The authors have no acknowledgements.

1.检测混合物准备<ol><li>请为100μmol/ L浓度的股票在分子级的水全部引物和探针在TE pH值为8的缓冲[肠F1A，肠R1，GPL813TQ 12; HF183-1，BthetR1，BthetP1 3]。 注:探头的两个目标是荧光标记用不同的荧光7作为材料/设备清单表示。 </li><li>通过混合计划准备主结构，每反应，12微升数字PCR混合物（2倍的股票，见材料/设备清单）每前进，0.216微升和反向引物，0.06微升每个荧光探针和5.016微升无核酸酶的水（终浓度:900纳米每个底漆，250纳米每个探头）。吸管上下的至少10倍，同时服用小心不要在溶液中引入气泡来混合。 </li><li>吸取18μl反应混合液（步骤1.2）成常规PCR管或板，在6微升DNA模板混合（提取描述页上一页诡13），使液滴产生测定混合物。如果运行样品一式两份，吸管预混的36微升至12微升DNA模板到每口井。离开相应的重复井上盘空。包括阳性对照（见材料/设备的列表）和无模板对照（NTC）（ 即作为模板分子级水）。 注:正控制是必要的，以确保该测定正常运行和NTC必须确保有在板内没有污染，并在数据分析以后设置荧光灯基线。第一次建议用户同时使用未稀释和稀释的DNA样本。 </li></ol> 2.滴生成和PCR板设置<ol><li>通过使用多管移液器吹打同比下降约15倍混合测定混合物。确保枪头内保持液态，以避免在混合物中做过多的泡沫。 </li><li>插件ERT盒1（含8个孔）到白色盒保持器，并点击盒保持器关闭。墨盒1现在是牢固就位，并且不能从持有人在产生飞沫脱落。使用多通道移液管，轻轻转移20微升测定混合物的进入暗盒标记为"样品"的中间位置，而不引入气泡。 </li><li>吸管70微升液滴生成油到盒的左侧（标记为"油"）。 </li><li>盖盒带垫片确保垫圈是平坦的，由4均匀地保持蜱朝盒的边缘。按下液滴生成绿色照明按钮，打开并放置盒。再次按下绿色照明按钮，关闭发电机。 注意:一旦门关闭，按钮是灰色的，门不能被重新打开和液滴产生立即开始约1分钟，继续。 </li><li>如果在第二个做超过8反应，地方盒2白盒保持器和以相同的方式作为盒1制备而产生液滴正在进行节省总建立时间。 </li><li>打开液滴生成门口的时候朦胧亮按钮变成绿色表明液滴形成的完成。取出墨盒含1白盒支架，预留和地点盒2到液滴发生器。 </li><li>从盒中取出1垫圈并丢弃。轻轻地从盒（标记为"小滴"）的第三列到最终的PCR板（保持在室温）下的热循环传送的总体积（大约40微升）中产生的液滴。 注意:不要取消勾选白盒支架的动作可能打破新产生的飞沫​​;只有后液滴已转移到最终的板打开盒保持器。 </li><li>重复步骤2.1-2.7额外的样品。 </li><li>当所有的液滴都在最后的PCR板，放置一个可刺穿在板的顶部电化铝盖，并将其放置在板盖。坐落在封口机封口机到180°C，按"播放"，让密封，持续10秒。 </li></ol> 3.热循环和液滴阅读<ol><li>放置密封板在热循环仪，一用在步骤2.7中使用的最终的PCR板，和2.0℃/秒的温度变化速度相容。 </li><li>在98℃（可选在94℃在95℃10分钟，接着30秒的40个循环，在60℃下60秒，接着是10分钟保持:运行热方案如下，在4最终保持或15℃）。 </li><li>在循环结束后，在各孔中板转移到液滴读者为荧光的自动测量在每个液滴。可替代地，存储在4℃的板对液滴读数之前3天。确保液滴在室温下是有阅读，然后再继续。 <ol><li>打开附带的软件设置液滴读数。在含一个空的96孔板的示意图默认"设置"菜单，双击以及A1打开包含三个部分菜单:"样品"，"试验1"和"2测定" </li><li>在"示例"部分输入样品编号为标有"名"，然后按回车或检查框标记右边的方框"应用"。接下来，单击下拉菜单中标​​记为"实验"，并选择"红色"（罕见的事件检测），并单击进入。 </li><li>移动到表示"分析1."一节在"名称"部分填写的检测（ 如 肠球菌 ），然后单击进入。在下面标有"类型"框单击下拉菜单并选择"通道1未知"，然后单击进入。 </li><li>移动到表示"检测2."一节在"名称"部分填写的检测（ 如 HF183），然后单击进入。在下面标有"类型"框单击下拉菜单中d选择&#39;频道2未知"，然后单击进入。所有的步骤的信息3.3.2 3.3.4现在出现在A1孔。 </li><li>名称含有水滴所有后续井。为了节省安装时间总共，点击"转移"或"Ctrl键"，可以同时选择多个井。 </li><li>当板的数字描述反映了物理盘，按"OK"在菜单的右下角。在出现在板原理图顶部的新菜单，在"模板"部分中选择"另存为"，并将其命名并保存板。 </li><li>在屏幕的左侧，单击"运行"，然后在弹出的"运行选项"窗口中选择合适的染料组。数据收集起始并在软件中实时显示。 </li></ol></li></ol> 4.数据分析和报告<ol><li>当阅读器完成，一个箱子出现，说明"运行完成"，然后单击"确定"。 注:软洁具显示在该软件的"分析"部分中的最后一个数据文件。这种观点认为，该软件对所有的板块中选择的井中，默认为"2D振幅"数据图。 </li><li>检查正和负的液滴之间的距离。确保在NTC井的所有液滴的荧光接近基线。 </li><li>单击按钮标记为"事件"。所呈现的直方图右侧单击名为"总计"栏框和底部名为"单"，显示接受液滴总数每口井的框。排除包含&lt;万滴7按住Ctrl键并单击井（S），以排除任何井。 </li><li>点击表示为"1D振幅&#39;在为两种靶的NTC井负液滴上方约一个标准差（500-700荧光单位）来设置荧光阈的按钮。在最左侧的屏幕联合国明镜"自动分析"显示两个门槛按钮，选择图标，有通过它运行了坚实的粉红色水平线的权利。 </li><li>点击下每个幅度图的框"设置门槛"的左侧，并输入相应的荧光阈值。每微升反应复制的目标浓度，然后自动计算。 注意:阈值不需要是对于不同的目标是相同的。 </li><li>通过点击1D幅画面的左上角的"导出"按钮，导出.csv文件的结果。在.csv文件，乘以4的出口目标浓度从每微升反应目标复印件（23S基因肠球菌，或者HF183标记）转换的目标，每微升DNA模板进行复制。 </li></ol>

