作者要感谢谁做这项工作可能所有的同事，尤其是玛雅人Balamane，伊丽莎白·约翰斯顿白，沙龙Sjoblom，格雷格 - 奥尔丁Zakhona Gumede，Xolile Kineri，Phindile Mabaso，Lungisa Ndwandwe，詹姆斯·加维，加文·科布，铣三Maphanga，Terusha切提，Kevi奈杜，安德鲁Skingsley，凯瑟琳·斯托特和Lungani Ndwandwe。作者还要感谢卫生署和谁工作赫拉比撒艾滋病治疗和护理方案非洲中心人员的全体人员。

<html> 1。乙二胺四乙酸（EDTA）的全血处理中注：血液可以收集可以被存储在4℃下不超过24小时后，立即进行处理。 <ol><li>工作在一个生物安全柜，让EDTA全血样品达到室温。 </li><li>对于每个样品，标记足够的冷冻管与样品标识（ID），存储材料（血浆）和日期。 </li><li>离心样品10分钟，在1000×g下。不要使用刹车停止离心机。这将产生3层（从顶部到底部）：血浆，白细胞（血沉棕黄层） - 一个非常薄的层 - 和红细胞，包括血小板。 </li><li>小心吸出上清液（血浆），并等分500毫升到每个离心管。小心不要破坏细胞层（血沉棕黄层）或转让任何细胞。 </li><li>储存于-80℃直至提取RNA或立即进行RNA提取。 </li></ol>ve_title“&gt; 2。RNA提取<ol><li>准备提取工作表与要提取的样本，包括正面和负面的血浆控件的ID。 </li><li>每个样品要被提取，标号1.5ml的无菌微量离心管中的样本ID，提取日期和“RNA”。还标记的组装柱和收集管，以及含有与从提取工作表相应的数字工作裂解溶液2ml微量离心管中。 </li><li>工作中的生物安全柜，加入200μl样品的工作裂解液相应的2 ml离心管。 </li><li>涡流井后，于室温下10分钟。 </li><li> 10分钟后，离心反应管短暂。 </li><li>加入800毫升的无水乙醇中，以每管中。 </li><li>脉冲涡流和短暂离心混匀。 </li><li>转移600μl的该溶液中，以相应的列/集合管组件。离心6,000×克1分钟。 </li><li>转塔到一个新的收集管中，并丢弃含有滤液的老收集管。两次重复上述步骤2.8（上图）。 </li><li>加入500μl洗涤缓冲液AW1到每一列和离心机在6000×g离心1分钟。 </li><li>弃去滤液和收集管和柱转移至新的收集管。 </li><li>加入500μl为缓冲AW2和离心20,000 xg离心3分钟。重复步骤2.11。 </li><li>离心机在一个新的收集管中以20,000×g离心额外的2分钟。 </li><li>在1.5 ml离心管弃去滤液，将柱。 </li><li>加60微升缓冲液AVE（无RNA酶的水）的柱以确保不分配液体在塔的一侧的中间。 </li><li>在室温下孵育1分钟。 </li><li>离心机在6000×g离心2分钟。 </li><li>丢弃柱和盖1.5 ml离心管中。 </li><li>样品现在已准备好丽花SE转录。 </li><li>如果测试是要立即长达6小时完成，储存在4℃。但是，如果测试是要推迟然后立即放置于-80℃。注意：不要冻结/解冻的样品超过3倍。 </li></ol> 3。试剂制备的反转录<ol><li>在开始之前，计算每个需要被处理，包括阳性和阴性对照血浆样品的数量的试剂的体积。同时添加的试剂控制。 </li><li>从步骤3.1使用计算的体积（上图），准备脱氧核苷酸三磷酸（dNTP的）引物组合在一个无尘，无菌的200微升PCR管后短暂脉冲涡流。每个样品应该具有0.5微升反向引物RT21和0.5微升的dNTP， 见表2。 </li><li>分装1.0微升dNTP的引物组合，以200微升PCR管。 </li><li>制备反转录酶（RT）的酶混合物中加入1μ;升的10×逆转录缓冲液，1微升的0.1M DTT和2微升的25mM的MgCl 2至无菌试管随后通过涡旋和短暂离心， 见表3。 </li><li>将每个酶RNAseOUT和三标逆转录酶的酶混合物管的0.5微升然后点选管轻轻混匀。 </li><li>保持管与冷块上的dNTP，引物混合物和酶混合物，并移动到RNA站。 </li></ol> 4。反转录<ol><li>加入6微升的RNA样品到的dNTP，引物混合物管随后简要地涡旋混合。 </li><li>在添加的RNA后，移动到PCR房间，两个的dNTP /底漆/ RNA的结构和RT酶混合物管内的冷块或冰块上。 </li><li>短暂离心的dNTP /引物/ RNA的结构管（步骤4.2），并将其放置在一个热循环。 </li><li>热，在65℃下进行5分钟以变性RNA。 </li><li>迅速冷却至4℃，保持2分钟。 </li><li>暂停的热循环，同时仍然在4℃;取出离心管。 </li><li>很快，同时保持冷块上管加入5微升的酶混合物。 </li><li>通过敲击管轻轻混匀，然后短暂离心管中，并返回到热循环。 </li><li>持所述管在50℃进行60分钟反转录的RNA随后酶变性，在85℃下进行5分钟以停止反转录。 </li><li>冷却至37°C。一旦温度到达37℃时，暂停并采取管从PCR仪。 </li><li>快速加入0.5微升RNA酶H的该管子并返回到热循环。 </li><li>保持在37℃20分钟，然后冷却至4°C。 </li><li>将互补DNA（cDNA）可立即使用，或者可以被存储在-20℃或更低的温度，直到需要。然而，长期存储的cDNA应于-80℃。 </li></ol> 5。试剂制备用于PCR <ol><li> Befo重新出发，计算出每个需要的样本被处理和控制的数量的试剂的量。除了三个控件（正，负，和试剂），您还可以添加一个PCR对照（HIV DNA）。在第一和第二轮PCR混合物可同时制备和第二主混合物储存于-20℃直至需要。混合物可以被存储为约8小时。 </li><li>添加18.4微升水，2.5微升10X缓冲液，1.0微升的MgCl 2，0.5微升dNTPs和0.25微升各引物的如列在表4和旋涡。 </li><li>加入0.1微升的白金Taq聚合酶（和耐热），并通过点击它轻轻混匀管。 </li><li>分装23μL母液以200微升PCR管。 </li><li>用冷块或冰块移动到PCR房间母液管。 </li></ol> 6。巢式PCR <ol><li>加入2μlcDNA的23微升的第一轮PCR MASTE的ř混合。 </li><li>关闭该管后，把样品中的热循环，并使用以下PCR循环条件为：94℃2分钟，95℃30个循环持续30秒，58℃20秒，72℃进行2分钟，随后在72℃最终延伸10分钟，如在图1中 。 </li></ol><img alt="图1" fo:content-width="5in" fo:src="/files/ftp_upload/51242/51242fig1highres.jpg" src="/files/ftp_upload/51242/51242fig1.jpg" /> 图1。巢式PCR循环条件。 <a href="https://www.jove.com/files/ftp_upload/51242/51242fig1highres.jpg" target="_blank">点击这里查看大图。</a> <ol start="3"><li>继续第二轮的PCR阶段或在-20℃或更低的温度，直到在稍后阶段需要存储第一轮PCR产物。 </li><li>对于第二轮PCR，加入2μl的第一轮PCR产物，以23微升第二轮PCR扩增预混一个的D使用上图1相同的PCR程序。 </li></ol> 7。凝胶电泳<ol><li>凝胶法制备<ol><li>添加0.5克琼脂糖平板中，以250毫升的玻璃烧瓶中，并加入50毫升1×TBE缓冲液的烧瓶中。 </li><li>热火在微波到沸腾;经常搅动（约每30秒），直至完全溶解。使用硅胶握把或硅胶烤箱手套，以把握热点烧瓶中。琼脂糖溶液可以熬出来的烧瓶很容易如此密切监测这一过程。 </li><li>在室温下冷却约10分钟。 </li><li>倒入琼脂糖为含有适当大小的梳子凝胶浇注托盘;凝胶是准备在使用约20-30分钟。 </li><li>放置在凝胶电泳室，并运行所推荐的制造商。 </li></ol></li><li>凝胶电泳和可视化。 <ol><li>新型涡汁，持续10秒才能使用。 </li><li>稀释1微升新型果汁与5微升的DNA样本和组合。 </li><li>稀3微升新型果汁与3微升分子量标记和混合。 </li><li>从第7.2.2和7.2.3（以上）装入混合和运行凝胶在100 V和400 mA为40分钟，以评估PCR扩增。 </li><li>阳性扩增可在UV光下显现为1315 bp的片段， 图2。 </li></ol><img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51242/51242fig2highres.jpg" src="/files/ftp_upload/51242/51242fig2.jpg" /> 图2。用1％琼脂糖凝胶电泳和200 bp梯度PCR扩增的凝胶确认。 <a href="https://www.jove.com/files/ftp_upload/51242/51242fig2highres.jpg" target="_blank">点击这里查看大图。</a> <ol start="6"><li>应的负和试剂对照中没有扩增，从而表明没有污染。 </li></oL&gt; </li></ol> 8。 PCR产物清理<ol><li>在筹备测序反应，阳性第二轮PCR产物使用的PureLink PCR纯化试剂盒清理。 </li><li>加入80微升的工作结合缓冲高截止（B3）项至20μlPCR产物和吸管组合。 </li><li>加样品与结合缓冲液，以在集合管的旋转柱混合。 </li><li>离心柱以10,000×g离心1分钟。柱转移至新的收集管中。 </li><li>用650微升洗涤液用乙醇洗柱。 </li><li>离心柱以10,000×g离心1分钟。