这项工作是由美国国立卫生研究院（NIH）根据授予RC3 AI089106支持。 耐多药结核病核酸是由联合国儿童基金/联合国开发计划署/世界银行/世界卫生组织特别规划热带病（TDR），日内瓦，瑞士提供的研究和培训。 我们感谢美国疾病控制和预防的汤姆Shinnick博士对特定基因的突变和指导在原型试验，以包括。

对于遵循通用的PCR预防措施实验室，在运作上更有效率，包括几个放大芯片和基板每垫圈和在散装容器同时清洗所有放大芯片，如下所述。耗材格式可用于执行在一个完全密封的，封闭的扩增试验扩增后芯片的洗涤步骤，因为在别处30,33报。 1。设置<ol><li>提取并在适当的生物安全条件，选择的方法纯化核酸从样品。的要求是，核酸具有足够的纯度为不对称的，多重PCR扩增。 </li><li>擦拭表面和设备净化解决方案做准备的工作区。 </li><li>将扩增试剂在冰或冷水块。 </li><li>标注无菌1.5毫升microfuge管扩增预混，并标记无菌0.2毫升离心TU对于每个样品。 </li><li>后的试剂被解冻，简要地涡旋，并收集内容到管的底部用脉冲自旋在微型离心机。不要旋涡振荡Taq聚合酶。轻轻一抖管混合，并按照与脉冲自旋在微型离心机。 </li><li>使用表1中所示的每个样品的反应体积制备的扩增母液。为了考虑可能的移液误差，准备至少一个以上的反应体积比被处理的样本的总数。请注意，主混合物包含额外的Taq聚合酶，通过与以上所提供的muliplex PCR缓冲液。只有放大/抑制控件添加到主混音的所有其他试剂结合后，股市试剂管返回到存储。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 试剂 </td><td> 每个样品体积（μl） </td><td> &lt;STRONG&gt;决赛[] </td></tr><tr><td> Mulitplex PCR缓冲液与HotStar加Taq酶</td><td> 25 </td><td> 1X </td></tr><tr><td>牛血清白蛋白（BSA） </td><td> 0.55 </td><td> 0.6毫克/毫升 </td></tr><tr><td>甲酰胺</td><td> 3.8 </td><td> 7.6％ </td></tr><tr><td>额外的Taq聚合酶</td><td> 0.8 </td><td> 单位/μL（4台总） </td></tr><tr><td>耐多药结核病的引物组合</td><td> 15.75 </td><td> - </td></tr><tr><td>无RNA酶H 2 O的</td><td> 2.1 </td><td> - </td></tr><tr><td>放大/抑制控制</td><td> 1 </td><td> 5.0 FG /μL </td></tr><tr><td>总</td><td> 49 </td><td></td></tr></tbody></table> 表1。耐多药结核病的放大芯片预混成分。 <ol start="7"><li>涡流的主混合物，并收集内容到管的一个脉冲自旋在微型离心机的底部。 </li><li>结合49μl反应混合液和1μlM的杆菌 DNA的每个步骤1.4，上述样品管。对于外部，无模板对照（NTC），使用1微升的分子生物学级水代替M的肺结核 DNA样本。涡流和脉冲旋转时完成管（S）。 </li></ol> 2，载入扩增微阵列<ol><li>加入48微升每个主混音/样品到各自的微阵列的中心，小心不要触摸芯片本身。 </li><li>广场上的芯片垫片和密封件的顶部盖玻片，注意不要捕获任何气泡在微阵列室。气泡可以扩增效率产生负面影响并可能造成假象或在随后的微阵列图像噪声。 </li></ol> 3，热循环<ol><li>一旦所有的芯片都装有样品，放置在基板的平坦块热循环仪。确保加热盖选项是打开“关闭”。从Akonni生物系统获得的装备就已经通过资格预审的使用。否则，它是非常重要的，以验证该平坦块热循环仪（次），得到（S）均匀的，一致的加热在所有微阵列基板。 </li><li>打开热循环仪软件，选择相应的程序，输入“50微升”的反应体积，并开始运行。