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The authors declare that they have no competing financial interests.

有在协议中的几个关键步骤。首先，由于主结构比传统的qPCR预混液较粘稠的分​​析混合物混合是很困难的。简单涡旋可能导致混合不充分，从而导致对模板DNA的非均匀分布在测定混合物和随后的液滴。在协议中所描述的混合逐移液技术必须遵循以确保准确的DPCR定量。其次，重要的是要确保液滴在一个均匀的形状和大小产生。谨记场合时的液滴发生器花费来完成液滴上产生一个墨盒是比通常短。这可能表示不理想的液滴的产生。如果需要的话，每一个盒上的液滴产生时间可以被记录为故障排除。第三，对整个40微升产生液滴从盒转移到最终的PCR板没有剪切是很重要水滴，避免没有足够的液滴进行量化。以下技术可以被用于传输:1）将多通道移液管向40微升和插入的尖端到盒（标记为"小滴"）中的45°角的右侧部分; 2）吸取水滴慢慢地，并确保所有液滴被滴了（请注意，如果任何液滴留下，需要吸取了第二轮）; 3）一旦液滴被去除，通过将吸管尖靠在井壁大约一半时将其转移到最终板并缓慢排出液滴。 该EntHF183复式DPCR试剂盒在现有的个人qPCR实验为同一分析物的巨大优势。首先，双工DPCR试 ​​验并不需要标准曲线量化未知数，从而消除与标准7变异相关的定量PCR定量偏差。第二，双工DPCR测定可以容忍禁止或浓度的数量级比由相应的qPCR实验7耐受更高的一到两个数量。这一特点使双工DPCR法分析环境样品（ 如雨水样本），往往含有抑制PCR以物质非常有吸引力。第三，容量来同时量化两个肠球菌以及在一个双工DPCR测定中HF183标记（相对于具有在两个qPCR实验分别量化它们）是更有成本效益7和通过避免与相关的累计吸移变性提高数据量进行两个独立的实验（vs.单次双面DPCR法）。第四，相比于相应的qPCR实验双工DPCR测定也表现出更高的精度和可重复性，使得在比较研究7前一个很好的选择。 然而，对于该方法存在限制（优点和局限性详细描述别处7）。第一，每反应接受液滴的数量决定的上定量限DPCR的（UQL）。在这项工作中所用的DPCR仪器（参考材料/设备的列表）已经报道了具有每反应10 5拷贝一个UQL。这对应于5×10 5 肠球菌细胞和2×10 6个 ，每100毫升水中HF183拷贝假设先前7所述DNA提取协议之后，得到100μl的DNA（ 即每DNA提取在洗脱步骤）100毫升的水没有之前DPCR 7样品处理过程中的任何损失。环保水一般不超过该浓度肠球菌和HF183的。然而，样品稀释应考虑高浓度的样品，如动物粪便，未经处理的污水和水样采集立即跟随污水溢出。其次，高浓度的总DNA]（2004NG目标和非靶DNA）可与液滴分区过程干涉。它不建议通过限制使用超过66纳克总DNA无需预处理酶7。第三，虽然EntHF183双工DPCR法反对抑制比的qPCR显著更健壮，此法还是可以产生假阴性的结果，如果严重的抑制防止PCR扩增的发生。抑制控制措施，因此仍应执行。如先前所描述，可用于评估抑制7标准加成或稀释方法。最后，双工法只被证实在一个DPCR平台，方法验证应在使用前其他数字PCR平台进行。 这是第一次公布的双工数字PCR分析能够同时量化被广泛接受的水质量指示符和粪便污染7流行主机相关标记。所表现出的优势的EntHF183双工DPCR法承诺在微生物源跟踪的数字PCR技术的其他有用的应用程序，检测的病原体和抗生素抗性基因。例如，一般的粪便指示（ 肠球菌属，大肠杆菌，普通Bacteroidales）和一个主机相关粪便标记（HF183，HumM2，CowM2，LeeSeagull）2可以类似地配对（如在EntHF183双工DPCR测定）在双工DPCR测定为更具成本效益和一般与宿主相关的粪便污染的准确定量。定位相同的源（或不同的来源）微生物源跟踪标记也可以在双面DPCR测定配对，以增加在检测粪便来源置信度（或同时获得多个粪便来源的信息）。对PCR抑制剂更高的耐受性也可以允许更高的样本量分析，以增加具有低感染剂量，但仅在ENVIR目前检测的病原体的可能性onmental水在低浓度。在定量PCR数字PCR的方法相似的优点也可以适用于开发抗生素抗性基因定量数字PCR检测。