柱转移至新的收集管中。 </li><li>以最大速度离心柱2-3分钟，以除去任何残留的洗涤缓冲液。 </li><li>将离心柱与试剂盒提供一个干净的1.7毫升洗脱管。 </li><li>加入40微升的洗脱缓冲液对柱的中心，并孵育柱在室温温度场e表示1分钟。 </li><li>离心柱以最大的速度持续2分钟（&gt; 10,000×g离心）。 </li><li>洗脱管包含您的纯化PCR产物准备测序。丢弃的列。 </li><li>确定使用NanoDrop中的DNA的浓度和质量。 </li><li>如果没有内部测序设备的情况下，将纯化的PCR产物可以被发送到一个商业测序实验室在这个阶段。 </li></ol> 9。测序反应<ol><li> PCR产物使用的是大染料终止试剂盒3.1版和4的引物对每个样品（2正向和两个反向）进行测序。引物序列示于表2中 。因此，测序运行后，每个样品将有四个​​序列装配成重叠群。 </li><li>设置如表5中所示的每一个的四个引物的测序反应。 </li><li>在使用之前涡旋混合排序缓冲区和引物。 </li><li>混合水，通过添加大染料测序之前缓冲液和引物。涡旋混匀。 </li><li>颠倒离心管或轻轻拍打它加入大测序染料混合后，轻轻地混合母液。 </li><li>等分试样9微升主混合物到96孔光学板的。 </li><li>为了运行24个样品/盘，立盘所示， 图3所示。 </li></ol><img alt="图3" fo:content-width="5in" fo:src="/files/ftp_upload/51242/51242fig3highres.jpg" src="/files/ftp_upload/51242/51242fig3.jpg" /> 图3。 96孔板12患者样本进行测序，每个4引物（RTC1F，RTC2R，RTC3F和RTC4R）的计划表示。 <a href="https://www.jove.com/files/ftp_upload/51242/51242fig3highres.jpg" target="_blank">点击这里查看大图。</a> <ol start="8"><li>加入1.0微升的DNA样本（〜20-40纳克）的，覆盖板用N粘合剂铝盖，然后轻轻地混合。 </li><li>离心机在3000×g离心1分钟。除去铝盖，并添加一个橡胶密封垫。 </li><li>将板在热循环仪中，并运行在图4所示的下列循环程序。 </li></ol><img alt="图4" fo:content-width="5in" fo:src="/files/ftp_upload/51242/51242fig4highres.jpg" src="/files/ftp_upload/51242/51242fig4.jpg" /> 图4。 PCR循环条件进行测序 。 <a href="https://www.jove.com/files/ftp_upload/51242/51242fig4highres.jpg" target="_blank">点击这里查看大图。</a> <ol start="11"><li>当在PCR结束后，立即清理测序产物。 </li></ol> 10。测序清理<ol><li>对于每个测序反应中，混合50微升无水乙醇和5μl3M乙酸钠。 </li><li>使用多道移液器，加55＆＃956;升的乙酸钠/乙醇溶液，以每孔中。 </li><li>密封井用胶粘剂铝盖，确保每个井妥善密封。 </li><li>离心机在3000×g离心20分钟。 </li><li> 20分钟后，取下盖​​子，翻转板，以平滑的动作，到折叠实验室组织（请勿一鼓作气摆脱上清因为这将赶走颗粒！）。 </li><li>离心倒板在相同的组织中，在150×g离心2分钟。 </li><li>立即加入150μl的70％冷乙醇。不要拖延，加入乙醇在这一步。 </li><li>轴封采用相同的粘接铝盖和旋涡。 </li><li>离心机在3000×g离心5分钟。 </li><li>翻转板到一个新的折叠纸巾和离心机倒在150×g离心1分钟。 </li><li>离心后，将发现在热循环仪中，在50℃下进行2分钟干燥。 </li><li>一旦该板块是干的，用胶粘薄膜覆盖密封，包装箔及金属储存在-20˚C，直到准备进行无线TH测序电泳。 </li><li>当准备序列，溶解清洗测序产物在10毫升高迪甲酰胺，变性和负载电泳。 </li></ol> 11。生物信息学<ol><li>序列组装<ol><li>启动该程序Geneious。 </li><li>创建工作文件夹来存储序列。 </li><li>进口依照该测序仪产生的使用导入工具的工作文件夹ABI的文件。 Geneious将分配比例的质量分数为导入的每个序列。 </li><li>打开序列质量分数&gt; 70％通过双击它们。 </li><li>每个文件都应该在一个新的窗口中打开。该软件将指示在质量，使用淡蓝色条序列的质量色谱图的每个核苷酸位置。指示条越长，效果越好底座通话质量。 </li><li>使用光标，选择序列的离开了两端的中间部分，其质量较差一般。 </li&gt; <li>点击提取按钮来提取该地区具有良好的品质序列。 </li><li>选择每个样品所有四个序列中提取并将它们组装与参考序列。 </li><li>检查组装顺序，以确保你在正确的阅读框。如果你是在正确的读码框，蛋白酶的开始应该从以下氨基酸：PQITLW。 RT年初将开始与PISPIE。 </li><li>提取重叠群区域覆盖公关的开始到第300 RT密码子。在此过程中，还要检查插入或缺失。 </li><li>经过共识提取的重叠群的序列，识别任何含糊之处，并通过质量检验的基本通话（对称，高度，背景和侧翼区的肩膀）验证混合基地的位置。 </li><li>选择保守序列，然后单击提取按钮，从四个引物建立的共识序列的一个单独的文件和拉贝升了适当。 </li><li>顺序导出到计算机或网络文件夹上的备份存储文件夹。 </li></ol></li><li>序列质量评估（HIVDB） <ol><li>使用HIVDB节目在分析序列<a href="http://hivdb.stanford.edu">http://hivdb.stanford.edu</a> 。 </li><li>检查是否有缺失和插入的摘要数据，并确保该序列覆盖了所有的99蛋白酶（PR）的密码子，并且第一300 RT密码子。 </li><li>检查是否有任何突出显示的质量保证（QA）问题，同时在公关和RT地区，如终止密码子，移码，模棱两可的立场和不同寻常的残留物。 </li></ol></li><li>测序质量控制<ol><li>对爆炸从以前的运行本地序列数据库中的新序列。 </li><li>如果新的序列类似于数据库中的任何序列&gt; 97％，该协议的所有阶段应审查，开始与序列分析和回溯到RNA提取到ensurË有没有混合起坐（采样开关，贴错标签）或污染。 </li><li>如果没有发现问题，则重复从RNA提取阶段旧的和新的样本进行分析。 </li><li>如果序列仍&gt; 97％相似，回顾病史，评估对个人之间的流行病学联系。 </li></ol></li><li>系统发育分析<ol><li>从排列在Geneious使用的ClustalW程序数据库中的所有序列。 </li><li>手动检查对齐对齐序列，缺失和插入和编辑相应。 </li><li>构建系统发育树使用PHYML，Geneious树构建器或其他树建设者Geneious。 </li><li>检查树与短支长的样品。 </li><li>复查样品短支长的可能的污染。 </li></ol></li></ol> 12。 REGA DB资讯<ol><li>序列上传<ol><li>登录到REGADB使用一个唯一的用户名和密码。 </li><li>在下拉菜单中，根据患者ID，选择“开始使用”。 </li><li>新增病人ID，然后选择其基因型是要上载的个人。 </li><li>在菜单的左边，选择“病毒隔离”。 </li><li>从下病毒分离株的选项中选择“添加”。 </li><li>输入采样日期，样品编号，序列号和序列日期。 </li><li>选择“选择文件”，然后导航至要上传的序列的FASTA文件。 </li><li>选择要上传的FASTA文件后，点击上传。 </li><li>一旦上传的顺序出现在核苷酸框下的序列识别和日期，单击窗口右下角的OK按钮。 </li><li>检查PR和RT蛋白排列按一下按钮蛋白质及选择公关或RT。 </li><li>检查是否有耐药性突变通过点击阻力按钮。这使你从三种算法的耐药谱：ANRS，斯坦福HIVDB和RegaDB。 </li></ol></li><li>使用REGA报告生成<ol><li>登录到RegaDB使用唯一的用户名和密码。 </li><li>在下拉菜单中，根据病人ID，选择“开始使用”。 </li><li>新增病人ID，然后选择其报告将产生个人。 </li><li>在菜单右边，选择病毒分离。 </li><li>从下病毒分离株的选项点击“视图”。 </li><li>双击您要创建报告的病毒株。 </li><li>对病毒分离株窗口中，单击病毒分离株的报告选项卡上。 </li><li>选择算法的基因型从下拉菜单中的解释，然后选择报表模板使用。 </li><li>一旦算法和模板被选中，点击按钮“生成”​​。 </li><li>下载生成的RTF文档。 </li><li>打开RTF做cument为Word文档。 </li><li>调整治疗史图。 </li><li>图表后，添加部分“临床图表和阻力的解释”。 </li><li>使用电阻表和临床图表上的数据，添加患者的电阻曲线的开始与患者的治疗史的描述，​​以及药物其中病毒分离物具有抗性。还添加了患者的病毒载量的描述，并从图表CD4 +细胞计数型材。 </li><li>发送报告，传染病（ID）专家审查和对未来的病人管理的建议。这一过程也是非常重要的质量保证阶段。在治疗史的基因型或出现不一致的任何错误，病毒学和免疫学资料，可以识别和审查的最终报告发送之前，所有的建议，给临床医生管理患者。 </li></ol></li></ol>

