This protocol development was supported by the Bill and Melinda Gates Foundation Grand Challenges in Global Health grant 37774. Real-time PCR reagents and advice were provided by Abbott Molecular Inc. Des Plaines, IL. 8E5-LAV cells were provided by the Virology Quality Assurance Laboratory, Rush Presbyterian; St. Luke's Medical Center. HIV-1 negative blood was provided by Core Lab, NorthShore University HealthSystems, Evanston, IL. Thanks to Mark Fisher for photography help.

伦理声明:在本研究中使用的全血标本不被认为是涉及人类受试者的研究。用于临床诊断目的，这是满足了获得的样本，并为FINA研究测定提供这些试样的剩余部分。试样进行编码，使得研究者不能够容易地确定个人的身份。 1.国际泳联样品制备模块的研制<ol><li>准备用切割35×35毫米2.6 平方毫米厚的100％化妆棉吸水垫。切标本每一个焊盘。 </li><li>制备塑料石蜡膜与纸背衬带通过切割片石蜡膜（25毫米（H）乘50毫米（宽）），然后冲压在用锤子驱动打孔器磁带的中心的7.14毫米直径的孔。准备每个试样一个磁带。 </li><li>冲切8.35毫米直径的圆准备开往玻璃血液分离膜（捕获膜）盘用锤子驱动打孔。冲床每个试样一个磁盘。该盘具有2.3微米的颗粒截留能力。 </li><li>通过将捕获磁盘中使用镊子的印迹垫的中心组装FINA样品制备模块。放置在捕获磁盘石蜡膜带，使得石蜡带的孔离开露出的捕获盘的中心。 </li><li>通过稍微拉伸石蜡胶带以覆盖印迹垫并按下石蜡胶带牢固地将其粘到周围的磁盘的所有边缘的印迹垫确保磁盘和印迹垫之间的最大接触。 </li><li>作为实验所需或储存以备将来使用使尽可能多的模块。 </li></ol> 2.定量PCR管的制备<ol><li>制备5.1毫米磁带磁盘，将细胞捕获膜锚定在定量PCR管的一侧通过冲压双涂聚合α-的圆，以防止膜堵塞荧光检测STER诊断胶带从胶带的片材锤驱动冲头。准备每个试样一个磁带磁盘。 </li><li>移除胶带的贴衬垫的一侧。磁带放置到200μl的PCR管，粘着其上的一侧和朝向管的底部。删除第二个贴纸衬垫。管是现在准备好接收准备磁盘。 </li><li>作为实验所需或储存以备将来使用产生尽可能多的管子。 </li></ol> 3.图谋血液标本注意:如果正在准备一个真正的测试样品，继续步骤4。 <ol><li>解冻8E5-LAV细胞窝藏15从病毒学质量保证实验室获得的HIV-1前病毒的一个副本（VQA;拉什长老/圣路加医疗中心，芝加哥，IL）。 注意:它们被存储为在4000个细胞/μl浓度的冷冻细胞沉淀。如前面9中所述细胞计数进行了验证。 </li><li>准备SER胶质稀释在冷冻培养基（90％胎牛血清，10％二甲亚砜），以浓度范围从1至400个细胞/微升。 </li><li>吸管10微升细胞从各系列稀释成离心管中。添加100μl的新鲜的HIV-1阴性EDTA处理的全血样品的每个管来创建8E5-LAV拷贝数10-4,000的一个标准曲线。通过轻弹管的底部5次混合。 </li></ol> 4.执行国际泳联提取<ol><li>裂解血细胞中加入的Triton-X100），以1％（11微升10％的Triton-X100至110微升全血和来自步骤3.3的细胞）的最终浓度。 </li><li>通过轻弹管的底部5次混合。血应该把半透明的红色。 </li><li>吸取所有的血样到捕获盘。 注:石蜡带和捕获膜之间的水密封确保了血液流经膜，和捕获微米之间良好的接触embrane和印迹垫组件可确保快速样品排汗。 </li><li>允许样品通过过滤器浸泡。 注意:血液溶胞产物灯芯入印迹垫由于毛细管作用，捕捉俘获磁盘的表面上的基因组DNA。当它出现雾的裂解物已浸透到磁盘。 </li><li> 600-1000微升10毫米氢氧化钠逐滴加入到采集磁盘。 注意:洗涤缓冲液还可以加入作为本体加入600微升。缓冲器将形成疏水性石蜡的磁带上的微滴和通过在大约20秒的孔到盘芯吸。该过滤器会从红色变为血红蛋白的白色显示的间隙。 </li><li>分开用钳子印迹垫盘和盘适用于制备PCR管的胶带。如果有任何红色留在过滤器中添加50-100微升洗涤缓冲液在管放置之前清除过滤器。 注意:不经常，编写过程中施加过大压力从印迹垫分离期间在膜中的层的分区的FINA模块的结果。丢弃这些磁盘，并准备一个新鲜的样本。 </li><li>该过滤器被放置在PCR管后，分析样品的时候了。可替代地，在一个包含框硫酸钙干燥剂干燥过夜，然后盖和地点与二氧化硅干燥剂和存储的箔袋，直到准备用于分析。 </li></ol> 5.定量PCR反应<ol><li>使用HIV特异性引物和探针如前面所述9,11每准备100反应的qPCR微升主结构。包括内部放大控制并监控扩增抑制剂的存在和验证一个负样品是真阴性。确保过滤器完全覆盖主混合物和该过滤器保持固定到管的侧面整个反应。 </li><li>使用实时PCR装置如前面所述9执行放大。 </li></ol>