定量PCR（qPCR的）的方法已被广泛地用于娱乐水质监测和微生物源跟踪应用，因为它们是更快速，更灵活，相对于传统的基于培养的方法的具体。因此，定量PCR被推荐用于实现一般粪便指标如肠球菌快速水质监测结果。在修订后的USEPA娱乐用水质量标准1。许多qPCR实验也已开发并验证用于识别环境水2粪便污染的来源。这其中，HF183标记测定法是最常用的用于识别人的相关粪便污染3。 然而，定量PCR的结果常常可以因为它们依赖于用于定量的标准曲线偏置。通过定量PCR从斯坦的插补周期量化（CQ）量化样品中未知浓度的目标描述Cq的和一组系列稀释的标准的数量之间的线性关系准曲线。因此缺乏可靠和一致的标准参考材料可以大大地偏置的qPCR结果。研究表明的qPCR结果由来自不同供应商4大约一半日志使用标准不同，并通过使用标准的不同批次来自相同卖方5 2倍。 数字PCR（DPCR）技术，通过量化大量微型PCR的由分割批量定量PCR成均匀的纳升或皮升的反应6十万或上百万计数产生积极的频率未知样品。如果分区是水包油滴（ 即 ，本文），或在一个芯片上的小室被称作液滴数字PCR（ 即 ，这种物品）或室数字PCR，分别。较高的样品浓度将导致靶DNA存在（因此阳性PCR）分区中的比例较高，通过泊松分布近似的关系。因此，二进制的结果（阳性或阴性的PCR）被记录并且正面的液滴的频率用于通过泊松近似值直接计算未知样品浓度，从而消除了如在定量PCR的标准曲线的需要。 这个二进制数字PCR定量性质的qPCR超过7提供了更多的优势。因为延迟放大仍然可以产生阳性PCR，定量DPCR反对抑制降低扩增效率更加强劲。同样，DPCR量化不Cq的值是定量PCR，导致DPCR的更高的精度相比的qPCR 8-10受变性。 此外，该分割过程确保靶DNA存在的极少量在每个液滴，有效地减少基板竞争（amplificatio期间不同的DNA靶）是在定量PCR实现双面打印（ 即化验同时测量两个DNA目标）常见的障碍ñ。正因为如此，无论是在双工或单工运行（ 即一个目标是在一个单一的测定法测量）DPCR产生两种靶7,11近区分定量。 本文中所描述的数字PCR测定（EntHF183 DPCR）的目标是获得一般粪便指示器肠球菌的同时直接定量。和具有改善的精度，重现性和对抑制鲁棒性人类粪便相关HF183标记相比，其定量PCR同行。该测定中已被成功地用于在环境淡水，海水，以及各种排泄物7定量粪便污染，但也可以应用于任何其它类型的水和废水。此法适用于分析沉积物或土壤中，由于它巨大潜力秒超耐受性抑制，但进一步的测试，因为复杂性所需抑制剂在这些类型的样品混合。

