Name: development of a quantitative recombinase polymerase amplification assay with an internal positive control
Uploaded: 2015-03-30T15:00:00.000+00:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control at JoVE.com

该研究是由来自比尔和梅林达·盖茨基金会通过大挑战全球健康计划的资助。

1.程序的实时qRPA反应的热循环仪<ol><li>创建在热循环仪软件的新协议。 <ol><li>插入预孵育步骤:39℃，1分钟。 </li><li>添加第二个步骤:39℃，20秒，接着是板读。 </li><li>最后，插入一个"GO TO"，重复第二步59次以上。 </li><li>保存的协议。 </li></ol></li><li>在软件的"板块编辑器"选项卡中创建一个新的板块。选择上板孔对应于​​RPA反应的位置（这里，利用井A1，A4-A8，B1和B4-B8）。 注:是否指定井（ 例如，"标准"，"NTC"）的样品类型，使用自定义脚本来完成数据分析并不重要。 <ol><li>对于含有HIV-1 DNA只实验，选择"HEX"荧光所有井。对于含有HIV-1 DNA和内部积极合作实验ntrol，同时选中"HEX"和"FAM"荧光所有井。存盘。 </li></ol></li></ol> 2.准备HIV-1 qRPA实验<ol><li>组装RPA反应在含有指定移液器，移液管尖端，涡流混合器，和微型离心机，作为用于定量PCR指定的前置放大工作区。如RPA可由幅度高达10个数量扩增DNA的单拷贝（数据未示出），处理和处置含有DNA后的扩增产物在一指定的后扩增区管。 <ol><li>防止所有预扩增试剂和扩增后的产品之间的接触。之前和所有实验后喷预放大工作空间和设备，用50％的漂白剂。用纸巾擦去多余的漂白剂。 </li></ol></li><li>购买正向引物序列5&#39;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3&#39;和反向引物序列5&#39;-CCC GAA AAT TTT GAA TTT TTG TAA TT牛逼GTT TTT G-3&#39;从商业DNA合成。购买探针序列5&#39;-TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA / HEX] GC [THF] C [胸苷BHQ1] GTA CCC CCC AAT CCCç-3&#39;从商业渠道。存储在等份所有引物和探针在4℃以在使用之前为10μM的浓度存在。 注:qRPA法放大了独特的HIV-1的序列。用于概念验证的该试验中，使用由萨顿等人 ，其中包含与靶序列16描述的质粒pHIV-IRES-eYFPΔEnvΔVifΔVpr。对于qRPA实验中，质粒被保存在4℃，在含有在含10mM Tris，0.1毫摩尔EDTA在pH 8.0的缓冲液每μl的1，10，10 2，10 3，和10 4份等分试样，和1纳克/微升的人类基因组DNA的载体（360486）。该载体DNA浓度相当于从用于HIV-1的诊断17商用血清DNA提取试剂盒获得的DNA浓度。这些HIV-1稀释液用作qRPA反应的模板，以建立一个标准曲线。 </li></ol> 3.装配的HIV-1 qRPA标准曲线<ol><li>之前组装qRPA反应，如在步骤2.1.1中所述制备的预放大的工作区。放置在存储96孔冷块在4℃。 </li><li>准备试剂12反应: <ol><li>移动乙酸镁，补液缓冲液，和12 RPA反应粒料到预放大的工作区。 </li><li>解冻的乙酸镁，在室温下再水化缓冲液中。 </li><li>等分32.5微升乙酸镁（每反应2.5微升）到一个离心管中。 </li><li>准备一个13倍母液。加入383.5微升补液缓冲液（每反应29.5微升），以一个单独的离心管中的主结构。添加41.6微升的核酸自由水（每反应3.2微升）的主结构。 VORTEX的主结构。 </li><li>返回镁aceta德和再水化缓冲液储存于-20℃。 </li><li>移动从冰箱到预扩增区域中的引物和探针的等分试样，从而避免过度曝光，以减少光漂白。 </li><li>添加27.3微升各为10μM的正向和反向引物（每个反应的引物2.1微升）到主混合物。 </li><li>加入7.8微升10μMHEX标记的HIV-1 DNA探针（每反应0.6微升）的主结构。 </li><li>返回引物和探针存储。 </li></ol></li><li>匹配在1.2节中描述的板块布局，放置1酶颗粒中的每个最左边管和1酶颗粒在每2低层8孔PCR管带5极右管。 </li><li>仔细增加37.5微升主混合物含有一种酶颗粒每管，采用了新的枪头每个管，轻轻地搅拌预混和球团用刀尖直到沉淀完全溶解。