这里所描述的热循环程序包括： <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 热循环步骤 </td></tr><tr><td> 1 </td><td> 88°C </td><td> 5分钟</td></tr><tr><td> 2 </td><td> 88°C </td><td> 30秒</td></tr><tr><td> 3 </td><td> 55°C </td><td> 1分钟</td></tr><tr><td> 4 </td><td> 65°C </td><td> 30秒</td></tr><tr><td> 5 </td><td>重复步骤（2-4）50次</td></tr><tr><td> 6 </td><td> 65°C </td><td> 3分钟</td></tr><tr><td> 7 </td><td> 55°C </td><td> 3小时</td></tr></tbody></table></li><li>热循环的总数和55℃扩增后的保持时间为独立变量，用户可以修改，根据需要或适合于特定的实验。因为MDR-TB的主混合物产生不对称（主要是单链的）扩增子，它通常是有用的，以包括更多的热循环比否则可能被用于常规的，指数的PCR扩增方案。 </li><li>当热循环和杂交程序完成，结束程序，取出放大芯片，关闭该软件，并关闭热循环仪（S）。</li></ol> 4，清洗和干燥<ol><li>对于同时清洗多达24个基板，准备至少250毫升的1X SSPE - 0.1％的Triton X-100洗涤缓冲液，和两个容器至少250毫升去离子或Milli-Q系统级水各。洗涤缓冲液可以提前准备。 </li><li>小心取出盖玻片和垫圈使用平头镊子每个扩增微阵列腔， 这一步是在哪个点有物理损坏凝胶元素的数组的最大风险。使用合适的PCR污染管制，以防止扩增核酸的蔓延内的工作环境。 </li><li>将微阵列基板的组织学幻灯片夹，并将所有滑入含有洗涤缓冲液洗仓。覆盖容器用盖。 </li><li>为10分钟，在室温下轻轻搅动洗涤微阵列。 </li><li>浸微阵列在去离子的两个连续洗涤各三次或Milli-Q系统级的水，和空气干燥。 </li></ol> 5。成像不对称的耐多药结核病的引物组合产生Cy3标记的扩增子。凝胶元素微阵列可以成像与任何标准的微阵列成像仪成像能力的Cy3的。下面的成像过程是具体的耐多药结核病预混，Dx2100现场便携式成像仪，并自动分析软件由Akonni提供。 <ol><li>确保从成像器将以太网电缆连接到计算机。 </li><li>打开成像器。电源开关位于仪器的后面板上。当成像仪电源打开热像仪的前面的蓝色LED指示灯会亮起。 </li><li>开启计算机。 </li><li>在计算机桌面上，双击软件图标启动自动图像分析软件。 </li><li>等待建立与成像相机连接的软件。一旦连接建立，分析按钮变为可用，而出现“连接成功”将出现在屏幕的左下角。 </li><li>将干燥的扩增微阵列与微阵列面向远离用户，并且向物镜。如果芯片被不正确地定向相对于所述透镜和照相机，自动分析软件将不能放置一个网格中的荧光图像上。 </li><li>关闭检修门。预览图像将可在计算机屏幕上。 </li><li>从下拉菜单中选择耐多药结核病的分析脚本，并输入所需的曝光时间（以毫秒为单位）。一个典型的起点凝胶元素放大芯片是100-500毫秒。 </li><li>在红色的饱和像素将被显示在预览图像。调节曝光时间以个别斑点内的饱和像素的数量最小化。 </li><li>单击分析以获取和分析的荧光图像。该软件会自动将一个耐多药结核病检测特定的网格上的图像，提取积分强度和背景值，并报告耐药性及基因分型结果。自动分析通常需要几秒钟。对于包含刮痕，灰尘，荧光文物，或微阵列功能的物理损坏挑战性的图像，该软件可能需要长达1分钟即可完成分析。 </li><li>一旦分析完成后，点击保存，文件名中选择目标文件夹，类型，然后单击确定。底层芯片数据被保存为。xls文件，并可以导出用于离线分析，如果需要的话。 </li><li>一旦分析完成后，从成像器取出放大芯片，并单击新建以启动下一个分析。 </li><li>当收集完数据后，关闭软件，关闭计算机，并关闭相机。 </li></ol>