<table><tbody><tr><td>Low Bind Microtubes</td><td>Costar</td><td>3207</td><td>For storage of reagents, samples/production of master mixes</td></tr><tr><td>Nuclease Free Water</td><td>FisherSci</td><td>BP2484-50</td><td></td></tr><tr><td>TE pH 8 buffer</td><td>FisherSci</td><td>BP2473-100</td><td></td></tr><tr><td>Hardshell 96 Well Plate</td><td>BioRad</td><td>HSP-9601</td><td>For initial master mix and sample inoculation</td></tr><tr><td>Aluminum Sealing Film</td><td>BioRad</td><td>359-0133</td><td>To seal sample plate</td></tr><tr><td>Droplet Generator</td><td>BioRad</td><td>186-3002</td><td></td></tr><tr><td>Droplet Generation Oil</td><td>BioRad</td><td>186-3005</td><td></td></tr><tr><td>Cartridge</td><td>BioRad</td><td>186-4008</td><td></td></tr><tr><td>DG8 Cartridge Holder</td><td>BioRad</td><td>186-3051</td><td></td></tr><tr><td>Gasket</td><td>BioRad</td><td>186-3009</td><td></td></tr><tr><td>20 &amp;#956;l pipet tips</td><td>Rainin</td><td>GP-L10F</td><td>For tansferring sample/master mix to cartidge</td></tr><tr><td>200 &amp;#956;l pipet tips</td><td>Rainin</td><td>GP-L200F</td><td>For transferring droplets to final Twin.Tec Plate</td></tr><tr><td>Twin.Tec 96 Well Plate</td><td>Eppendorf</td><td>951020320</td><td>For final droplets thermal cycling and reading</td></tr><tr><td>Pierceable Heat Seal Foil</td><td>BioRad</td><td>181-4040</td><td>To seal Twin.Tec plate before thermalcycling</td></tr><tr><td>PX1 PCR Plate Sealer</td><td>BioRad</td><td>181-4000</td><td></td></tr><tr><td>CFX96 Thermalcycler</td><td>BioRad</td><td>CFX96 and C1000</td><td>Only the thermal cycler is needed, no optics</td></tr><tr><td>QX100 Droplet Reader</td><td>BioRad</td><td>186-3001</td><td></td></tr><tr><td>Droplet Reader Oil</td><td>BioRad</td><td>186-3004</td><td></td></tr><tr><td>Droplet PCR Supermix&amp;nbsp;</td><td>BioRad</td><td>186-3024</td><td>&lt;em&gt;i.e.&lt;/em&gt; the digital PCR mix in manuscript</td></tr><tr><td>QuantaSoft software (v1.3.2)</td><td>Quantasoft&amp;nbsp;</td><td>QX100&amp;nbsp;</td><td>For viewing, analyzing, and exporting ddPCR data</td></tr><tr><td>Entero Forward Primer (Ent F1A)</td><td>Operon</td><td>GAGAAATTCCAAACGAACTTG</td><td>Alternative vendor can be used</td></tr><tr><td>Entero Reverse Primer (Ent R1)</td><td>Operon</td><td>CAGTGCTCTACCTCCATCATT</td><td>Alternative vendor can be used</td></tr><tr><td>Entero Probe (GPL813TQ)</td><td>Operon</td><td>[6-FAM]-TGG TTC TCT CCG AAA TAG CTT TAG GGC TA-[BHQ1]</td><td>Alternative vendor can be used, but the florophore has to be FAM</td></tr><tr><td>HF183 Forward Primer (HF183-1)</td><td>Operon</td><td>ATCATGAGTTCACATGTCCG</td><td>Alternative vendor can be used</td></tr><tr><td>HF183 Reverse Primer (BthetR1)</td><td>Operon</td><td>CGTAGGAGTTTGGACCGTGT</td><td>Alternative vendor can be used</td></tr><tr><td>HF183 Probe (BthetP1)</td><td>Operon</td><td>[6-HEX]-CTGAGAGGAAGGTCCCCC&lt;br/&gt; ACATTGGA-[BHQ1]</td><td>Alternative vendor can be used, but the florophore has to be HEX (if using VIC, then the appropriate matric compensation must be chosen.&amp;nbsp;</td></tr><tr><td>Positive control</td><td></td><td></td><td>Mixture of &lt;em&gt;E. faecalis&lt;/em&gt; genomic DNA and HF183 standard plasmid (ordered from IDT). For detailed methods in culturing &lt;em&gt;E. faecalis&lt;/em&gt; and sequences of the ordered HF183 plasmid, please see Cao &lt;em&gt;et al.&lt;/em&gt; 2015 (doi:10.1016/j.watres.2014.12.008). Commercilaly available &lt;em&gt;Enterococcus&lt;/em&gt; DNA standards (ATCC 29212Q-FZ) can also be used in the positive control in place of lab-prepared &lt;em&gt;E. faecalis&lt;/em&gt; genomic DNA.</td></tr></tbody></table>

a duplex digital pcr assay for simultaneous quantification of the enterococcus spp. and the human fecal-associated hf183 marker in waters

这份手稿描述了肠球菌的同时定量，和人类粪便相关HF183标记的全双工数字PCR检测（EntHF183 DPCR）。该EntHF183复式DPCR（简称EntHF183 DPCR批文）检测使用相同的引物和探针序列作为其公布的个别定量PCR（定量PCR）同行。同样，如之前的qPCR进行同样的水过滤和DNA提取过程之前运行DPCR遵循。然而，双工DPCR法拥有的qPCR实验几个优点。最重要的是，DPCR测定省去了运行的标准曲线，因此，相关的偏差和变异性，由它的目标直接定量。此外，虽然双工中的qPCR（ 即同时定量） 肠球菌和HF183往往导致的样品中的低丰度目标的严重低估，DPCR同时提供目标的一致定量，磨她在同一反应单独或同时定量。该DPCR测定也能容忍的PCR抑制剂浓度是幅度比那些通过qPCR耐受更高的一到两个数量级。这些优势使得EntHF183 DPCR测定特别有吸引力的，因为它同时提供了在环境水一般和人力相关粪便污染准确和可重复的信息，而不需要运行两个独立的qPCR测定。尽管它的优点在定量PCR中，DPCR测定与现有仪器的上定量限是约幅度比可实现的通过qPCR下部的四个数量。因此，需要一种用于高浓度的靶标生物体，如那些以下污水外溢通常观察到的测量稀释。