轻轻搅拌以防止泡沫formati，并小心地取出笔尖​​防止反应体积的任何损失。 </li><li>毕竟酶颗粒已经水化与主结构，检索4℃保存96孔冷块。将PCR管带中冷块寒意的主结构。 </li><li>而主混合物被冷却，装入热循环仪软件与协议和板在第1节中所述。 </li><li>切成2片的透明塑料微密封粘合剂为比PCR管稍宽，并检索2扁平PCR管条盖。 </li><li>从存储器中检索模板6等份（含有的每微升的HIV-1的质粒0，1，10 1，10 2，10 3，或10 4个拷贝）。 </li><li>加入10微升每个模板，以指定的模板浓度管。使用新的枪头，每个管，轻轻搅动以尖端混合主结构和模板的解决方案。请务必将模板添加到正确的位置，以米ATCH第1.2节中描述的平板布局。 </li><li>确保PCR管带适配器的离心到位。 </li><li>分配2.5微升乙酸镁进每个管盖，将被放置在含有qRPA反应的管。 </li><li>轻轻地放在盖子上的PCR管，使得它们没有完全密封的，并且可以被去除的顶部。乙酸镁将坚持盖和清晰可见从上面。 </li><li>插入每个PCR管中带材用盖子到PCR管夹持器的每一侧。关闭minifuge的盖，旋乙酸镁出盖子并进入RPA反应，引发RPA反应。离心直至所有气泡被消除，并且所有的液体被收集在每个管（约10秒）的底部。 </li><li>快速去除PCR管条，并将其返回到冷块停止qRPA反应。 </li><li>小心取下盖子，以防止气溶胶的形成和密封每个PCR管带用切个明确的微密封膜。 </li><li>快速步行到热循环仪并加载两个PCR管带成相匹配的板布局（井A1-B8）的位置的热循环仪。关闭热循环仪的盖子，然后单击"开始→运行"，并指定一个文件名实验。 注意:虽然在制造商建议5分钟温育后，搅拌该样品，该步骤不是必需为这个特定测定和可被省略。 </li><li>运行完毕后，选择"设置"，"基线设置"，"没有基线减法"，以显示原始荧光数据。通过选择将数据导出到一个电子表格程序"导出"，"导出所有数据表到Excel中。"A文件增编"定量扩增结果"将创建包含原始的实时荧光数据。 </li></ol> 4.制定一个内部阳性对照<ol><li>选择从一个物种，是不太可能的目标样品中被发现的DNA序列。该序列需要比目标扩增子得较长，以便产生靶序列的是有利的。 注意:对于这个测定法中， 小隐孢子虫 ，肠寄生虫，被选为充当模板用于产生对IPC序列，因为这种微生物是不太可能在血液中被发现并且是可在从另一项目中的实验室。要生成IPC序列，提取和纯化C. DNA 孢子虫卵囊其他地方18所描述。 </li><li>购买前（5&#39;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G / ATC TAA GGA AGG CAG CAG GC-3&#39;）和反向（5&#39;-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G / TGC TGG AGT ATT CAA GGC ATA-3&#39;）从商业来源获得的引物。在该试验中所使用的IPC的PCR引物示于图1，并包含一个区域是互补的IPC的模板ðNA（ 例如，微小隐孢子虫 ，紫色在图1）和一个区域是互补的靶生物（ 例如，HIV-1，蓝色于图1）。 </li><li>进行PCR使用引物和IPC模板DNA。 <ol><li>组装根据制造商的说明使用的Phusion高保真DNA聚合酶试剂盒的试剂50μl的PCR反应，但不包括聚合酶。使用2微升DNA模板的Phusion缓冲HF的。 </li><li>添加的Phusion聚合酶到每个反应热启动在98℃，接着60秒，在98℃，随后通过15秒的40个循环后，在98℃，30秒，62℃，和30秒72℃下用在72℃最终延伸5分钟。热循环后，保持在4℃的样品。 </li><li>分析通过凝胶电泳的样品在2％TAE琼脂糖凝胶上，然后提取和使用根据微量协议附带QIAquick凝胶提取试剂盒纯化DNA试剂盒中。 </li></ol></li><li>屏幕IPC候选人为他们的使用qRPA放大能力。 <ol><li>准备qRPA反应，如在第3节所述，使用HIV-1引物，一个FAM标记探针具体到IPC（5&#39;-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [胸苷BHQ1 ] GGT TTT GTA ATT GGA甲-3&#39;），以及总共0，10，10 2，10 3，10 4或10 5个拷贝每反应各IPC的候选，检验所有浓度一式两份的。 </li><li>选择IPC候选人，放大最一致，并具有最低的极限的检测。 </li></ol></li><li>共扩增的HIV-1和IPC来确定的IPC DNA的最佳浓度。 <ol><li>类似于第3节，结合下面的每50微升反应:29.5微升的补液缓冲区，2.1微升正向引物[10μM]的RPA的，2.1微升RPA的反向引物[10μM]，0.6微升的HIV-1 HEX标记的探针[10μM]，10微升HIV-1的DNA的不同浓度，2.5微升乙酸镁，以及1酶沉淀。代替加入3.2微升不含核酸酶的水作为在步骤3.2.4完成，加入0.6微升的IPC FAM标记的探针[10μM]的，和2.6微升的IPC的DNA。使用IPC浓度是极限- - 的检测限（LOD）时qRPA与IPC DNA的单独（定义为最低浓度，其中荧光信号的产生的量是大于由通过与样本产生的荧光所产生的LOD执行没有目标DNA）。 </li><li>孵育使用1.1​​节中描述的协议的样品。 </li><li>如果IPC不每个样品中扩增，考虑重复实验幅度更高的IPC浓度一个数量级。 IPC DNA的HIV-1检测的最佳浓度为10000拷贝/微升。虽然样本被认为是有效的，如果有任何IPC或HIV-1 DNA扩增，理想的IPC具有检测扩增每一个反应。 </li></ol></li></ol> 5.建立来自多个实验标准曲线<ol><li>确保数据在第3.13节描述的每个实验已出口到电子表格程序。 </li><li>打开并运行脚本"JoVE_qRPA_standard_curve.m"。所有的输入会自动提示，在命令窗口。被启动的脚本后，用户被自动提示指定"的数据文件数"用于构建标准曲线。 </li><li>在命令窗口中，键入要使用的文件的数量来构建标准曲线，然后按回车。 </li><li>在命令窗口中键入的样本数和重复在每个训练文件数（按下每个条目后进入）。 注:在1.2节中描述的板布局，有6个样品不同浓度和2次重复每个样品）。 </li><li>请在命令窗口中最低的DN用于构建标准曲线（以log10拷贝），然后按回车浓度（在1.2节中描述的板块布局，最低模板浓度为1 log10拷贝）。 </li><li>在命令窗口中输入每个标准间的浓度差（以log10拷贝），然后按回车键（在1.2节中描述的板块布局，集中时间间隔为1 log10拷贝）。 </li><li>提示后，请在命令窗口中的"斜率阈值"，然后按Enter。 </li><li>在命令窗口中，键入"号（Z）的背景正阈值以上的标准偏差"，然后按回车键。典型值z范围从1至5。 </li><li>指定使用热循环仪（&#39;1&#39;）中的数据是否被收集，* .TIFF显微镜图像（&#39;2&#39;），或* .JPG显微镜图像（&#39;3&#39;），通过输入数字"1"，"2"，或&#39;3&#39;和对ressing进入。 </li><li>选择设置新的门槛IPC或键入分别为&#39;Y&#39;或&#39;N&#39;，出现提示时使用的阈值的默认值。 </li><li>接下来，选择每个用于构建标准曲线的电子表格文件。 注:随后，脚本会自动导入HEX和FAM荧光数据;确定各反应是否是有效的（ 即，无论是荧光信号超过阈值，HEX或FAM通道）;确定第r 2系数的指数，线性和二阶多项式拟合;暗算"log10拷贝与循环门槛"，每用于构建标准曲线样品;并创建包含基本变量来重建标准曲线进行验证实验，而不需要用户重新输入所有输入参数的再一个电子表格文件。 </li></ol> 6.试验验证和未知样品使用的量化标准曲线<ol><li>使用的脚本"JoVE_qRPA_validation_and_quantification.m"通过使用样品与已知浓度来估计测定的精度和准确度，或用它来量化样品与未知浓度。 注:因为之前发表的报告显示，指数拟合得到最好的R平方系数18，这个脚本利用一个指数拟合的标准曲线。如果不同类型的合适的是理想的，该脚本可以被相应地修改。 <ol><li>打开并运行脚本"JoVE_qRPA_validation_and_quantification.m"。启动脚本后，该用户将自动提示输入所有输入到命令窗口。 </li><li>输入"1"用标准曲线与已知样品的浓度来验证试验或输入"2"量化未知样品。 </li><li>当系统提示，要么选择一个保存的标准曲线或进入日建一个新的E资料是在命令窗口中自动搜罗（即所需"JoVE_qRPA_standard_curve.m"剧本从第5相同的信息）。 </li><li>类型的实验数量（ 即电子表格文件）来分析进行验证/定量。 </li></ol></li></ol> 7.筹备数据收集使用荧光显微镜和加热芯片<ol><li>确保荧光显微镜有一个阶段，加热器和1通道，高精度，高稳定性温度控制器到位。 </li><li>确保荧光显微镜有一个过滤器立方体激发并从每个荧光收集荧光。激发和收集通过GFP过滤器（激发BP四十分之四百七十○纳米，发射BP五十〇分之五百二十○纳米）IPC DNA扩增过程中产生的FAM荧光。激发和收集通过HEX过滤器（BP激发波长530/30，排放BP40分之575nm）的HIV-1 DNA扩增过程中产生的HEX荧光。 </li><li>使用显微镜脚本编辑软件来创建一个自动算法来复制的热循环仪的数据收集。 <ol><li>第一插入一个"设置"步骤以关闭快门。下一步添加一个"曝光"一步曝光设置为60秒。将"捕捉"执行60秒的延迟，它实现了1分钟前的潜伏期。添加一个"设置"步骤的工作组更改为GFP过滤器。插入另一张"暴露"的步骤来调整曝光，以200毫秒。使用GFP或HEX滤波器立方体所有曝光将被设置为200毫秒。 </li><li>之后，"曝光"的步骤，添加一个"快照"，指定该图像将采取相机。使用另一个"设置"的步骤，关闭快门和"保存图像"的步骤将图像保存到一个用户定义的临时文件夹。 </li><li>使用另一个"设置"的步骤和TH接下来切换到HEX过滤立方体恩添加"曝光"在200毫秒，一个"快"和"保存图像"的步骤。 </li><li>重复此过程59次20秒而不是60（靠近快门的初始延迟，等待20秒，切换到GFP滤块，设置曝光为200毫秒，扣，关闭快门，保存，切换到HEX滤块，设置曝光至200毫秒，SNAP）。 </li></ol></li><li>创建一个芯片将举行qRPA反应。 <ol><li>为使用显微镜的实验中，使用文件JoVE_qRPA_base.ai于使用激光切割器设定为100％的功率，10％的速度从1/8"厚的黑色丙烯酸树脂的片材切割为40×15毫米矩形基。 </li><li>使用文件JoVE_qRPA_well.ai切割反应以及由一个10×10平方毫米的具有从1.5毫米厚的透明的丙烯酸片材一个5mm直径的圆形孔切割。 </li><li>连上三个井用强力胶凝胶一个基地。 </li><li>干燥过夜。 </li></ol></li></ol> 8.数据收集和ANALYSIS使用荧光显微镜<ol><li>通过装载自动数据收集脚本JoVE_AxioVision_Script.ziscript准备显微镜进行数据收集。 <ol><li>设置阶段加热器至48℃和预热上段加热器的反应芯片为约20分钟。 48℃的温度设定保持39°C的反应以及温度（数据未显示）。 </li><li>使用10X，0.45 NA物镜，着​​眼于反应很好地与代替GFP过滤器的内边缘的顶部。丙烯酸类的边缘激光切割后自身荧光，并且可以很容易地可视化。对焦后，不调整显微镜阶段的高度，直到所有的数据收集。 </li></ol></li><li>组装qRPA预混在指定的前置放大工作区，如步骤4.5.1描述。对于每个样本，混合酶颗粒，主控组合，并在离心管中HIV-1 DNA模板。加入2.5微升的醋酸镁至第一reaction和简要旋涡。 </li><li>迅速输送含有qRPA样品荧光显微镜的离心管中。 <ol><li>认真吸取30微升的RPA反应到反应良好，没有产生气泡。放置一滴的PCR级矿物油在RPA反应以防止蒸发。 </li><li>定位以及直接的显微镜物镜下，注意图像只有中央的很好，没有任何自动荧光亚克力边缘。井被定位后，运行自动化的脚本7.脚本中使用FAM过滤立方体和使用HEX滤块每20秒1 JPEG图像生成一个JPEG图像。 </li></ol></li><li>剧本完成后，运行其他样品之前，这些转移出来的图像临时存储文件夹中。 <ol><li>每个荧光基团（ 即 "十六进制"和"FAM"）1:创建2个文件夹。存储在同一个父文件夹这两个文件夹。在每一个荧光文件夹中创建标记存在于样品（ 例如，0，10，等等 ）中的DNA拷贝数的文件夹。 </li><li>转移的前缀"HEX"所有文件到相应的子文件夹中的"HEX"文件夹中。转移在"FAM"文件夹中的前缀"GFP"进入相应的子文件夹中的所有文件。 </li></ol></li><li>对于qRPA反应以0，10，10 2，10 3，10 4，和10 5的HIV-1 DNA拷贝重复部分8.2-8.4。 </li><li>收集在一个实验中所有样品qRPA数据后，加载脚本"JoVE_real_time_intensity_to_excel.m。"当脚本提示时，选择含有2荧光的文件夹，这反过来又包含子文件夹中的每个浓度的图像的父文件夹。 注:该脚本将自动生成在其中HEX荧光数据将被保存在一个表南的父目录中的电子表格文件主编"HEX"，和FAM荧光数据将被保存在一个名为"FAM"表。在每个片材中，循环数被列在B列，和用于增加模板的浓度的实时荧光数据中列C，D 等中给出。每个实时荧光数据是在该时间点拍摄的图像的平均荧光强度。 </li><li>使用默认的散点图功能的电子表格程序绘制单元格B2:在"HEX"和"FAM"片H61形象化实时荧光并产生图类似于图2A，2B，3A和3B。 注:在步骤8.6中产生的电子表格，也可以在脚本"JoVE_qRPA_standard_curve.m"和"JoVE_qRPA_validation_and_quantification.m"使用。 </li></ol>