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	<li>Shao, Y., Williamson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+HIV-1+epidemic:+low-+to+middle-income+countries.">The HIV-1 epidemic: low- to middle-income countries.</a> Cold Spring Harbor Persp. Med. 2, (2012).</li><li>Mutevedzi, P. C., 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=Scale-up+of+a+decentralized+HIV+treatment+programme+in+rural+KwaZulu-Natal,+South+Africa:+does+rapid+expansion+affect+patient+outcomes.">Scale-up of a decentralized HIV treatment programme in rural KwaZulu-Natal, South Africa: does rapid expansion affect patient outcomes.</a> Bull. World Health Organ. 88, 593-600 (2010).</li><li>Houlihan, C. F., 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=Cohort+profile:+Hlabisa+HIV+treatment+and+care+programme.">Cohort profile: Hlabisa HIV treatment and care programme.</a> Int. J. Epidemiol. 40, 318-326 (2011).</li><li>Tanser, F., Barnighausen, T., Grapsa, E., Zaidi, J., Newell, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+coverage+of+ART+associated+with+decline+in+risk+of+HIV+acquisition+in+rural+KwaZulu-Natal.">High coverage of ART associated with decline in risk of HIV acquisition in rural KwaZulu-Natal.</a> South Africa. Science. 339, 966-971 (2013).</li><li>Bor, J., Herbst, A. J., Newell, M. L., Barnighausen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increases+in+adult+life+expectancy+in+rural+South+Africa:+valuing+the+scale-up+of+HIV+treatment.">Increases in adult life expectancy in rural South Africa: valuing the scale-up of HIV treatment.</a> Science. 339, 961-965 (2013).</li><li>Montaner, J. S., 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+case+for+expanding+access+to+highly+active+antiretroviral+therapy+to+curb+the+growth+of+the+HIV+epidemic.">The case for expanding access to highly active antiretroviral therapy to curb the growth of the HIV epidemic.</a> Lancet. 368, 531-536 (2006).</li><li>Granich, R. M., Gilks, C. F., Dye, C., De Cock, K. M., Williams, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+voluntary+HIV+testing+with+immediate+antiretroviral+therapy+as+a+strategy+for+elimination+of+HIV+transmission:+a+mathematical+model.">Universal voluntary HIV testing with immediate antiretroviral therapy as a strategy for elimination of HIV transmission: a mathematical model.</a> Lancet. 373, 48-57 (2009).</li><li>Johnson, L. F., 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=Life+expectancies+of+South+african+adults+starting+antiretroviral+treatment:+collaborative+analysis+of+cohort+studies.">Life expectancies of South african adults starting antiretroviral treatment: collaborative analysis of cohort studies.</a> PLoS Med. 10, (2013).</li><li>Blower, S., Ma, L., Farmer, P., Koenig, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+the+impact+of+antiretrovirals+in+resource-poor+settings:+preventing+HIV+infections+whilst+controlling+drug+resistance.">Predicting the impact of antiretrovirals in resource-poor settings: preventing HIV infections whilst controlling drug resistance.</a> Curr. Drug Targets. 3, 345-353 (2003).</li><li>Geretti, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+antiretroviral+drug+resistance+in+drug-naive+persons.">Epidemiology of antiretroviral drug resistance in drug-naive persons.</a> Curr. Opin. Infect. Dis. 20, 22-32 (2007).</li><li>Larder, B. A., Darby, G., Richman, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV+with+reduced+sensitivity+to+zidovudine+(AZT)+isolated+during+prolonged+therapy.">HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy.</a> Science. 243, 1731-1734 (1989).</li><li>Erice, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brief+report:+primary+infection+with+zidovudine-resistant+human+immunodeficiency+virus+type+1.">Brief report: primary infection with zidovudine-resistant human immunodeficiency virus type 1.</a> N. Engl. J. Med. 328, 1163-1165 (1993).</li><li>Williams, I., 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=British+HIV+Association+guidelines+for+the+treatment+of+HIV-1-positive+adults+with+antiretroviral+therapy.">British HIV Association guidelines for the treatment of HIV-1-positive adults with antiretroviral therapy.</a> HIV Med. 13 Suppl 2, 1-85 (2012).</li><li>DHHS, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> US Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. , (2012).</li><li>Vandamme, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+recommendations+for+the+clinical+use+of+HIV+drug+resistance+testing:+2011+update.">European recommendations for the clinical use of HIV drug resistance testing: 2011 update.</a> AIDS Rev. 13, 77-108 (2011).</li><li>DHHS, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> US Panel on Antiretroviral Therapy and Medical Management of HIV-Infected Children. Guidelines for the Use of Antiretroviral Agents in Pediatric HIV Infection. , (2012).</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=Department+of+Health.">Department of Health.</a> Clinical guidelines for the management of HIV &amp; AIDS in adults and adolescents. , (2010).</li><li>Conradie, F., 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+2012+southern+African+ARV+drug+resistance+testing+guidelines.">The 2012 southern African ARV drug resistance testing guidelines.</a> S. Afr. J.HIV Med. 13, 162-167 (2012).</li><li>Manasa, J., 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=Primary+Drug+Resistance+in+South+Africa:+Data+from+10+Years+of+Surveys.">Primary Drug Resistance in South Africa: Data from 10 Years of Surveys.</a> AIDS Res. Hum. Retroviruses. 28, 558-565 (2012).</li><li>Tshabalala, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveillance+of+transmitted+antiretroviral+drug+resistance+among+HIV-1+infected+women+attending+antenatal+clinics+in+Chitungwiza,+Zimbabwe.">Surveillance of transmitted antiretroviral drug resistance among HIV-1 infected women attending antenatal clinics in Chitungwiza, Zimbabwe.</a> PLoS ONE. 6, (2011).</li><li>Bartolo, I., 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=Antiretroviral+drug+resistance+surveillance+among+treatment-naive+human+immunodeficiency+virus+type+1-infected+individuals+in+Angola:+evidence+for+low+level+of+transmitted+drug+resistance.">Antiretroviral drug resistance surveillance among treatment-naive human immunodeficiency virus type 1-infected individuals in Angola: evidence for low level of transmitted drug resistance.</a> Antimicrob. Agents Chemother. 53, 3156-3158 (2009).</li><li>Bartolo, I., 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=HIV-1+genetic+diversity+and+transmitted+drug+resistance+in+health+care+settings+in+Maputo,+Mozambique.">HIV-1 genetic diversity and transmitted drug resistance in health care settings in Maputo, Mozambique.</a> J. Acquir. Immune Defic. Syndr. 51, 323-331 (2009).</li><li>Hamers, R. 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=HIV-1+drug+resistance+in+antiretroviral-naive+individuals+in+sub-Saharan+Africa+after+rollout+of+antiretroviral+therapy:+a+multicentre+observational+study.">HIV-1 drug resistance in antiretroviral-naive individuals in sub-Saharan Africa after rollout of antiretroviral therapy: a multicentre observational study.</a> Lancet Infect. Dis. 11, 750-759 (2011).</li><li>Hamers, R. 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=HIV-1+Drug+Resistance+Mutations+Are+Present+in+Six+Percent+of+Persons+Initiating+Antiretroviral+Therapy+in+Lusaka,+Zambia.">HIV-1 Drug Resistance Mutations Are Present in Six Percent of Persons Initiating Antiretroviral Therapy in Lusaka, Zambia.</a> J. Acquir. Immune Defic. Syndr. , (2010).</li><li>Nwobegahay, J., Selabe, G., Ndjeka, N. O., Manhaeve, C., Bessong, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+prevalence+of+transmitted+genetic+drug+resistance+in+a+cohort+of+HIV+infected+naive+patients+entering+antiretroviral+treatment+programs+at+two+sites+in+northern+South+Africa.">Low prevalence of transmitted genetic drug resistance in a cohort of HIV infected naive patients entering antiretroviral treatment programs at two sites in northern South Africa.</a> J. Med. Virol. 84, 1839-1843 (2012).</li><li>Iweriebor, B. C., 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=Molecular+epidemiology+of+HIV+in+two+highly+endemic+areas+of+northeastern+South+Africa.">Molecular epidemiology of HIV in two highly endemic areas of northeastern South Africa.</a> Arch. Virol. 157, 455-465 (2012).</li><li>Parboosing, R., Naidoo, A., Gordon, M., Taylor, M., Vella, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+antiretroviral+drugs+in+newly+diagnosed,+young+treatment-naive+HIV-positive+pregnant+women+in+the+province+of+KwaZulu-Natal+South+Africa.">Resistance to antiretroviral drugs in newly diagnosed, young treatment-naive HIV-positive pregnant women in the province of KwaZulu-Natal South Africa.</a> J. Med. Virol. 83, 1508-1513 (2011).</li><li>Nwobegahay, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+of+antiretroviral+drug+resistance+mutations+and+HIV-I+subtypes+among+newly-diagnosed+drug-naive+persons+visiting+a+voluntary+testing+and+counselling+centre+in+northeastern+South+Africa.">Prevalence of antiretroviral drug resistance mutations and HIV-I subtypes among newly-diagnosed drug-naive persons visiting a voluntary testing and counselling centre in northeastern South Africa.</a> J. Health Popul. Nutr. 29, 303-309 (2011).</li><li>Nwobegahay, J., 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=Prevalence+of+drug-resistant+mutations+in+newly+diagnosed+drug-naive+HIV-1-infected+individuals+in+a+treatment+site+in+the+waterberg+district,+limpopo+province).">Prevalence of drug-resistant mutations in newly diagnosed drug-naive HIV-1-infected individuals in a treatment site in the waterberg district, limpopo province).</a> S. Afr. Med. J. 101 (2011), 335-337 (2011).</li><li>Rosen, S., Long, L., Sanne, I., Stevens, W. S., Fox, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+net+cost+of+incorporating+resistance+testing+into+HIV/AIDS+treatment+in+South+Africa:+a+Markov+model+with+primary+data.">The net cost of incorporating resistance testing into HIV/AIDS treatment in South Africa: a Markov model with primary data.</a> J. Int. AIDS Soc. 14, 24 (2011).</li><li>Dalai, S. C., 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=Evolution+and+molecular+epidemiology+of+subtype+C+HIV-1+in+Zimbabwe.">Evolution and molecular epidemiology of subtype C HIV-1 in Zimbabwe.</a> AIDS. 23, 2523-2532 (2009).</li><li>Chen, J. H., 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=In-house+human+immunodeficiency+virus-1+genotype+resistance+testing+to+determine+highly+active+antiretroviral+therapy+resistance+mutations+in+Hong+Kong.">In-house human immunodeficiency virus-1 genotype resistance testing to determine highly active antiretroviral therapy resistance mutations in Hong Kong.</a> Hong Kong Med. 18, 20-24 (2012).</li><li>Lee, C. 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=An+in-house+HIV+genotyping+assay+for+the+detection+of+drug+resistance+mutations+in+Southeast+Asian+patients+infected+with+HIV-1.">An in-house HIV genotyping assay for the detection of drug resistance mutations in Southeast Asian patients infected with HIV-1.</a> J. Med. Virol. 84, 394-401 (2012).</li><li>Aitken, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pragmatic+Approach+to+HIV-1+Drug+Resistance+Determination+in+Resource-Limited+Settings+by+Use+of+a+Novel+Genotyping+Assay+Targeting+the+Reverse+Transcriptase-Encoding+Region+Only.">A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only.</a> J. Clin. Microbiol. 51, 1757-1761 (2013).</li><li>Zhou, Z., 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=Optimization+of+a+low+cost+and+broadly+sensitive+genotyping+assay+for+HIV-1+drug+resistance+surveillance+and+monitoring+in+resource-limited+settings.">Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings.</a> PLoS One. 6, (2011).</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=Life+Technologies.">Life Technologies.</a> Life Technologies and SATuRN Collaborate to Increase Access to HIV Testing in Africa. , (2012).</li><li>Lihana, R. W., Ssemwanga, D., Abimiku, A., Ndembi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+HIV-1+diversity+in+Africa:+a+decade+in+review.">Update on HIV-1 diversity in Africa: a decade in review.</a> AIDS Rev. 14, 83-100 (2012).</li></ol>