<ol>
	<li>Byrnes, S. 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=One-step+purification+and+concentration+of+DNA+in+porous+membranes+for+point-of-care+applications.">One-step purification and concentration of DNA in porous membranes for point-of-care applications.</a> Lab Chip. 15 (12), 2647-2659 (2015).</li><li>Connelly, J. T., Rolland, J. P., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Paper+Machine"+for+Molecular+Diagnostics.">"Paper Machine" for Molecular Diagnostics.</a> Anal Chem. 87 (15), 7595-7601 (2015).</li><li>Govindarajan, A. V., Ramachandran, S., Vigil, G. D., Yager, P., Bohringer, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+low+cost+point-of-care+viscous+sample+preparation+device+for+molecular+diagnosis+in+the+developing+world;+an+example+of+microfluidic+origami.">A low cost point-of-care viscous sample preparation device for molecular diagnosis in the developing world; an example of microfluidic origami.</a> Lab Chip. 12 (1), 174-181 (2012).</li><li>Linnes, J. 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=Paper-based+molecular+diagnostic+for.">Paper-based molecular diagnostic for.</a> RSC Adv. 4 (80), 42245-42251 (2014).</li><li>Rodriguez, N. 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=Paper-Based+RNA+Extraction,+in+Situ+Isothermal+Amplification,+and+Lateral+Flow+Detection+for+Low-Cost,+Rapid+Diagnosis+of+Influenza+A+(H1N1)+from+Clinical+Specimens.">Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens.</a> Anal Chem. 87 (15), 7872-7879 (2015).</li><li>Peeling, R. W., Holmes, K. K., Mabey, D., Ronald, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+tests+for+sexually+transmitted+infections+(STIs):+the+way+forward.">Rapid tests for sexually transmitted infections (STIs): the way forward.</a> Sex Transm Infect. 82, 1-6 (2006).</li><li>Mabey, D., Peeling, R. W., Ustianowski, A., Perkins, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostics+for+the+developing+world.">Diagnostics for the developing world.</a> Nat Rev Microbiol. 2 (3), 231-240 (2004).</li><li>Jangam, S. R., Agarwal, A. K., Sur, K., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+point-of-care+PCR+test+for+HIV-1+detection+in+resource-limited+settings.">A point-of-care PCR test for HIV-1 detection in resource-limited settings.</a> Biosens Bioelectron. 42, 69-75 (2013).</li><li>Jangam, S. R., Yamada, D. H., McFall, S. M., Kelso, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+point-of-care+extraction+of+human+immunodeficiency+virus+type+1+proviral+DNA+from+whole+blood+for+detection+by+real-time+PCR.">Rapid point-of-care extraction of human immunodeficiency virus type 1 proviral DNA from whole blood for detection by real-time PCR.</a> J Clin Microbiol. 47 (8), 2363-2368 (2009).</li><li>Kelso, D. M., Jangam, S., Yamada, D., McFall, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (2012).</li><li>McFall, S. 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=A+Simple+and+Rapid+DNA+Extraction+Method+from+Whole+Blood+for+Highly+Sensitive+Detection+and+Quantitation+of+HIV-1+Proviral+DNA+by+Real-Time+PCR.">A Simple and Rapid DNA Extraction Method from Whole Blood for Highly Sensitive Detection and Quantitation of HIV-1 Proviral DNA by Real-Time PCR.</a> Journal of Virological Methods. 214, 37-42 (2015).</li><li>Boom, R., 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+and+simple+method+for+purification+of+nucleic+acids.">Rapid and simple method for purification of nucleic acids.</a> J Clin Microbiol. 28 (3), 495-503 (1990).</li><li>Radstrom, P., Knutsson, R., Wolffs, P., Lovenklev, M., Lofstrom, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-PCR+processing:+strategies+to+generate+PCR-compatible+samples.">Pre-PCR processing: strategies to generate PCR-compatible samples.</a> Mol Biotechnol. 26 (2), 133-146 (2004).</li><li>Schrader, C., Schielke, A., Ellerbroek, L., Johne, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR+inhibitors+-+occurrence,+properties+and+removal.">PCR inhibitors - occurrence, properties and removal.</a> J Appl Microbiol. 113 (5), 1014-1026 (2012).</li><li>Folks, T. M., Powell, D. M., Martin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Google Patents. , (1991).</li><li>Violari, 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=Early+Antiretroviral+Therapy+and+Mortality+among+HIV-Infected+Infants.">Early Antiretroviral Therapy and Mortality among HIV-Infected Infants.</a> N Engl J Med. 359 (21), 2233-2244 (2008).</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=.">.</a> World Health Organization. , (2010).</li><li>Gruner, N., Stambouli, O., Ross, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dried+blood+spots--preparing+and+processing+for+use+in+immunoassays+and+in+molecular+techniques.">Dried blood spots--preparing and processing for use in immunoassays and in molecular techniques.</a> J Vis Exp. (97), (2015).</li><li>Maiers, T. 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=An+investigation+of+fingerstick+blood+collection+for+point-of-care+HIV-1+viral+load+monitoring+in+South+Africa.">An investigation of fingerstick blood collection for point-of-care HIV-1 viral load monitoring in South Africa.</a> S Afr Med J. 105 (3), 228-231 (2015).</li></ol>