<ol>
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A., Richards-Kortum, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+HIV-1+DNA+using+Real-Time+Recombinase+Polymerase+Amplification.">Quantification of HIV-1 DNA using Real-Time Recombinase Polymerase Amplification.</a> Analytical Chemistry. 86 (12), (2014).</li><li>Sollis, K. 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=Systematic+review+of+the+performance+of+HIV+viral+load+technologies+on+plasma+samples.">Systematic review of the performance of HIV viral load technologies on plasma samples.</a> PloS one. 9 (2), e85869 (2014).</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> Guidelines for the use of antiretroviral agents in HIV-1-infected adults and adolescents. , 1-166 (2011).</li></ol>

The authors declare they have no competing financial interests.

为了获得使用MATLAB算法有意义的量化数据，系统提示时，用户必须选择合适的输入值。开始在第5和第6的每个脚本之后，所有的输入变量自动征求在命令窗口中，并自动生成输出。在第5.7节中提示用户选择斜率阈值。的斜率阈值影响契合的相关系数（R 2）的平方。当使用从热循环仪导出原始荧光数据，在2.0和5.0的值趋向于产生高R 2系数。在第5.8节，用户必须指定背景以上的标准偏?...

定量核酸扩增是用于检测环境，食源性和水生病原体，以及用于临床诊断的一项重要技术。实时定量聚合酶链反应（qPCR的）可以灵敏，特异和定量检测的核酸， 例如，HIV-1的病毒载量检测，检测的细菌病原体，并筛选许多其他生物体1的金标准方法- 3。在实时定量PCR，引物扩增在周期病原体的DNA和荧光信号被产生成比例的扩增的DNA的样​​品，在每个循环中的量。将含有未知浓度的病原体的DNA的样品可以使用涉及标准样品的初始DNA浓度和在该荧光信号达到一定的阈值的时间（ 即，循环阈值，或（C T））的标准曲线进行定量。 4。这些平台通常提供结果更快和扩增核酸在一个较低的，单一的温度，其可以实现与更便宜，更简单的设备。 RPA，其用于护理点的应用特别有吸引力的，放大的DNA在几分钟内，需要低放大温度（37℃），并且在杂质5,6存在下保持有效。 RPA试验已经开发了用于范围广泛的应用，包括食品分析，病原体检测，癌症药物筛选，并检测生物威胁因子7 - 12。然而，使用的RPA的用于核酸定量一直局限于13,14。 在以往的工作，这是笑WN的实时定量RPA（qRPA）是可行的15。这里，更详细的协议提供了一种用于使用实时定量RPA量化使用标准曲线中，一种方法使用qPCR即类似于量化未知样品。本协议描述了如何执行在热循环的RPA反应检测HIV-1 DNA作为概念证明的，以及如何开发一个内部阳性对照（IPC），以保证系统正常运行。使用热循环仪或显微镜和数据分析使用训练数据构建的标准曲线数据收集也详细叙述。最后，该方法用于定量用标准曲线与自定义脚本未知样品被证实。此qRPA技术使具有未知浓度的样品进行定量，并具有比传统的实时定量PCR的许多优点。

