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>

<ol>
	<li>Boehm, A. B., 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=Performance+of+forty-one+microbial+source+tracking+methods:+A+twenty-seven+lab+evaluation+study.">Performance of forty-one microbial source tracking methods: A twenty-seven lab evaluation study.</a> Water Res. 47 (18), 6812-6828 (2013).</li><li>Layton, B. 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=Performance+of+human+fecal+anaerobe-associated+PCR-based+assays+in+a+multi-laboratory+method+evaluation+study.">Performance of human fecal anaerobe-associated PCR-based assays in a multi-laboratory method evaluation study.</a> Water Res. 47 (18), 6897-6908 (2013).</li><li>Cao, Y., 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=Effect+of+platform,+reference+material,+and+quantification+model+on+enumeration+of+Enterococcus+by+quantitative+PCR+methods.">Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.</a> Water Res. 47 (1), 233-241 (2013).</li><li>Sivaganesan, M., Siefring, S., Varma, M., Haugland, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MPN+estimation+of+qPCR+target+sequence+recoveries+from+whole+cell+calibrator+samples.">MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.</a> J. Microbiol. Methods. 87 (3), 343-349 (2011).</li><li>Huggett, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Digital+MIQE+Guidelines:+Minimum+Information+for+Publication+of+Quantitative+Digital+PCR+Experiments.">The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments.</a> Clin. Chem. 59 (6), 892-902 (2013).</li><li>Cao, Y., Raith, M. R., Griffith, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+digital+PCR+for+simultaneous+quantification+of+general+and+human-associated+fecal+indicators+for+water+quality+assessment.">Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment.</a> Water Res. 70, 337-349 (2015).</li><li>Sanders, R., Huggett, J. F., Bushell, C. A., Cowen, S., Scott, D. J., Foy, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+Digital+PCR+for+Absolute+DNA+Quantification.">Evaluation of Digital PCR for Absolute DNA Quantification.</a> Anal. Chem. 83 (17), 6474-6484 (2011).</li><li>Whale, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microfluidic+digital+PCR+and+conventional+quantitative+PCR+for+measuring+copy+number+variation.">Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation.</a> Nucleic Acid Res. 40 (11), 82-89 (2012).</li><li>Hindson, C. 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=Absolute+quantification+by+droplet+digital+PCR+versus+analog+real-time+PCR.">Absolute quantification by droplet digital PCR versus analog real-time PCR.</a> Nature Methods. 10 (10), 1003-1005 (2013).</li><li>Morisset, D., &#352;tebih, D., Milavec, M., Gruden, K., &#381;el, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+food+and+feed+aamples+with+droplet+digital+PCR.">Quantitative analysis of food and feed aamples with droplet digital PCR.</a> PLoS One. 8 (5), e62583 (2013).</li><li>Cao, Y., 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=Evaluation+of+molecular+community+analysis+methods+for+discerning+fecal+sources+and+human+waste.">Evaluation of molecular community analysis methods for discerning fecal sources and human waste.</a> Water Res. 47 (18), 6862-6872 (2013).</li></ol>