The authors have nothing to disclose.

EID联系到快速治疗的访问已被证明是降低婴儿死亡率由于HIV感染16。由于婴儿的血液母源抗体的持久性，快速的艾滋病病毒抗体测试已确定HIV暴露婴儿的状态用处有限。世界卫生组织建议，出生HIV-1阳性母亲的婴儿都应该在4-6周龄用病毒学测试17进行测试。我们已经报道HIV-1的原病毒DNA的全血标本中检测和定量，能够单拷贝检测与证实100％的敏感性和特异性的61的中国婴儿11的小字段的评价的测定法的发展。一百微升或更多的全血可以通过手指或脚跟棒被收集并经由FINA模块18,19进行处理。这些血液样品然后可以扩增使这种方法的ide之前存放至少一个月人测试是远离实验室的婴儿。 在图3中 ，人为标本从HIV-1的负的全血的标准曲线呈现的研究与解冻细胞携带的HIV-1前病毒DNA被描绘尖刺。一个警告这项研究的是，一些加入到样品中的细胞可能已裂解由于冷冻/解冻，这可能潜在地提高靶的检测水平。更严格的实验设计本来已经使用做作这将有更准确地模仿全血新鲜培养细胞标本。 国际泳联的样品制备方法是专为纳入一个EID POC的qPCR装置（开发中）在有限的资源设置8使用;然而，这里描述的手动格式也可以是一个有益的补充实验室的样品制备产品组合。国际泳联不使用绑定，洗净，洗脱在DRI使用策略ED血斑点等纸质提取系统的稀释提取的核酸，可能导致次优的检测灵敏度5。国际泳联系统非常灵活，可方便地适用于除原病毒HIV DNA检测的其他基因测试。的血液或其他生物样品降低体积可为丰富的目标和更高的产量被处理不太丰富的。在我们最初的FINA系统的实施方案中，我们捕获使用血液分离膜从全血中的细胞，然后通过加入10mM的氢氧化钠9的裂解它们。该版本具有少一个用户的步骤的优点，但是，可以被处理的细胞数/血液体积更小。除了本报告中所使用的结合的玻璃膜，其他血液分离或血液收集滤纸已成功地与该方法中使用，并且几种不同的定量PCR的仪器也已显示为放大嵌入的F模板ILTER盘9。唯一的要求是，过滤器不会阻止实时PCR仪的荧光读数。 FINA提取的显著限制是，没有对捕获膜的直径的尺寸约束。用膜大于9毫米的磁盘无法被放置在一个200微升的qPCR管没有在这限制暴露于反应混合物中的表面区域的管膜重叠。此外，盘的直径越大，则需要更多的qPCR反应体积以覆盖这增加了成本的过滤器和周转时间的测定。如果过滤器太小而有效地捕获在样品中的DNA，它可能阻塞预防有效洗涤。 使用结合的玻璃捕获膜，我们能够从全血样本只捕获HIV-1原病毒DNA，而不是病毒RNA（未示出数据）。我们不知道，如果替代的膜或缓冲剂可以用于吨ø促进RNA的隔离代替或除了的DNA。如果读者想只从试样分离DNA这个协议不需要酶促去除一些协议所需的RNA。在临床样品中检测两个HIV-1的RNA和DNA将改善EID的灵敏度和这种缺乏RNA检测的是我们的诊断测试的限制。 在适应的FINA过程到一个新的测定中最关键的步骤是验证过滤器的大小，以适应的DNA， 即，细胞的数目的量，在试样进行测试。一般FINA提取与细胞的更小的数字样本的效果最好，使更小的捕获磁盘都可以使用。确定过滤器的结合能力，只是第二滤波器磁盘添加到堆叠在收集膜下方的FINA模块。加样品和洗膜如步骤4.1-4.5。每个DNA捕获磁盘放入单独的定量PCR管和放lify。使用标准曲线，确定每个过滤器捕获输入DNA的百分比。过滤器的尺寸可以被优化到测定的需要。对于这里描述的FINA原病毒DNA测定中，当细胞裂解物添加到过滤器（未发表的观测）使优良的检测能力的人类基因组DNA的99％以上被捕获。 除了EID，FINA的未来的应用可以包括检测和前病毒DNA的定量，作为HIV-1的疾病监测的生物标记物与谁已经由于抗逆转录病毒疗法取得病毒抑制的患者。旨在消灭艾滋病毒治疗的监测可以通过除外周血的HIV-1感染细胞的水库筛选其他投入来执行。 HIV前病毒检测也可以用来筛查疫苗试验科目为真正的感染。将血样于FINA模块储存期间是稳定的，并且可以购买装运到CE字段的条件下被收集ntral实验室PCR分析做流行病学研究这种方法的理想。除了HIV-1的检测，这种高度灵活的方法也可用于扩增在生物样品中发现，如药物遗传学筛选或其它SNP检测或用于动物模型的快速基因分型精密药的目标。等温扩增的方法，如解旋酶依赖性扩增（HDA）或环介导等温扩增（LAMP）也可以与FINA用于提取模板或该DNA嵌入在过滤器上，可作为模板常规PCR扩增。