<table><tbody><tr><td>qRPA Assay</td><td></td><td></td><td></td></tr><tr><td>HIV-1 forward primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>HIV-1 reverse primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G-3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>HIV-1 probe</td><td>BioSearch Technologies&amp;nbsp;</td><td>custom DNA oligos</td><td>5&amp;rsquo;- TGC TAT TAT GTC TAC TAT TCT TTC CCC [SIMA/HEX] GC [THF] C [dT-BHQ1] GTA CCC CCC AAT CCC C -3&amp;rsquo;&amp;nbsp;</td></tr><tr><td>IPC probe</td><td>BioSearch Technologies&amp;nbsp;</td><td>custom DNA oligos</td><td>5&amp;rsquo;-AGG TAG TGA CAA GAA ATA ACA ATA CAG GAC [FAM] T [THF] T [dT-BHQ1] GGT TTT GTA ATT GGA A -3&amp;rsquo;</td></tr><tr><td>RPA exo reagents (pellets, rehydration buffer, magnesium acetate</td><td>TwistDx</td><td>TwistAmp exo</td><td></td></tr><tr><td>PCR tube strips</td><td>BioRad</td><td>TLS0801</td><td></td></tr><tr><td>PCR flat cap tube strips</td><td>BioRad</td><td>TCS0803</td><td></td></tr><tr><td>Micro-seal adhesive</td><td>BioRad</td><td>558/MJ&amp;nbsp;</td><td></td></tr><tr><td>HIV-1 target (pHIV-IRES- eYFP&amp;Delta;Env&amp;Delta;Vif&amp;Delta;Vpr)</td><td></td><td></td><td>custom plasmid, see: Segall, H. I., Yoo, E. &amp;amp; Sutton, R. E. Characterization and detection of artificial replication-competent lentivirus of altered host range. Molecular Therapy 8, 118&amp;ndash;129, doi:10.1016/S1525-0016(03)00134-5 (2003).</td></tr><tr><td>Human male genomic DNA</td><td>Applied Biosystems</td><td>360486</td><td></td></tr><tr><td>96 well cold-block</td><td>Cole Parmer</td><td>EW-36700-48</td><td></td></tr><tr><td>Thermal cycler</td><td>BioRad</td><td>CFX96</td><td></td></tr><tr><td>MiniFuge</td><td>VWR</td><td>93000-196</td><td></td></tr><tr><td>Vortex</td><td>VWR</td><td>58816-121</td><td></td></tr><tr><td>Tris buffer 1.0 M, pH 8.0</td><td>EMD Millipore</td><td>648314</td><td></td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Promega</td><td>V4321</td><td></td></tr><tr><td>Nuclease free water</td><td>Ambion</td><td>AM9937</td><td></td></tr><tr><td>IPC Development</td><td></td><td></td><td></td></tr><tr><td>Cryptosporidium parvum IPC template</td><td>Waterborne Inc</td><td>P102C</td><td>It is also possible to order a double stranded synthetic target from IDT if the user is unequipped to work with C. parvum (a BSL-2 infectious agent). PCR and RPA primers for C. parvum were designed using GenBank accession number AF115377.1</td></tr><tr><td>PCR long forward primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;-TGG CAG TAT TCA TTC ACA ATT TTA AAA GAA AAG G/ ATC TAA GGA AGG CAG CAG GC-3&amp;rsquo;</td></tr><tr><td>PCR long reverse primer</td><td>Integrated DNA Technologies</td><td>custom DNA oligos</td><td>5&amp;rsquo;- CCC GAA AAT TTT GAA TTT TTG TAA TTT GTT TTT G/ TGC TGG AGT ATT CAA GGC ATA -3&amp;rsquo;</td></tr><tr><td>Phusion High-Fidelty DNA Polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>Qiaquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>TAE 10X buffer</td><td>EMD Millipore</td><td>574797</td><td></td></tr><tr><td>Agarose</td><td>Sigma