<div>加强卫生系统和艾滋病病毒治疗失败（HIV-：这项工作是由威康信托基金会（082384/Z/07/Z），欧盟（2007年147-790 SANTE），美国中心疾病支持通过CAPRISA（项目名称控制TFC）），以及瑞士南非联合研究计划（SSJRP）有权授予研究“瑞士普罗特/南非：蛋白质生物信息资源开发的重要的健康相关的病原体”。RL是支持的威康信托基金（批准号090999 / Z / 09 / Z轴）的资助者在研究设计，数据收集和分析，发布的决定，或准备稿子没有作用。作者宣称没有竞争的财务权益。 </div>

几个低成本的内部方法已经在努力，力图使艾滋病病毒耐药性基因分型更实惠33，34，36被描述。有需要对药物的抗药性测试融入护理的连续性就抗逆转录病毒疗法在资源有限的环境个人毫无疑问的。然而，大多数报道的方法集中于药物抗性基因分型中的耐药性在群体水平的监视应用程序。土星/ Life Technologies公司的基因分型方法是监测和耐药性监测一个完全集成的协议。这种方法被设计成一个负担得起的协议落实耐药性，为临床管理报告和生成的解释大多是开源和开放获取生物信息资源。 它是通过与FDA批准的Viroseq基因分型方法是比较示在确定由ANRS能力验证样品的面板，在面板的实验室样品已成功扩增100％耐药突变准确。精度也评估对C亚型病毒，在南部非洲最主要的亚型的临床标本。该方法是准确的C亚型的样品，因为它是对亚型A和B，但是，如果该方法将在世界上CRF02_AG是普遍存在的其它部分中使用，有必要对引物，因为该方法的修改未能扩增被证明具有CRF02_AG面板样品之一。可替换地，简并引物组对所有M组病毒33敏感，36可以使用在区域，进行亚型分布更加异构38。 反转录和PCR的灵敏度可提高从更高容量的血浆，如500毫升提取的RNA。等离子体可以centrifuged在21000×g离心90分钟，在进行所描述的那样的QIAamp病毒RNA提取试剂盒小型的协议之前以浓缩的病毒颗粒。 如图所示，该新方法具有它产生对个体患者的管理的综合报告一个附加的优点。这些报告是一个整合的基因型，免疫学和病毒学监测数据以及临床和治疗史从RegaDB。这是伴随着电阻档的详细解释化验后病人的临床病史同样详细的审查，以及治疗建议。使用一个专科医师审阅报告及患者提供治疗建议提供了急需的导师为执业护士以及临床医生经验不足，谁正越来越多地提供抗逆转录南非作为对任务转变世界卫生组织建议的一部分。这些临床报告已经证明是有效的教具为临床医生提供在药物抗性管理很少或没有经验。从患者的角度来看，我们的方法减少了前往中央网站访问专科艾滋病毒服务。 因此，作为一个整体所描述的协议提供了一个很好的平台，让艾滋病病毒耐药性的管理可以整合，以低廉的成本，为照顾爱滋病病毒感染人士未能艺术的统一体。可用于流行病学目的所产生的数据，以评估药物抗性，在社会的发展和传播。产生的POL片段的大小是比较复杂的系统发育分析，将产生更好的了解疫情在居群水平不够好。