若干报告已经与使精美灵敏度和分子诊断的特异性可用于所有的目的讨论了点- -关心（POC）装置1-5的造纸或膜-基于提取方法中使用的开发。世界卫生组织（WHO）性传播疾病诊断倡议创造ASSURED（价格实惠，敏感，特异，用户界面​​友好，快速和强大，装备自由并交付给那些谁需要它）来描述理想的POC的特征词测试6。这些准则的免费装备，特点是特别具有挑战性，以实现分子诊断。然而，在该领域的每一次创新将推动实现这些最需要的目标，并有通过调整现有的技术7是希望短期内改善测试性能。 这里介绍一个简单的协议，用于从全血中提取DNA不需要复杂的化学或实验室仪器。国际泳联（核酸过滤隔离）的样品制备方法最初是提取全血白细胞DNA，以检测HIV-1前病毒定量PCR样品到答案点保健（POC）的一部分（定量PCR）婴儿早期诊断（EID）平台，在资源有限的设置8-11使用。 FINA提取不同于其中使用二氧化硅膜或氧化硅-包被的顺颗粒在离液剂12的存在下可逆地结合DNA常规纯化方法。相反，国际泳联采用立式过滤通过分离膜直接提取全血细胞DNA。含有包埋的DNA的膜可以直接在PCR管放置并且立即在购买扩增9干燥的PCR反应或空气中。没有离液剂，苯酚或醇是在样品提取使用，elimina婷去除提取过程13,14衍生有力的qPCR抑制剂需要大量洗涤步骤。 国际泳联膜可以捕获样品被添加到膜之前通过细胞裂解11解放或细胞9或基因组DNA。用于小区捕获，全血直接加入到该膜。细胞随后在膜中加入10毫摩尔的NaOH裂解。这种方法的优点是，它仅涉及3个步骤:1）加入样品; 2）细胞裂解/洗3）过滤盘放置在定量PCR管。这种方法的缺点是，该膜只能容纳一个定义单元成正比的膜盘的直径的数量。为了达到为EID所需的检测的限制，将100μl的全血需要这需要一个过滤器，过大，被放置在qPCR的管样品输入。裂解血细胞用洗涤剂添加样品到集合前膜增加了一个处理步骤，但允许对同一样本大小使用较小的滤波器。我们能够证明高再现性，单拷贝检测，并从100微升的血液使用这种测试配置11的HIV-1原病毒DNA的少至10个拷贝的定量。 在这份报告中，我们描述最初实验室使用开发的国际泳联协议。称为FINA样品制备模块的膜/滤膜夹层可以大批量制备，并储存起来以备后用。当试样被提取这个过程需要2分钟，并可以在不同大小的批次进行。在定量PCR可立即运行或包含嵌入DNA中的过滤器可以被存储直到它方便执行定量PCR。这种方法是用于在低和高资源设置标本常规分析非常方便。