Aldrich</td><td>A9539</td><td></td></tr><tr><td>Microscope Experiments</td><td></td><td></td><td></td></tr><tr><td>Upright fluorescence microscope</td><td>Zeiss</td><td>Zeiss Imager.J1</td><td></td></tr><tr><td>Stage heater</td><td>Bioscience Tools</td><td>TC-GSH</td><td></td></tr><tr><td>1-Channel Precision High Stability Temperature Controller</td><td>Bioscience Tools</td><td>TC-1100S</td><td></td></tr><tr><td>FAM/GFP filter cube</td><td>Zeiss</td><td>filter set 38 (000000-1031-346)</td><td>excitation BP 470/40 nm, emission BP 520/50 nm</td></tr><tr><td>HEX filter cube</td><td>Chroma</td><td>49014</td><td>excitation BP 530/30 nm, emission BP 575/40 nm</td></tr><tr><td>Laser cutter</td><td>Engraver&#039;s Network</td><td>VLS3.60</td><td></td></tr><tr><td>1/8&quot; black acrylic</td><td>McMaster Carr</td><td>8505K113</td><td></td></tr><tr><td>1.5 mm clear acrylic</td><td>McMaster Carr</td><td>PD-72268940&amp;nbsp;</td><td></td></tr><tr><td>Super glue</td><td>Office Depot</td><td>Duro super glue&amp;nbsp;</td><td></td></tr><tr><td>PCR grade mineral oil</td><td>Sigma Aldrich</td><td>M8662-5VL</td><td></td></tr><tr><td>Data Analysis</td><td></td><td></td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr><tr><td>MATLAB</td><td>MATLAB</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_qRPA_standard_curve.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_qRPA_validation_and_quantification.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>MATLAB script: &quot;JoVE_real_time_intensity_to_excel.m&amp;rdquo;</td><td>Included in SI</td><td></td><td></td></tr><tr><td>Adobe Illustrator</td><td>Adobe</td><td></td><td></td></tr><tr><td>JoVE_qRPA_well.ai</td><td>Included in SI</td><td></td><td></td></tr><tr><td>JoVE_qRPA_base.ai</td><td>Included in SI</td><td></td><td></td></tr><tr><td>AxioVision software</td><td>Zeiss</td><td></td><td></td></tr><tr><td>JoVE_AxioVision_Script.ziscript</td><td>Included in SI</td><td></td><td></td></tr></tbody></table>

development of a quantitative recombinase polymerase amplification assay with an internal positive control

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to quantify DNA sample concentration using a standard curve. In this manuscript, a detailed protocol for developing and implementing a real-time quantitative recombinase polymerase amplification assay (qRPA assay) is provided. Using HIV-1 DNA quantification as an example, the assembly of real-time RPA reactions, the design of an internal positive control (IPC) sequence, and co-amplification of the IPC and target of interest are all described. Instructions and data processing scripts for the construction of a standard curve using data from multiple experiments are provided, which may be used to predict the concentration of unknown samples or assess the performance of the assay. Finally, an alternative method for collecting real-time fluorescence data with a microscope and a stage heater as a step towards developing a point-of-care qRPA assay is described. The protocol and scripts provided may be used for the development of a qRPA assay for any DNA target of interest.