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Microbiol. 45, 2802-2807 (2007).</li><li>Chandler, D. P., Bryant, L., 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=Integrated+amplification+microarrays+for+infectious+disease+diagnostics.">Integrated amplification microarrays for infectious disease diagnostics.</a> Microarrays. 1, 107-124 (2012).</li><li>Vincent, V., Rigouts, L., 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=The+TDR+Tuberculosis+Strain+Bank:+a+resource+for+basic+science,+tool+development+and+diagnostic+services.">The TDR Tuberculosis Strain Bank: a resource for basic science, tool development and diagnostic services.</a> Int. J. Tuberc. Lung Dis. 16, 24-31 (2012).</li><li>Golova, J. B., Chernov, B. K., 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=Non-volatile+copolymer+compositions+for+fabricating+gel+element+microarrays.">Non-volatile copolymer compositions for fabricating gel element microarrays.</a> Anal. Biochem. 421, 526-533 (2012).</li><li>Cooney, C. G., Sipes, D., Thakore, N., Holmberg, R., Belgrader, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plastic,+disposable+microfluidic+flow+cell+for+coupled+on-chip+PCR+and+microarray+detection+of+infectious+agents.">A plastic, disposable microfluidic flow cell for coupled on-chip PCR and microarray detection of infectious agents.</a> Biomed. Microdevices. 14, 45-53 (2012).</li><li>Lane, S., Evermann, J., Loge, F., Call, 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=Amplicon+secondary+structure+prevents+target+hybridization+to+oligonucleotide+microarrays.">Amplicon secondary structure prevents target hybridization to oligonucleotide microarrays.</a> Biosens. Bioelectron. 20, 728-735 (2004).</li><li>El-Hajj, H. H., Marras, S. A. 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=Use+of+sloppy+molecular+beacon+probes+for+identification+of+Mycobacterial+species.">Use of sloppy molecular beacon probes for identification of Mycobacterial species.</a> J. Clin. Microbiol. 47, 1190-1198 (2009).</li><li>Boehme, C. C., Nabeta, P., 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=Rapid+molecular+detection+of+tuberculosis+and+rifampin+resistance.">Rapid molecular detection of tuberculosis and rifampin resistance.</a> N. Engl. J. Med. 363, 1005-1015 (2010).</li></ol>

作者宣称没有竞争的财务权益。

复用的扩增微阵列的程度最终是由多重不对称PCR的效率，而不是在微阵列所决定的。在我们的经验，10-12独特的引物对能够容易地复用在一个扩增微阵列格式。因此，常规的引物和探针的设计准则适用于新的测定法，不同之处在于1也需​​要考虑溶液相的核酸和固定化微阵列的探针，该热循环仪的热效率，并且在PCR缓冲液探针杂交行为之间可能的相互作用也含有PCR引物和低分子量的扩增假象。高?...