<table><tbody><tr><td>Fusion 5</td><td>GE Healthcare Life Sciences</td><td>8151-9915</td><td></td></tr><tr><td>707 blotting pad</td><td>VWR International</td><td>28298-014</td><td></td></tr><tr><td>PARAFILM M</td><td>VWR International</td><td>52858-000</td><td></td></tr><tr><td>3M Double-Coated Polyester Diagnostic Tape&amp;nbsp;</td><td>3M Medical Specialties</td><td>9965</td><td></td></tr><tr><td>200 &amp;micro;l qPCR strip tubes</td><td>Agilent</td><td>401428</td><td></td></tr><tr><td>optical strip caps</td><td>Agilent</td><td>401425</td><td></td></tr><tr><td>Mx3005p qPCR System</td><td>Agilent</td><td>401456</td><td></td></tr><tr><td>sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>A.C.S. Reagent</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td>BioXtra</td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma-Aldrich</td><td>D8418</td><td>for molecular biology</td></tr><tr><td>fetal bovine serum, certified, U.S. origin</td><td>Thermo Fisher Scientific</td><td>16000-044</td><td></td></tr><tr><td>Hammer driven small hole punch 3/16&quot; hole diameter (5.1 mm)</td><td>McMaster Carr</td><td>3424A16</td><td></td></tr><tr><td>Hammer driven small hole punch 1/4&quot; hole diameter (7.14 mm)</td><td>McMaster Carr</td><td>3424A19</td><td></td></tr><tr><td>Hammer driven small hole punch 5/16&quot; hole diameter (8.35 mm)</td><td>McMaster Carr</td><td>3424A23</td><td></td></tr></tbody></table>

filtration isolation of nucleic acids:  a simple and rapid dna extraction method

FINA，核酸过滤分离，是通过分离膜和吸收垫利用垂直过滤提取从全血细胞的DNA，在不到2分钟的新颖的提取方法。血液检体与洗涤剂，短暂混合处理，并用移液管施加到分离膜。溶解物芯吸入印迹垫由于毛细管作用，捕捉分离膜的表面上的基因组DNA。期间的简单的洗涤步骤，其中PCR抑制剂是恶进入吸湿印迹垫提取的DNA被保留在膜上。然后将含有包埋的DNA的膜被加入到没有进一步纯化的PCR反应。这种简单的方法不需要实验室设备，并可以用廉价实验室用品很容易地实现。在这里，我们描述了从100微升全血中HIV-1前病毒DNA的高度敏感的检测和定量的协议作为一种模式在早期艾滋病毒的FANT诊断，可以容易地适用于其他遗传靶。