艾滋病疫情在南部非洲已迅速发展1与抗逆转录病毒疗法（ART）的个人伴随指数增长，尤其是在南非的2，3。作为大型的治疗方案在降低发病率4，增加在资源有限的环境预期寿命（RLS）5流行病学影响的证据不断积累，努力提高技术覆盖面将愈演愈烈。指引对检测和治疗方案下使用的治疗作为预防工具6，7的演变意味着在处理个人的绝对数量将进一步增加。大量个人的将是艺术的更长的时间作为艺术个体的平均寿命接近，艾滋病毒感染人群8。艾滋病病毒耐药性的发展和传播具有ALWAYS被认为是艺术9-12所取得的成就构成了威胁。因此，有必要更严格监视和监测药物抗性作为多的人正在发起到ART。 基因型耐药性检测（GRT）已成功地应用在发达国家，无论是监视，以及在接受ART个人监测的HIV-1耐药性。在这些情况下，GRT已经融入护理对HIV-1感染者的统一体。大多数国际指南推荐GRT成人或儿童患者未能艺术（一线和二线）13-15，儿童患者暴露预防母亲向婴儿传播（PMTCT）方案，但后来感染16，并在与设置高级别之间急性感染者13-15传播耐药性。然而，成本，技术和基础设施的要求也限制了implementation的类似的方法在RLS的耐药性监测。 南非艾滋病毒治疗和监测指南不建议目前在艺术指导选择使用GRT的个人失败的一线药物17。个人主要基于病毒学（HIV-1 RNA病毒负载）参数切换。然而，在2012年，南部非洲艾滋病临床医师学会出版了第一本南部非洲抗逆转录病毒药物耐药性检测的准则18。这些指南推荐所有成人未能一线和二线抗逆转录和感染的婴儿和儿童接触到预防艾滋病母婴传播18 GRT测试。然而，GRT不建议用于18急性感染的个体，因为没有证据显示目前在南部非洲19-29高水平的传播耐药性。预计其中一些建议将被整合随着时间的推移进入全国特雷各个国家在该地区的atment和监控指引。目前，在2013年南非治疗指南，现在有GRT的在成人二线故障时间，并在为儿童30一线或二线PI为基础的治疗失败时间的建议。 它已经表明，整合GRT到南非治疗指南的话会有潜在的成本无关。考虑到第二线疗法的药物相对比一线药物更加昂贵的成本，使用粒度，以确定病人谁真正需要切换到第二线治疗不会造成任何额外费用给程序。此外，GRT还可以找出其他原因失效，节约治疗方案，并产生对新兴的耐药模式31的信息。因此，有必要减少甚至进一步，以提高访问，护理的质量耐药性监测方法的成本ð结果。 在这里，我们提出了一个GRT方法设计为使用泛型（开放源码）引物进行反转录聚合酶链反应（PCR）和测序（ 表1），以及用于耐药性的解释大多是开源软件。临床管理，该协议是由一个全面的检讨和报告的方法与实验耐药性报告，严格遵循国家治疗指南的专家解释称赞。该协议被分成四个不同的部件：1）HIV核糖核酸（RNA）提取，2）反转录聚合酶链反应（PCR）扩增的病毒目标，3）测序和4）的生物信息学方法，色谱图中，对应的分析，策展和序列数据的解释。

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			<td style="width:372px;">Biomatters</td>
			<td style="width:119px;"></td>
			<td>Sequence analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;"></td>
			<td style="width:372px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Internet Access</td>
			<td style="width:372px;"></td>
			<td style="width:119px;"></td>
			<td>Preferrably high speed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;"></td>
			<td style="width:372px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">Web resources</td>
			<td style="width:372px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">hivdb.stanford.edu</td>
			<td style="width:372px;">Stanford University</td>
			<td style="width:119px;"></td>
			<td>Drug reistance analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">http://bioafrica.mrc.ac.za:8080/regadb-ui/RegaDB</td>
			<td style="width:372px;">SATuRN</td>
			<td style="width:119px;"></td>
			<td>database</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:372px;">http://bioafrica.mrc.ac.za/tools/pppweb.html</td>
			<td style="width:372px;">SATuRN</td>
			<td style="width:119px;"></td>
			<td>Sequence quality tool</td>
		</tr>
	</tbody>
</table>

an affordable hiv-1 drug resistance monitoring method for resource limited settings

HIV-1耐药性有可能会严重危及抗逆转录病毒疗法（ART）的有效性和影响。在撒哈拉以南非洲艺术活动继续扩大，对艺术的个人应密切关注耐药性的出现监控。传播耐药性跟踪病毒株，以艺术已经耐药的传播监测也很关键。不幸的是，耐药性测试仍然没有在资源有限的设置容易获得，因为基因分型是昂贵的，需要先进的实验室和数据管理基础设施。开放获取基因型耐药性的监测方法来管理个人和评估传播耐药性描述。该方法使用药物的耐药模式的解释和个别病人报告的生成免费的开源软件。基因分型协议具有大于95％的用于与AV血浆样品的扩增率iral负荷&gt; 1,000 HIV-1 RNA拷贝/毫升。敏感度显著的病毒载量&lt;1,000 HIV-1 RNA拷贝/毫升下降。这里描述的方法验证对测试经美国食品和药物管理局（FDA）的Viroseq基因分型方法的HIV-1耐药性的方法。这里描述的方法的局限性包括的事实，它不是自动的，并且它也未能扩增来自样品的验证面板的循环重组形式CRF02_AG，虽然它扩增亚型A和B在相同的面板。