一个好的EntHF183双工DPCR运行应导致相对高一些接受滴（ 约 10,000-17,000）和荧光值相差比较大（ 约 5000）的消极和积极的液滴7。液滴的不寻常的低数目可能表示在上述液滴生成过程中的问题，和10,000液滴的截止是基于本文中所使用的液滴DPCR系统的经验数据表明。正面和负面的液滴之间疲软的分离（ 即小荧光差异）可能表明抑制或降低探头7。 总体而言，当定量PCR被校正为与标准相关联的偏差和不存在抑制7 EntHF183双工DPCR测定产生高度可比的结果，从单纯的qPCR。从EntHF183双工DPCR试验和心病结果响应单纯DPCR测定法是高度一致的，通常这两种格式7（ 图1）之间的区别。此外，该DPCR测定也可以容忍抑制剂浓度数量级大于由它的qPCR同行7（ 图2）的耐受性更高的一到两个数量。 <img alt="图1" src="/files/ftp_upload/53611/53611fig1.jpg" /> 图 1: 由EntHF183双工DPCR测定相应单纯DPCR测定左粪便和水样和面板和右面板的定量分别显示肠球菌和HF183量化，与双工和单工的结果之间的对应的相关系数（P &lt;0.001）。实线表示回归线和灰色阴影表明相应的标准误差。符号表示不同类型的样品（圆，三角<html> S和十字架表示粪便，淡水和海洋水样，分别）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53611/53611fig1large.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/53611/53611fig2.jpg" /> 图2:单工的qPCR和双工洁污DNA的HF183标记DPCR量化飙升随PCR抑制剂浓度（腐植酸）三角形和X交叉盘表示DPCR和qPCR量化分别。在不存在抑制剂的预期HF183量化被定义为95％置信区间（ 即 ，两个水平虚线之间）。 "-1"的结果表明未检出。 肠球菌定量显示了EntHF183双工DPCR法同样高的耐受性抑制剂比相应的qPCR分析7。="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53611/53611fig2large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters at JoVE.com

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

该原稿描述了一种双工数字PCR分析可以用于同时定量肠球菌和HF183遗传标记在娱乐用水一般与人类相关的粪便污染的指标。

双工数字PCR方法的同时定量<em&gt;肠球菌。</em&gt;和人的粪便相关HF183标记的水域

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

a-duplex-digital-pcr-assay-for-simultaneous-quantification

Nanyang Technological University

Research

JoVE Journal

Environment

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

9.7K 次观看.  Southern California Costal Water Research Project Authority. 该原稿描述了一种双工数字PCR分析可以用于同时定量肠球菌和HF183遗传标记在娱乐用水一般与人类相关的粪便污染的指标。 

