如​​果PCR产物样品中高度丰富，峰值可能出现双穗。在这种情况下，阅读的高峰期形成的中心。 （参见图6，病人6）。 数据管理可以为这种类型的工作任务十分艰巨。重要的是要记录所有相关信息，如所有的实验:一个任务或程序的日期，运营商，带/板地图，FLA的订单，PCR反应条件和配方，凝胶和照片的日期，储存温度，DNA模板稀释的FLA电子文件的存储位置，等良好的组织和数据管理可以节省小时的时间花在寻找特定的信息，在未来。 这里描述的实验室工作已经持续了数年在科罗拉多州立大学和世界各地的其他地点。所有收集到的数据的手段，怎么可能将来使用大图片开始出现。图10A和 10B展示如何将这些DNA指纹图谱可用于区分不同的 M.国家（10A）或什至家庭（10B）之间的麻风菌株。家庭和社区联系在一起案件已被证明携带M.麻风的相似或相同的VNTR应变类型。希望的是，它可能会获得进一步洞察到了麻风的传播模式（S），使早期检测系统的传输网络，可能会为那些人MOS开发在风险吨，而治疗性药物治疗可能会开始前做永久性的神经和皮肤的损害。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> DNeasy的血液和组织套件</td><td> Qiagen公司</td><td> 69504 </td><td></td></tr><tr><td>多重PCR套件</td><td> Qiagen公司</td><td> 206143 </td><td></td></tr><tr><td> DNA引物</td><td>各种</td><td></td><td></td></tr><tr><td>琼脂糖凝胶</td><td>各种</td><td></td><td></td></tr><tr><td> 5倍或6倍的凝胶样缓冲液</td><td> New England Biolabs公司</td><td> B7021S </td><td>食谱可让您自己* </td></tr><tr><td>溴化乙锭溶液</td><td>各种</td><td></td><td>从浓度稀释。 </td></tr><tr><td>高狄甲酰胺溶液</td><td>美国应用生物系统公司</td><td> 4311320 </td><td></td></tr><tr><td>基因扫描-500 LIZ </td><td>美国应用生物系统公司</td><td> 4322682 </td><td></td></tr></tbody></table> <a href="http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html">* http://biowww.net/buffer-reagent/6X-Gel-loading-buffer-bromophenol-blue-sucrose.html</a> <table border="1"><tbody><tr><th>设备</th><th>评论</th></tr><tr><td> 2冰箱</td><td> 4 ° C时:1 DNA样本，1对引物和多重试剂</td></tr><tr><td> 1微波炉</td><td>琼脂糖和水加热，使凝胶或合适的方式</td></tr><tr><td> 1高​​压灭菌器</td><td>或消毒材料压力锅</td></tr><tr><td> PCR仪</td><td>热循环</td></tr><tr><td> 2套移液器</td><td> 0.5-10μL，10-100μL，20-200μL，1000μL1引物，1套用于DNA </td></tr><tr><td>潜艇凝胶电泳中</td><td>准备凝胶形式和梳子。 </td></tr><tr><td>电泳电源</td><td>凝胶中的连接电缆</td></tr><tr><td>加热块</td><td>对于Eppendorf管</td></tr><tr><td>离心分离</td><td> Eppendorf管/条</td></tr><tr><td>毛细管电泳系统（遗传分析仪） </td><td>或访问一个机构，可以进行这种分析</td></tr><tr><td>塑料</td><td>一次性气溶胶的枪头，Eppendorf管中，8条，96孔板，一些光学质量</td></tr><tr><td>计算机与互联网连接</td><td></td></tr></tbody></table>

dna fingerprinting of mycobacterium leprae strains using variable number tandem repeat (vntr) - fragment length analysis (fla)

病原体以来， 麻风分枝杆菌 ，不能在实验室中培养了麻风的传播研究是特别困难的。细菌的唯一来源是麻风患者，实验感染的犰狳和裸鼠。因此，许多现代流行病学的方法不适用于麻风病的研究。尽管一个广泛的全球麻风病药物治疗方案，实施由世界卫生组织 1，麻风病仍然在许多国家流行的约25万，每年新发病例。整个 M。 麻风杆菌基因组已经被映射到3,4和许多位点已经确定有2个或更多的碱基对重复段（称为微型和小卫星 ）5 M.临床株麻风许多在这些位点（短串联重复序列，STR）的串联重复片段的数量可能会有所不同 。5,6,7可变数目串联重复序列（VNTR）5分析已经被用来区分不同麻风杆菌菌株。一些位点，似乎比其他人更稳定，显示重复次数少的变化，而其他人似乎更迅速地改变，有时在同一病人。虽然某些VNTRs的变化带来了关于是否适合他们的应变输入7,8,9的问题，新兴的数据表明，分析多个基因位点，这是其稳定性不同，可作为一种宝贵的流行病学工具使用。多个位点的VNTR分析（MLVA）10已被用来研究麻风病的演变，并在几个国家，包括中国 11,12， 马拉维 8， 菲律宾 10,13，和巴西 14传输。 MLVA涉及多个步骤。首先，细菌的DNA是从临床活检或缝皮肤涂片（SSS）与东道国组织DNA提取沿10所需的位点，然后从提取的DNA，通过聚合酶链反应（PCR）扩增。每个反应均采用荧光标记的引物为4-5个不同的基因位点，18个位点共有4个反应扩增 。10 PCR产物可能会受到琼脂糖凝胶电泳验证所需的DNA片段的存在，然后提交的荧光片段长度分析，利用毛细管电泳（FLA）的。从犰狳的DNA作为阳性对照菌传与已知数量的每个位点的重复副本。 FLA色谱研究使用山顶的扫描仪软件和片段长度转换为VNTR副本（等位基因）的数量。最后，VNTR单体型的模式进行了分析，并与病人的临床数据相结合，可用于跟踪应变类型的分布。