用于提取从8E5-LAV细胞掺入的全血前病毒DNA的工作流程示于图1， 图2示出了FINA示例模块和制备的qPCR管。这种方法允许从8E5-LAV细胞以不同拷贝数的HIV-1原病毒的有效扩增，如图做作标本（ 图3）的标准曲线。 PCR具有103％的效率，如从效率= 1 + 10（-1 /斜率）计算。线的方程为y = -3.25x + 27.95，与R 2 = 0.996的相关性。艾滋病毒原病毒DNA的标准曲线进行复制也有高度可重复性的扩增，如表1看出。另外，500份的内部对照羟基丙酮酸还原酶的存在是为了表明，有不存在PCR抑制从具有FINA提取血液所得的，独立的数目前HIV-1前病毒的副本（图4）。所有测试的18个样品中被有效地获得的IC扩增，具有21.92±0.25平均CQ和1％的CV值。 <img alt="图1" src="/files/ftp_upload/54289/54289fig1.jpg" /> 图1:工作流，用于检测从全血中病毒DNA掺入8E5-LAV细胞的qPCR。 （A）中从左至右，将制备的FINA模块，血液加入到捕获膜，并加入洗涤缓冲液后后。加入粘双面胶带的qPCR预混捕获磁盘（B）定量PCR管。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54289/54289fig2.jpg" /> 图2:FINA在内部模块和qPCR管制备。 </st荣&gt;（A）中的8.35毫米直径的捕获膜磁盘夹在正方形707印迹垫和包含在中心毫米直径7.14孔石蜡带的薄片之间，使得所述石蜡带的孔集中于通过吸管直接应用的裂解血液采集磁盘。 （B）5.1毫米双涂聚酯诊断胶带粘在200微升的qPCR管的一侧。带的第二衬垫以暴露粘物表面的准备时，将捕获磁盘。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54289/54289fig3.jpg" /> 图3:HIV-1前病毒DNA标本做作标准曲线从100微升4（A）得到扩增曲线复制HIV-1 NEgative全血飙升与4000，400，40和10 8E5-LAV细胞500拷贝/内部控制的实线=扩增曲线的反应;虚线=阈值。 Y轴是荧光单元和X轴是定量PCR循环数。 Cq的值（B）的标准曲线从扩增曲线对每100微升全血8E5-LAV细胞日志拷贝数来计算。该行的方程:Y = -3.25x + 27.95; R 2 = 0.996; PCR效率= 103.23％，通过计算效率= -1 + 10（-1 /斜率） <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig3large.jpg" target="_blank">点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54289/54289fig4.jpg" /> 图4: 内部控制指示无抑制的qPCR 500拷贝每反应加入羟基丙酮酸还原酶放大控制质粒。固体线=扩增曲线;虚线=阈值。 IC的平均Cq的= 21.92±0.25。 CQS介于21.21至22.32。变异（CV％）的系数为1％; N = 18， <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54289/54289fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table fo:keep-together.within-page="1"><tbody><tr><td colspan="4"> 8E5-LAV细胞/ </td></tr><tr><td> 100微升WB </td><td> 大道。 CQ </td><td> SD </td><td> 简历 （％） </td></tr><tr><td> 4000 </td><td> 16.27 </td><td> 0.14 </td><td> 1 </td></tr><tr><td> 400 </td><td> 19.54 </td><td> 0.15 </td><td> 1 </td></tr><tr><td> 40 </td><td> 22.56 </td><td> 0.3 </td><td> 1 </td></tr><tr><td> 10 </td><td> 24.83 </td><td> 0.15 </td><td> 1 </td></tr></tbody></table>表1: 平均CQ，标准差（SD）和变异系数（CV％）为4000，400，40，并与FINA提取和HIV-1特异性引物和8E5-LAV标准曲线的10人副本探头。N = 4，WB =全血。