验证的方法是与先前报导的方法20的变形例。该Viroseq基因分型方法，这已被FDA批准，作为在验证的参考方法。从用于研究艾滋病和病毒性肝炎（ANRS）法国国家机构获得能力验证样品的面板中使用了两种方法之间的主要比较。这两种基因分型方法是100％一致确定所有重要的临床药物耐药相关突变的诠释HIVDB方案已成功通过两种方法扩增的样品。如表6所示，三对的核苷酸序列分别为99.5％相同。预测的氨基酸序列分别为100％的同一性。一个样本出来五个不可能由Viroseq被成功扩增。除了未通过Viroseq扩增样品中，在内部方法未能扩增将其所示的第二样品通过Viroseq是一个循环的重组病毒（CRF02_AG）。三个样品，与这两个方法分别扩增B亚型（两个样本）和A亚型（一个样本）。 <img alt="图5" fo:content-width="4in" fo:src="/files/ftp_upload/51242/51242fig5highres.jpg" src="/files/ftp_upload/51242/51242fig5.jpg" /> 图5。使用完成作为序列的质量保证一部分HKY邻接树的。有序列很短的遗传距离4双/集群。 RES655和RES655_1之间的遗传距离（同一样品测序在不同的日子）为0.003。的是与RES637_1/RES638对一个潜在误差作为其遗传距离太短（0.075），用于从不同的流行病学无关联的个体的样品。有在树上的另一个RES637用时相比，RES638_1的距离为0.075。该CQ01/CQ02簇表明两种样品从面板是相同样品的重复。他们与B亚型参考序列，确认由REGA分型工具分配的亚型一起集群。 CQ05和CQ04集群与亚型A和G分别，而雷加分型工具分别分类为A和CRF02_AG。对于HIV亚型和重组的另一个有用的工具是SCUEL，这是可在http://www.datamonkey.org。 <a href="https://www.jove.com/files/ftp_upload/51242/51242fig5highres.jpg" target="_blank">点击这里查看大图。</a> 五个样品的面板是用来评估对内部方法的精度。生成各五个样品的十个复制基因型。使用16毛细管3130xl基因分析仪，分别来自24个运行产生48 50基因型，准备在同一天。对所有五个样本，预测的氨基酸序列是100％一致之间重复。对于核酸序列，第ERE的是&gt; 99％的成对相似性。 在最初的两年使用这种方法中，重复60样本随机RNA提取测序。有序列的质量分数和的复制品之间的混合碱基数差异无统计学显著差异。为60双两个核苷酸和氨基酸成对比较均大于99％相同。因此，耐药性突变为所有对分别为100％一致。 降低成本 反应体积为RT，PCR和测序分别减少了至少一半，相对于原来的方法20，32，在不影响所产生的序列的质量。这使在50％成本的RT和PCR阶段的降低。 这种新方法的最初目的是与六测序引物序列来工作<html>全部99个密码子的蛋白酶基因和前300个密码子的逆转录酶基因20，32的。类似的方法也可以使用6至8的引物33和34。一些最近发表的方法都用不到半年的引物，虽然有时测序蛋白酶和逆转录基因seprately 35，36。我们寻求减少的测序引物的数量从6到4，（ 图6） <img alt="图6" fo:content-width="5in" fo:src="/files/ftp_upload/51242/51242fig6highres.jpg" src="/files/ftp_upload/51242/51242fig6.jpg" /> 图6。从6比4的测序引物为1197 bp的pol序列可以覆盖所有99个HIV-1蛋白酶的密码子和第一300个密码子的逆转录酶基因的产生连续的序列的比较。242/51242fig6highres.jpg“目标=”_blank“&gt;点击这里查看大图。 从一组从六个引物产生的17个样品的序列进行了比较，排除两个引物（MAW46和RTY）后产生的序列。该亚型分别为14 C亚型，二B亚型和一个亚型A.有顺序的质量分数无显著差异。再次，在17对核酸之间的平均成对身份是在氨基酸​​水平为99％和100％。从而，降低了测序引物从6到4导致在测序成本降低了近三分之一。 在这个协议中使用的唯一的专有软件工具是Geneious为序列组装。耐药性的解释工具，以及报告生成工具都是免费的，开放的工具。这通过消除与使用专有软件的成本进一步降低成本。此外，collectivË谈判所允许的试剂，该协议被打包成自Life Technologies方便地访问一个工具包，可作为土星/生命技术基因分型方法37。此外，土星的会员可以以折扣价获得的试剂。 临床上 所描述的协议已在监测和耐药性监测在一个农村社区的夸祖鲁 - 纳塔尔省已经实施。共有604基因型从临床标本中产生的2010年12月2013年5月之间在95％与病毒载量&gt; 1,000 RNA拷贝/毫升样品的放大率。本临床艾滋病病毒耐药性研究经夸祖鲁 - 纳塔尔大学（参考BF052/10）的生物医学研究伦理委员会和卫生部的夸祖鲁 - 纳塔尔省部（参考HRKM十分之一百七十六）的健康研究委员会。生成并发送回诊所个别病人报告对病人的管理。 七十二（72）的基因型也产生了作为传播耐药性研究的监督的一部分，嵌套在一个大型的前瞻性人群为基础的HIV监测研究中。主要的样品收集在EDTA微管针挑破全血。在基因分型有79％的19放大率。从夸祖鲁 - 纳塔尔省生物医学研究伦理委员会的大学（参考BE066107）获得伦理委员会批准为样本，​​从监测研究的基因分型。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>引物名称</td><td>序列</td><td>长度</td><td>方向</td><td> HXB2位置</td><td></td></tr><tr><td> MAW-26 </td><td> TTGGAAATGTGGAAA GGAAGGAC </td><td> 23 </td><td>向前</td><td> 2028年至2050年</td><td>第一轮PCR </td></tr><tr><td> RT-21 </td><td> CTGTATTTCAGCTATC AAGTCCTTTGATGGG </td><td> 31 </td><td>反向</td><td> 3539-3509 </td><td>第一轮PCR </td></tr><tr><td>亲1 </td><td> TAGAGCCAACAGCCC CACCA </td><td> 20 </td><td>向前</td><td> 2147年至2166年</td><td>第二轮PCR </td></tr><tr><td> RT-20 </td><td> CTGCCAATTCTAATTC TGCTTC </td><td> 22 </td><td>反向</td><td> 3462-3441 </td><td>第二轮PCR </td></tr><tr><td> RTC1F </td><td> ACCTACACCTGTCAA CATAATTG </td><td> 23 </td><td>向前</td><td> 2486年至2508年</td><td>测序</td></tr><tr><td> RTC2R </td><td> TGTCAATGGCCATTG TTTAACCTTTGG </td><td> 27 </td><td>反向</td><td> 2630至2604年</td><td>测序</td></tr><tr><td> RTC3F </td><td> CACCAGGGATTAGAT ATCAATATAATGTGC </td><td> 30 </td><td>向前</td><td> 2956年至2994年</td><td>测序</td></tr><tr><td> RTC4R </td><td> CTAAATCAGATCCTAC ATACAAGTCATCC </td><td> 29 </td><td>反向</td><td> 3129-3101 </td><td>测序</td></tr><tr><td> RT-Y </td><td> GTGTCTCATTGTTTAT ACTAGG </td><td> 22 </td><td>反向</td><td> 2967年至2946年</td><td>测序</td></tr><tr><td> MAW-46 </td><td> TCCCTCAGATCACTC TTTGGCAACGAC </td><td> 27 </td><td>向前</td><td> 2251年至2277年</td><td>测序</td></tr></tbody></table> 表1中。