选择一个序列作为IPC在qRPA实验目标之前（HIV-1）的DNA，内部阳性对照（IPC）的候选人中产生和筛选它们放大的qRPA反应无HIV-1 DNA存在的能力。 IPC考生比目标（HIV-1）的DNA，以防止出竞争的HIV-1扩增子形成IPC形成更长的时间。 如图2A所示 ，两个C的代孢子虫 IPC候选人是由使 ​​用凝胶电泳415和435 bp的条带的存在验证。在qRPA反应，较短的IPC候选呈现小的扩增（ 图2B），<...

Watch this Scientific Journal Video about Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control at JoVE.com

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

一个定量聚合酶重组酶扩增法的发展与内部阳性对照

It was recently demonstrated that recombinase polymerase amplification (RPA), an isothermal amplification platform for pathogen detection, may be used to ...

development-quantitative-recombinase-polymerase-amplification-assay

Research

JoVE Journal

Biology

13.5K 次观看.  Rice University. Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

视频: Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

科研

遗传学

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

免疫与感染

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

开发和测试针对物种的定量PCR检测环境DNA应用

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

环境科学

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T细胞受体删除环同时定量（TRECs）和K-删除重组删除环（KRECs）通过实时PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Development and Validation of a Quantitative PCR Method for Equid Herpesvirus-2 Diagnostics in Respiratory Fluids

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 norm. After the development and first validation step, two distinct characterization steps were performed according to the AFNOR norm: (a) characterization of the qRT-PCR assay alone and (b) characterization of the whole analytical method. The validation of the whole analytical method included the portrayal of all steps between the extraction of nucleic acids and the final PCR analysis. 
Validation of the whole method is very important for virus detection by qRT-PCR in order to get an accurate determination of the viral genome load. Since the extraction step is the primary source of loss of biological material, it may be considered the main source of error of quantification between one protocol and another. For this reason, the AFNOR norm NF-U-47-600 recommends including the range of plasmid dilution before the extraction step. In addition, the limits of quantification depend on the source from which the virus is extracted. Viral genome load results, which are expressed in international units (IU), are easier to use in order to compare results between different laboratories. 
This new method of characterization of qRT-PCR should facilitate the harmonization of data presentation and interpretation between laboratories.

Here, we present a protocol for the development and validation of a quantitative PCR method used for the detection and quantification of EHV-2 DNA in equine respiratory fluids. The EHV-2 qRT-PCR validation protocol involves a three-part procedure: development, characterization of qRT-PCR assay alone, and characterization of the whole analytical method.

为马科动物疱疹病毒2诊断的呼吸道液体定量PCR检测方法的开发和验证

The protocol describes a quantitative RT-PCR method for the detection and quantification of EHV-2 in equine respiratory fluids according to the NF U47-600 ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

多重PCR和反向线杂交检测（MPCR / RLB）

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

生物学

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

医学

Open-Source Miniature Fluorimeter to Monitor Real-Time Isothermal Nucleic Acid Amplification Reactions in Resource-Limited Settings

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with integrated fluorescence detection of amplicons. Isothermal nucleic acid amplification technologies eliminate the need for thermal cycling; however, fluorescence-based detection of products is still required for real-time, quantitative results. Several portable isothermal heaters with integrated fluorescence detection are now commercially available; however, the cost of these devices remains a significant barrier to widespread adoption in resource-limited settings. Described here is a protocol for the design and assembly of a modular, low-cost fluorimeter constructed from off-the-shelf components. Enclosed in a compact 3D printed housing, the fluorimeter is designed to be placed atop a commercially available heat block holding a PCR tube. The fluorimeter described here was optimized to detect fluorescein isothiocyanate (FITC) dye, but the system can be modified for use with dyes commonly used as reporters in real-time nucleic acid amplification reactions. Clinical applicability of the system is demonstrated by performing real-time nucleic acid detection with two isothermal amplification technologies: recombinase polymerase amplification (RPA) for detection of positive control DNA provided in a commercial kit and reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of clinically meaningful levels of SARS-CoV-2 RNA.

Detailed instructions are provided to build an open-source, modular fluorimeter that is compatible with many low-cost heaters to perform real-time, quantitative isothermal nucleic acid amplification.

开源微型氟度计，以监测资源有限设置中的实时异热核酸放大反应

Traditional methods to detect and quantify nucleic acids rely on polymerase chain reaction (PCR) and require the use of expensive thermocyclers with ...

生物工程

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of ...