Watch this Scientific Journal Video about DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA at JoVE.com

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

麻风病，造成<em&gt;麻风分枝杆菌</em&gt;，在许多地方仍然流行。为了了解麻风病的传播蔓延和模式，重要的是要确定应变<em&gt; M。麻风</em&gt;已经感染的病人。可变数目串联重复（VNTR）分型是这样一种方法。

DNA指纹图谱麻风分枝杆菌株使用可变数目串联重复（VNTR） - 片段长度分析（FLA）

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory.  ...

dna-fingerprinting-mycobacterium-leprae-strains-using-variable-number

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

27.1K 次观看.  Colorado State University. 麻风病，造成麻风分枝杆菌，在许多地方仍然流行。为了了解麻风病的传播蔓延和模式，重要的是要确定应变 M。麻风已经感染的病人。可变数目串联重复（VNTR）分型是这样一种方法。

视频: DNA Fingerprinting of Mycobacterium leprae Strains Using Variable Number Tandem Repeat VNTR - Fragment Length Analysis FLA

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

发展的细胞类型特异性的抗艾滋病毒gp120基因的siRNA传递适配

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

科研

免疫与感染

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

样品制备<em&gt;结核分枝杆菌</em提取物对核磁共振的代谢组学研究

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

增长结核分枝杆菌生物膜

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

遗传操纵<em&gt;Δku80</em&gt;株功能基因组分析<em&gt;弓形虫</em

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Mycobacterium leprae, the causative agent of leprosy, does not grow in vitro. We describe an easy to follow protocol to prepare a bacillary suspension to ensure the maintenance of large quantities of M. leprae for a variety of applications. Protocols for propagation by mouse footpad inoculation, evaluation of viability, freezing and thawing bacillary stock are described in detail.

优化的协议<em&gt;麻风分枝杆菌</em&gt;应变管理:冷冻库存保持和维护在无胸腺裸鼠

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

分析18葡萄糖 PET/CT 成像作为研究结核分枝杆菌感染和治疗非人类灵长类动物的工具

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

报告酶荧光法检测小鼠结核分枝杆菌的影像学

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability to multiply inside such cells. These virulence traits render M. abscessus pathogenic, especially in vulnerable hosts with underlying structural lung disease, such as cystic fibrosis, bronchiectasis or tuberculosis. How patients become infected with M. abscessus remains unclear. Unlike many mycobacteria, M. abscessus is not found in the environment but might reside inside amoebae, environmental phagocytes that represent a potential reservoir for M. abscessus. Indeed, M. abscessus is resistant to amoebal phagocytosis and the intra-amoeba life seems to increase M. abscessus virulence in an experimental model of infection. However, little is known about M. abscessus virulence in itself. To decipher the genes conferring an advantage to M. abscessus intracellular life, a screening of a M. abscessus transposon mutant library was developed. In parallel, a method of RNA extraction from intracellular Mycobacteria after co-culture with amoebae was developed. This method was validated and allowed the sequencing of whole M. abscessus transcriptomes inside the cells; providing, for the first time, a global view on M. abscessus adaptation to intracellular life. Both approaches give us an insight into M. abscessus virulence factors that enable M. abscessus to colonize the airways in humans.

Identification of Virulence Markers of Mycobacterium abscessus for Intracellular Replication in Phagocytes

Here, we present two protocols to study Phagocyte-Mycobacterium abscessus interactions: the screening of a transposon mutant library for bacterial intracellular deficiency and the determination of bacterial intracellular transcriptome from RNA sequencing. Both approaches provide insight into the genomic advantages and transcriptomic adaptations enhancing intracellular bacteria fitness.

吞噬细胞内复制脓肿分枝杆菌毒力标志物的鉴定

What differentiates Mycobacterium abscessus from other saprophytic mycobacteria is the ability to resist phagocytosis by human macrophages and the ability ...

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally replicates through an episomal DNA intermediate; however, integration of HBV DNA fragments into the host genome has been observed, even though this form is not necessary for viral replication. The exact purpose, timing, and mechanism(s) by which HBV DNA integration occurs is not yet clear, but recent data show that it occurs very early after infection. Here, the in vitro generation and detection of HBV DNA integrations are described in detail. Our protocol specifically amplifies single copies of virus-cell DNA integrations and allows both absolute quantification and single-base pair resolution of the junction sequence. This technique has been applied to various HBV-susceptible cell types (including primary human hepatocytes), to various HBV mutants, and in conjunction with various drug exposures. We foresee this technique becoming a key assay to determine the underlying molecular mechanisms of this clinically relevant phenomenon.

Detection of Low Copy Number Integrated Viral DNA Formed by In Vitro Hepatitis B Infection

We describe here the in vitro generation of HBV DNA via a Hepatitis B virus infection system and the highly sensitive detection of its (1&#8211;2 copies) integration using inverse nested PCR.

乙型肝炎体外感染形成的低拷贝数综合病毒 dna 的检测

Hepatitis B Virus (HBV) is a common blood-borne pathogen causing liver cancer and liver cirrhosis resulting from chronic infection. The virus generally ...