早期发现和快速处理被认为是最有效的控制策略，以降低结核分枝杆菌 （MTB）传输1，和现在有在结核病的社区，一个点的护理（POC）或接近POC测试，同时诊断结核病的广泛共识和耐药性（DR）是必要的。技术，如Cepheid公司的的GeneXpert和其他核酸扩增试验的时间减少到诊断为许多结核病患者，并提供快速的读出指示的耐利福平或所选突变赋予对其他第一或第二线药物2。虽然实时性和等温核酸扩增试验的目的是确定药物的耐药突变，导致耐多药结核病，他们发现突变的频谱往往不足，设计对应于患者的耐药性分布的个体化药物治疗方案，及相关技术的限制光学串扰或扩增和报告的化学复杂性5月3日至7日限制位点或检测到的突变的数量。因此，检测技术具有较高的复用能力需要解决的差距称为耐多药结核病的POC诊断。 微阵列和世界卫生组织认可的海恩线性探针检测可以解决“多基因，多基因突变”诊断耐多药结核病8-29的挑战。不幸的是，这些杂交为基础的，复用检测平台使用多步骤，复杂的，需要显著培训和熟练掌握分子生物学技术的开放式扩增子协议。扩增芯片30的目的是要解决一些芯片的工作流程和运营的担忧。该简化的流体原则是扩增，杂交和单微流体腔室中检测靶核酸。用户引入了核酸扩增和马斯之三混合成流体室用吸管，并开始热循环程序。对于此处所示的批量处理方法，微阵列，其后洗涤散装溶液，干燥，并成像。这项研究表明，使用MDR-TB芯片测试的rpoB（30突变），katG的 （2突变） 的inhA（4突变） 的rpsL（2突变），embB（1突变），IS1245，IS6110扩增芯片的功能和一个内部的扩增和杂交的控制。至少有一个匹配的一对基因芯片的探针（野生型（WT）和单核苷酸突变（MU））被列入为每个感兴趣的突变。从耐多药M.纯化核酸结核病是由结核台湾存托凭证应变银行31。凝胶元素微阵列在玻璃基板上通过共聚如其他地方32描述制造基本上，除了我们用4％的单体和0.05毫每个探头在polymeriz通报BULLETIN混合物。阵列被包围在50ml的垫圈在使用之前。后的热循环，杂交和洗涤步骤，扩增微阵列上成像的Akonni便携式分析。背景校正，集成信号强度从原料取得，使用一个固定的圆的算法TIF图像。噪音对每个凝​​胶元素的计算公式为当地现货背景的三倍标准偏差。靶基因通常被认为是检测到的信号噪声比（SNR）值≥3。为了确定在每个基因的密码子或特定突变的存在或不存在下，生成的判别比是从SNR值作为（WT-MU）/（WT + MU）计算。判别比值&lt;0顷指示耐药突变的位点的，而比值&gt; 0是指示性的野生型序列中。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;">MDR-TB amplification microarrays, with applied gasket and pre-cut cover slips</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Multiplex PCR kit, containing 2X PCR buffer with HotStar Taq plus</td>
			<td>Qiagen</td>
			<td>#206143</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Taq polymerase</td>
			<td>Qiagen</td>
			<td>#201207</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">RNAse-free water</td>
			<td>Qiagen</td>
			<td>#206143&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Formamide</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP227-500</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">20 mg mL-1 non-acetlyated bovine serum albumin (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>#3B6917</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">5X concentrated MDR-TB primer mix</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">500 fg uL-1 amplification and inhibition control</td>
			<td>Akonni Biosystems</td>
			<td>Inquire</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">20X SSPE</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP1328-4</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Triton X-100</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#BP151-500</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Disinfecting Spray&#160;</td>
			<td>Current Technologies, Inc.</td>
			<td>#BRSPRAY128</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">70% Isopropyl Alcohol</td>
			<td>Decon Labs, Inc.</td>
			<td>#8416</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Equipment</td>
			<td>Company&#160;</td>
			<td>Catalog Number&#160;</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microarray imager, with automated image and data analysis software</td>
			<td>Akonni Biosystems</td>
			<td>100-20011</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Thermal cycler with flat block insert</td>
			<td>Akonni Biosystems</td>
			<td>100-10021</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">High-throughput wash station and slide holder</td>
			<td>ArrayIt</td>
			<td>HTW</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dissecting forceps</td>
			<td>Thermo Fisher Scientific, Inc.</td>
			<td>#10-300</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini Vortexer</td>
			<td>VWR</td>
			<td>#3365040</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Mini-centrifuge</td>
			<td>VWR</td>
			<td>#93000-196</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Airbrush Compressor Kit</td>
			<td>Central Pneumatic</td>
			<td>#95630</td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstrating a multi-drug resistant mycobacterium tuberculosis amplification microarray

简化工作流程芯片是一个用于创建可在低资源环境中经常使用的MDR-TB芯片为基础的诊断是必要的第一步。扩增基因芯片结合了不对称PCR扩增，目标尺寸选择，目标标签和芯片杂交在一个单一的解决方案，并成一个单一的微流体室。批次处理方法表现出了9复杂不对称的预混和低密度凝胶元素的微阵列基因分型耐多药结核分枝杆菌 （MDR-TB）。这里描述的协议可在6小时内完成，并提供正确的基因分型与基因组DNA的至少1000细胞当量。结合片上的洗涤步骤是可行的，这将导致在一个完全封闭的扩增子的方法和系统。复用的扩增微阵列的程度最终是由引物对的，能够被组合成一个单一的主米的数量受限九，并且仍然获得期望的灵敏度和特异性的性能度量，而不​​是被固定在阵列上的探针的数目。同样地，总分析时间可以缩短或加长取决于特定的预期用途，研究问题，并且检测期望的限制。然而，一般的方法显著简化了微阵列的工作流程，通过减少手动密集和费时的加工步骤的数量，最终用户，并且提供了一个简化的生物化学和微流体通道用于翻译的微阵列为基础的诊断成常规的临床实践。