Watch this Scientific Journal Video about Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method at JoVE.com

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

在这里，我们描述了一个简单，快速的基于纸张的DNA提取的HIV前病毒DNA的全血通过定量PCR检测方法。这种协议可以在检测其他遗传标记或使用替代的扩增方法被扩展为使用。

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

filtration-isolation-nucleic-acids-simple-rapid-dna-extraction

Nanyang Technological University

Research

JoVE Journal

Biology

18.1K 次观看.  Northwestern University. 在这里，我们描述了一个简单，快速的基于纸张的DNA提取的HIV前病毒DNA的全血通过定量PCR检测方法。这种协议可以在检测其他遗传标记或使用替代的扩增方法被扩展为使用。 

视频: Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

科研

遗传学

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

医学

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

免疫与感染

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

免电力，顺序的核酸和蛋白质的分离

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

生物学

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

使用洗涤剂和酶无酶方法从整个组织进行核分离

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

从冷冻哺乳动物组织中分离和分析单个细胞核

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.

一种简单、快速、部分自动化的方案，用于从冷冻哺乳动物组织中分离单细胞核，用于单细胞核测序

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they ...

通过富集低 MW DNA 评估环境 AMR 的高效技术

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water,&#160;isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.

作者聚焦：通过将基于培养的技术和基因组学相结合来了解和检测环境抗菌素耐药性

Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估 

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

核酸的分离过滤:一种简单快速的DNA提取方法

摘要

探索更多视频

此视频中的章节

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

免电力，顺序的核酸和蛋白质的分离

使用洗涤剂和酶无酶方法从整个组织进行核分离

一种简单、快速、部分自动化的方案，用于从冷冻哺乳动物组织中分离单细胞核，用于单细胞核测序

一种简单、快速、部分自动化的方案，用于从冷冻哺乳动物组织中分离单细胞核，用于单细胞核测序

一种简单、快速、部分自动化的方案，用于从冷冻哺乳动物组织中分离单细胞核，用于单细胞核测序

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估

从废水中增强低分子量 DNA 的提取，用于抗菌素耐药性的综合评估