逆转录，PCR，和在一个1197 bp的片段聚合酶覆盖所有99 HIV-1蛋白酶的密码子和第一300个密码子的反向trascriptase基因的产生用于测序的引物的自定义。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> RT21（5pmol/ml） </td><td> 0.5 </td><td> 0.2 </td></tr><tr><td>的dNTP（10mM的） </td><td> 0.5 </td><td> 0.4 </td></tr><tr><td>总</td><td> 1 </td><td></td></tr></tbody></table> 表2。的dNTP /底漆混合的逆转录反应。 <table border=“0”的cellpadding =“0”CELLSPACING =“0”&gt; <tbody><tr><td>试剂</td><td>体积（ml）/反应</td><td>浓度/反应</td></tr><tr><td>第一链缓冲液（10X） </td><td> 1 </td><td> 1 </td></tr><tr><td>的MgCl 2（25毫摩） </td><td> 2 </td><td> 4 </td></tr><tr><td> DTT（0.1M） </td><td> 1 </td><td> 0.008 </td></tr><tr><td> RNaseOUT（40单位/毫升） </td><td> 0.5 </td><td> 16 </td></tr><tr><td>的Superscript III反转录酶（200U/ml组） </td><td> 0.5 </td><td> 8 </td></tr><tr><td>总</td><td> 5 </td><td></td></tr></tbody></table> 表3。酶混合进行逆转录反应。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>试剂</td><td>体积（ml）/反应</td><td>最终浓度/反应</td></tr><tr><td> DEPC处理水</td><td> 18.4 </td><td> - &lt;/ TD&gt; </tr><tr><td> PCR缓冲液（10倍） </td><td> 2.5 </td><td> 1 </td></tr><tr><td> MGCI 2（50毫米） </td><td> 1 </td><td> 2 </td></tr><tr><td> dNTP混合物中（10mM） </td><td> 0.5 </td><td> 0.2 </td></tr><tr><td>刚愎底漆（5皮摩尔/毫升） </td><td> 0.25 </td><td> 0.05 </td></tr><tr><td>反向引物（5皮摩尔/ ml）的</td><td> 0.25 </td><td> 0.05 </td></tr><tr><td>白金Taq酶（5 U / ml的） </td><td> 0.1 </td><td> 0.02 </td></tr><tr><td>小计</td><td> 23 </td><td> - </td></tr></tbody></table> 表4。主结构的巢式PCR。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>试剂</td><td>体积（ml）/反应</td><td>浓度/反应</td></tr><tr><td> DEPC处理水</td><td> 6.1 </td><td></td></tr><tr><td>测序缓冲液（5X） </td><td> 2 </td><td> 1 </td></tr><tr><td>底漆（3.2皮摩尔/毫升） </td><td> 0.5 </td><td> 0.16 </td></tr><tr><td> Big Dye终止测序混</td><td> 0.4 </td><td> - </td></tr><tr><td>总</td><td> 9 </td><td></td></tr></tbody></table> 表5。主结构的测序反应。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td align="center" colspan="4"> Viroseq </td><td align="center" colspan="4">室内</td><td rowspan="2"> ％NA相似</td></tr><tr><td>样品编号</td><td>亚型</td><td>质量得分</td><td>公关突变</td><td> RT突变</td><td>亚型</td><td>质量得分</td><td>公关突变</td><td> RT突变</td></tr><tr><td> CQ01 </td><td>乙</td><td> 99.9 </td><td> M46L，I54L，V82A，L90M </td><td> D67N，T69D，K70R，M184V，T215V，K219Q </td><td>乙</td><td> 99.2 </td><td> M46L，I54L，V82A，L90M </td><td> D67N，T69D，K70R，M184V，T215V，K219Q </td><td> 100 </td></tr><tr><td> CQ02 </td><td>乙</td><td> 99.5 </td><td> M46L，I54L，V82A，L90M </td><td> D67N，T69D，K70R，M184V，T215V，K219Q </td><td>乙</td><td> 99.5 </td><td> M46L，I54L，V82A，L90M </td><td> D67N，T69D，K70R，M184V，T215V，K219Q </td><td> 100 </td></tr><tr><td> CQ03 </td><td>不适用</td><td>不适用</td><td></td><td></td><td>不适用</td><td>不适用</td><td></td><td>不适用</td><td>不适用</td></tr><tr><td> CQ04 </td><td> CRF02_AG </td><td> 98.4 </td><td> I54V，V82F，I84V </td><td> M41L，L74I，L210W，T215Y，V108I，Y181C </td><td>不适用</td><td>不适用</td><td>不适用</td><td>不适用</td><td>不适用</td></tr><tr><td> CQ05 </td><td>一</td><td> 99.7 </td><td></td><td> K103N </td><td>一</td><td> 93 </td><td></td><td> K103N </td><td> 100 </td></tr></tbody></table> 表6。比较雷苏尔TS从Viroseq基因分型方法，并在内部使用的样品由ANRS提供一个面板的方法之间的平行分析。