定性图像分析可以提供洞察实验噪音或变化的挑战是识别通过自动图像分析软件生成的数据表的来源。因此，它可以是有用的在视觉上确定：1）所有的凝胶元素都完好无损，2）全球背景是不受任何可能影响个人的信噪比（SNR）值荧光文物，3）没有证据气泡的形成或不均匀的放大/杂交在阵列中，和4），该软件可准确识别的微阵列图像上的所有点。例如MDR-TB的扩增微阵列图像示于图1，</strong...

Watch this Scientific Journal Video about Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray at JoVE.com

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

扩增微阵列结合的不对称PCR扩增和微阵列杂交到一个单一的腔室，其显著简化了微阵列的工作流程最终用户。简化工作流程芯片是一个用于创建可在低资源环境中经常使用的芯片为基础的诊断是必要的第一步。

展现着多药耐药<em&gt;结核分枝杆菌</em&gt;扩增基因芯片

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Research

JoVE Journal

Immunology and Infection

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

12.6K 次观看.  Akonni Biosystems, Inc.. 扩增微阵列结合的不对称PCR扩增和微阵列杂交到一个单一的腔室，其显著简化了微阵列的工作流程最终用户。简化工作流程芯片是一个用于创建可在低资源环境中经常使用的芯片为基础的诊断是必要的第一步。 

视频: Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Antimicrobial Susceptibility Testing of Mycobacterium Tuberculosis Complex for First and Second Line Drugs by Broth Dilution in a Microtiter Plate Format

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). Antimicrobial susceptibility testing (AST) of Mtb has traditionally been performed by the agar method of proportion or by 
macrobroth testing on an instrument such as the BACTEC (Becton Dickinson, Sparks, MD), VersaTREK (TREK Diagnostics, Cleveland, OH) or BacT/ALERT (bioM&eacute;rieux, Hazelwood, MO). The agar proportion method, while considered the &#x201C;gold&#x201D; standard of AST, is labor intensive and requires calculation of resistance by performing colony counts on drug-containing agar as compared to drug-free agar. If there is &ge;1% growth on the drug-containing medium as compared to drug-free medium, the organism is considered resistant to that drug. The macrobroth methods require instrumentation and test break point ("critical") drug concentrations for the first line drugs (isoniazid, ethambutol, rifampin, and pyrazinamide). The method described here is commercially available in a 96 well microtiter plate format [MYCOTB (TREK Diagnostics)] and contains increasing concentrations of 12 antimicrobials used for treatment of tuberculosis including both first (isoniazid, rifampin, ethambutol) and second line drugs (amikacin, cycloserine, ethionamide, kanamycin, moxifloxacin, ofloxacin, para-aminosalicylic acid, rifabutin, and streptomycin). Pyrazinamide, a first line drug, is not included in the microtiter plate due to its need for acidic test conditions. Advantages of the microtiter system include both ease of set up and faster turn around time (14 days) compared with traditional agar proportion (21 days). In addition, the plate can be set up from inoculum prepared using either broth or solid medium. Since the microtiter plate format is new and since Mtb presents unique safety challenges in the laboratory, this protocol will describe how to safely setup, incubate and read the microtiter plate.

Antimicrobial susceptibility testing of Mycobacterium tuberculosis is challenging but critical for patient care. This microtiter plate format offers testing of 12 antimycobacterial drugs and provides minimum inhibitory concentration (MIC) values, which will aid in appropriate treatment.

结核分枝杆菌综合肉汤稀释药敏试验微孔板格式为一线和二线药物

The rapid detection of antimicrobial resistance is important in the effort to control the increase in resistant Mycobacterium 
tuberculosis (Mtb). ...

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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

一种新型显微切割方法恢复<em&gt;结核分枝杆菌</em&gt;从福尔马林固定，石蜡包埋肺肉芽肿特异性转录

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

关于功能研究的三维人体肺组织模型<em&gt;结核分枝杆菌</em&gt;感染

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

高通量兼容检测，以评估对巨噬细胞传代药物疗效<em&gt;结核分枝杆菌</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

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 ...