视频: A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

科研

环境科学

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

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

遗传学

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better characterize the prevalence of these pathogens, EPA Method 1623 was developed and used to monitor levels of these organisms in US drinking water supplies 12. The method has three main parts; the first is the sample concentration in which at least 10 L of raw surface water is filtered. The organisms and trapped debris are then eluted from the filter and centrifuged to further concentrate the sample. The second part of the method uses an immunomagnetic separation procedure where the concentrated water sample is applied to immunomagnetic beads that specifically bind to the Cryptosporidium oocysts and Giardia cysts allowing for specific removal of the parasites from the concentrated debris. These (oo)cysts are then detached from the magnetic beads by an acid dissociation procedure. The final part of the method is the immunofluorescence staining and enumeration where (oo)cysts are applied to a slide, stained, and enumerated by microscopy.
Method 1623 has four listed sample concentration systems to capture Cryptosporidium oocysts and Giardia cysts in water: Envirochek filters (Pall Corporation, Ann Arbor, MI), Envirochek HV filters (Pall Corporation), Filta-Max filters (IDEXX, Westbrook, MA), or Continuous Flow Centrifugation (Haemonetics, Braintree, MA). However, Cryptosporidium and Giardia (oo)cyst recoveries have varied greatly depending on the source water matrix and filters used1,14. A new tangential flow hollow-fiber ultrafiltration (HFUF) system has recently been shown to be more efficient and more robust at recovering Cryptosporidium oocysts and Giardia cysts from various water matrices; moreover, it is less expensive than other capsule filter options and can concentrate multiple pathogens simultaneously1-3,5-8,10,11. In addition, previous studies by Hill and colleagues demonstrated that the HFUF significantly improved Cryptosporidium oocysts recoveries when directly compared with the Envirochek HV filters4. Additional modifications to the current methods have also been reported to improve method performance. Replacing the acid dissociation procedure with heat dissociation was shown to be more effective at separating Cryptosporidium from the magnetic beads in some matrices9,13 .
This protocol describes a modified Method 1623 that uses the new HFUF filtration system with the heat dissociation step. The use of HFUF with this modified Method is a less expensive alternative to current EPA Method 1623 filtration options and provides more flexibility by allowing the concentration of multiple organisms.

A Modified EPA Method 1623 that Uses Tangential Flow Hollow-fiber Ultrafiltration and Heat Dissociation Steps to Detect Waterborne Cryptosporidium and Giardia spp.

This protocol describes the use of a tangential flow hollow-fiber ultrafiltration sample concentration system and a heat dissociation as alternative steps for the detection of waterborne Cryptosporidium and Giardia species using EPA Method 1623.

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性<em&gt;隐</em&gt;和<em&gt;贾第虫属。</em

Cryptosporidium and Giardia species are two of the most prevalent protozoa that cause waterborne diarrheal disease outbreaks worldwide. To better ...

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species including invasive and (or) species at risk is the goal, typically accomplished by testing water and sediment for the presence of characteristic DNA signatures. Reliable and efficient procedures for the capture of eDNA are required, especially those that can be performed easily in the field by personnel with limited training and citizen scientists. The capture of eDNA using membrane filtration is widely used currently. This approach has inherent issues that include the choice of filter material and porosity, filter fouling, and time required on site for the process to be performed. Flocculation offers an alternative that can be easily implemented and applied to sampling regimes that strive to cover broad territories in limited time.

Capture of Environmental DNA eDNA from Water Samples by Flocculation

Environmental DNA (eDNA) is typically captured from water samples using alcohol precipitation, filtration, or flocculation. Presented here is an alternative protocol that uses lanthanum chloride to enable collection of dissolved and particulate-bound nucleic acids from water samples containing eukaryotic cells, prokaryotic cells, and virus.

The analysis of environmental DNA (eDNA) has become a widely used approach to problem solving in species management. The detection of cryptic species ...

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 ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

连续流PCR微流控芯片中 大肠杆 菌的扩增及毛细管电泳系统的检测

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

生物工程

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

双工数字PCR方法的同时定量

摘要

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此视频中的章节

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

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

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

开发和测试针对物种的定量PCR检测环境DNA应用

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

连续流PCR微流控芯片中大肠杆菌的扩增及毛细管电泳系统的检测

检测现场的

双工数字PCR方法的同时定量

摘要

探索更多视频

此视频中的章节

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

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

在复杂的临床和环境样本使用寡核苷酸加上荧光微多元检测细菌

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

一种改进的EPA方法1623使用切向流中空纤维超滤和热解离步骤检测水性

Capture of Environmental DNA (eDNA) from Water Samples by Flocculation

开发和测试针对物种的定量PCR检测环境DNA应用

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

连续流PCR微流控芯片中 大肠杆 菌的扩增及毛细管电泳系统的检测

检测现场的

连续流PCR微流控芯片中大肠杆菌的扩增及毛细管电泳系统的检测