Watch this Scientific Journal Video about An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings at JoVE.com

An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

耐药性检测为HIV-1感染的个体没有抗逆转录病毒疗法（ART）可以指导今后的治疗，提高治疗效果。在艾滋病高流行，但资源有限的环境优化个人和人群健康状况最终将需要负担得起的和可获得的耐药基因分型和解释方法。

对于资源有限设置一个负担得起的HIV-1耐药性监测方法

HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in ...

an-affordable-hiv-1-drug-resistance-monitoring-method-for-resource

Research

JoVE Journal

Medicine

18.4K 次观看.  University of KwaZulu-Natal, Durban, South Africa. 耐药性检测为HIV-1感染的个体没有抗逆转录病毒疗法（ART）可以指导今后的治疗，提高治疗效果。在艾滋病高流行，但资源有限的环境优化个人和人群健康状况最终将需要负担得起的和可获得的耐药基因分型和解释方法。 

视频: An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings

Method for Novel Anti-Cancer Drug Development using Tumor Explants of Surgical Specimens

The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined by current drug efficacy tests and the efficacy on patients. Thus, novel and reliable methods for evaluating drug efficacy are warranted in pre-clinical phase. In vitro culture of tumor tissues, including cell lines, has substantial phenotypic, genetic, and epigenetic alterations of cancer cells caused by artificial environment of cell culture, which may not reflect the biology of original tumors in situ. Xenograft models with the immunodeficient mice also have limitations, i.e., the lack of immune system and interspecies genetic and epigenetic discrepancies in microenvironment. Here, we demonstrate a novel method using the surgical specimens of malignant glioma as undissociated tumor blocks to evaluate treatment effects. To validate this method, data with the current first-line chemotherapeutic agent, temozolomide (TMZ), are described.


We used the freshly-removed surgical specimen of malignant glioma for our experiments. We performed intratumoral injection of TMZ or other drug candidates, followed by incubation and analysis on surgical specimens. Here, we sought to establish a tumor tissue explant method as a platform to determine the efficacy of novel anti-cancer therapies so that we may be able to overcome, at least, some of the current limitations and fill the existing gap between the current experimental data and the efficacy on an actual patient's tumor. This method may have the potential to accelerate identifying novel chemotherapeutic agents for solid cancer treatment.

Here, we established a method for drug efficacy testing with surgical specimens of brain tumors, termed &#x201C;tumor explant method&#x201D;. With this method, we can evaluate drug efficacy without breaking the microenvironment of solid tumors. To validate reliability of this method, we describe representative data with our glioma specimen treated with the current first-line chemotherapeutic agent, temozolomide.

新型抗癌药物使用手术标本肿瘤植的发展方法


The current therapies for malignant glioma have only palliative effect. For therapeutic development, one hurdle is the discrepancy of efficacy determined ...

科研

医学

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices currently entering the market. In order to evaluate the safety of these devices, in preclinical studies, reliable techniques are needed. However, the hard metal components utilised in some retinal implants are not compatible with traditional histological processes, particularly in consideration for the delicate nature of the surrounding tissue. Here we describe techniques for assessing the health of the eye directly adjacent to a retinal implant secured epiretinally with a metal tack.
Retinal prostheses feature electrode arrays in contact with eye tissue. The most commonly used location for implantation is the epiretinal location (posterior chamber of the eye), where the implant is secured to the retina with a metal tack that penetrates all the layers of the eye. Previous methods have not been able to assess the proximal ocular tissue with the tack in situ, due to the inability of traditional histological techniques to cut metal objects. Consequently, it has been difficult to assess localized damage, if present, caused by tack insertion.
Therefore, we developed a technique for visualizing the tissue around a retinal tack and implant. We have modified an established technique, used for processing and visualizing hard bony tissue around a cochlear implant, for the soft delicate tissues of the eye. We orientated and embedded the fixed eye tissue, including the implant and retinal tack, in epoxy resin, to stabilise and protect the structure of the sample. Embedded samples were then ground, polished, stained, and imaged under various magnifications at incremental depths through the sample. This technique allowed the reliable assessment of eye tissue integrity and cytoarchitecture adjacent to the metal tack.

Here we describe histological techniques for visualising ocular tissue directly adjacent to a metal epiretinal tack and retinal prosthesis.

为处理眼植入视网膜假体的局部病理组织学分析技术：第2部分视网膜前植入视网膜大头钉

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

实施<em&gt;体外</em&gt;耐药性检测：最大化的潜力，揭开临床有关的耐药机制

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

对小鼠淋巴瘤模型监测肿瘤进展和治疗效果生物发光基于肿瘤定量方法

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

在小鼠气管插管快速的改进方法

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

胰腺管输液：药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

经优化的 LC/ms 方法对囊性纤维化患者 Ivacaftor 及其主要代谢物和 Lumacaftor 的高通量分析

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

一种基于猪脑内皮细胞的体外血脑屏障模型的改进方法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...