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。

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			<td height="21" style="height:21px;width:265px;">Low Bind Microtubes</td>
			<td style="width:84px;">Costar</td>
			<td style="width:439px;">3207</td>
			<td style="width:575px;">For storage of reagents, samples/production of master mixes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Nuclease Free Water</td>
			<td style="width:84px;">FisherSci</td>
			<td style="width:439px;">BP2484-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">TE pH 8 buffer</td>
			<td style="width:84px;">FisherSci</td>
			<td style="width:439px;">BP2473-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Hardshell 96 Well Plate</td>
			<td style="width:84px;">BioRad</td>
			<td>HSP-9601</td>
			<td>For initial master mix and sample inoculation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Aluminum Sealing Film</td>
			<td style="width:84px;">BioRad</td>
			<td>359-0133</td>
			<td>To seal sample plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Droplet Generator</td>
			<td style="width:84px;">BioRad</td>
			<td style="width:439px;">186-3002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Droplet Generation Oil</td>
			<td style="width:84px;">BioRad</td>
			<td>186-3005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Cartridge</td>
			<td style="width:84px;">BioRad</td>
			<td>186-4008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">DG8 Cartridge Holder</td>
			<td style="width:84px;">BioRad</td>
			<td>186-3051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Gasket</td>
			<td style="width:84px;">BioRad</td>
			<td>186-3009</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">20uL pipet tips</td>
			<td style="width:84px;">Rainin</td>
			<td style="width:439px;">GP-L10F</td>
			<td>For tansferring sample/master mix to cartidge</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">200uL pipet tips</td>
			<td style="width:84px;">Rainin</td>
			<td style="width:439px;">GP-L200F</td>
			<td>For transferring droplets to final Twin.Tec Plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Twin.Tec 96 Well Plate</td>
			<td style="width:84px;">Eppendorf</td>
			<td style="width:439px;">951020320</td>
			<td>For final droplets thermal cycling and reading</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Pierceable Heat Seal Foil</td>
			<td style="width:84px;">BioRad</td>
			<td>181-4040</td>
			<td>To seal Twin.Tec plate before thermalcycling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">PX1 PCR Plate Sealer</td>
			<td style="width:84px;">BioRad</td>
			<td style="width:439px;">181-4000</td>
			<td>Only the thermal cycler is needed, no optics</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">CFX96 Thermalcycler</td>
			<td style="width:84px;">BioRad</td>
			<td style="width:439px;">CFX96 and C1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">QX100 Droplet Reader</td>
			<td style="width:84px;">BioRad</td>
			<td style="width:439px;">186-3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Droplet Reader Oil</td>
			<td style="width:84px;">BioRad</td>
			<td>186-3004</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Droplet PCR Supermix&#160;</td>
			<td style="width:84px;">BioRad</td>
			<td style="width:439px;">186-3024</td>
			<td>i.e. the digital PCR mix in manuscript</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QuantaSoft software (v1.3.2)</td>
			<td style="width:84px;">Quantasoft&#160;</td>
			<td style="width:439px;">QX100&#160;</td>
			<td>For viewing, analyzing, and exporting ddPCR data</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Entero Forward Primer (Ent F1A)</td>
			<td style="width:84px;">Operon</td>
			<td>GAGAAATTCCAAACGAACTTG</td>
			<td>Alternative vendor can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Entero Reverse Primer (Ent R1)</td>
			<td style="width:84px;">Operon</td>
			<td>CAGTGCTCTACCTCCATCATT</td>
			<td>Alternative vendor can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Entero Probe (GPL813TQ)</td>
			<td style="width:84px;">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 height="21">
			<td height="21" style="height:21px;width:265px;">HF183 Forward Primer (HF183-1)</td>
			<td style="width:84px;">Operon</td>
			<td>ATCATGAGTTCACATGTCCG</td>
			<td>Alternative vendor can be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">HF183 Reverse Primer (BthetR1)</td>
			<td style="width:84px;">Operon</td>
			<td>CGTAGGAGTTTGGACCGTGT</td>
			<td>Alternative vendor can be used</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:265px;">HF183 Probe (BthetP1)</td>
			<td style="width:84px;">Operon</td>
			<td>[6-HEX]-CTGAGAGGAAGGTCCCCC 
			ACATTGGA-[BHQ1]</td>
			<td style="width:575px;">Alternative vendor can be used, but the florophore has to be HEX (if using VIC, then the appropriate matric compensation must be chosen.&#160;</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:265px;">Positive control</td>
			<td style="width:84px;"></td>
			<td style="width:439px;"></td>
			<td style="width:575px;">Mixture of E.faecalius genomic DNA and HF183 standard plasmid (ordered from IDT). For detailed methods in culturing E. faecalius and sequences of the ordered HF183 plasmid, please see Cao et al 2015 (doi:10.1016/j.watres.2014.12.008). Commercilaly available Enterococcus DNA standards (ATCC 29212Q-FZ) can also be used in the positive control in place of lab-prepared E. faecalis 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

Determination of Microbial Extracellular Enzyme Activity in Waters, Soils, and Sediments using High Throughput Microplate Assays

Much of the nutrient cycling and carbon processing in natural environments occurs through the activity of extracellular enzymes released by microorganisms. Thus, measurement of the activity of these extracellular enzymes can give insights into the rates of ecosystem level processes, such as organic matter decomposition or nitrogen and phosphorus mineralization. Assays of extracellular enzyme activity in environmental samples typically involve exposing the samples to artificial colorimetric or fluorometric substrates and tracking the rate of substrate hydrolysis. Here we describe microplate based methods for these procedures that allow the analysis of large numbers of samples within a short time frame. Samples are allowed to react with artificial substrates within 96-well microplates or deep well microplate blocks, and enzyme activity is subsequently determined by absorption or fluorescence of the resulting end product using a typical microplate reader or fluorometer. Such high throughput procedures not only facilitate comparisons between spatially separate sites or ecosystems, but also substantially reduce the cost of such assays by reducing overall reagent volumes needed per sample.

Microplate based procedures are described for the colorimetric or fluorometric analysis of extracellular enzyme activity. These procedures allow for the rapid assay of such activity in large numbers of environmental samples within a manageable time frame.

在使用高通量微孔板测定水，土壤，沉积物和微生物测定胞外酶活性的

Much of the nutrient cycling and carbon  processing in natural environments occurs through the activity of extracellular  enzymes released by ...

科研

环境科学

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of agricultural waters. Here, water is filtered through sterile Modified Moore Swabs (MMS), which consist&#160;of a simple gauze filter enclosed in a plastic cartridge, to concentrate bacteria. Following filtration, non-selective or selective enrichments for the target bacteria are performed in the MMS. For colorimetric detection of the target bacteria, the enrichments are then assayed using paper-based analytical devices (&#181;PADs) embedded with bacteria-indicative substrates. Each substrate reacts with target-indicative bacterial enzymes, generating colored products that can be detected visually (qualitative detection) on the &#181;PAD. Alternatively, digital images of the reacted &#181;PADs can be generated with common scanning or photographic devices and analyzed using ImageJ software, allowing for more objective and standardized interpretation of results. Although the biochemical screening procedures are designed to identify the aforementioned bacterial pathogens, in some cases enzymes produced by background microbiota or the degradation of the colorimetric substrates may produce a false positive. Therefore, confirmation using a more discriminatory diagnostic is needed. Nonetheless, this bacterial concentration and detection platform is inexpensive, sensitive (0.1 CFU/ml detection limit), easy to perform, and rapid (concentration, enrichment, and detection are performed within approximately 24&#160;hr), justifying its use as an initial screening method for the microbiological quality of agricultural water.

Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water

A protocol involving integrated concentration, enrichment, and end-point colorimetric detection of foodborne pathogens in large volumes of agricultural water is presented here. Water is filtered through Modified Moore Swabs (MMS), enriched with selective or non-selective media, and detection is performed using paper-based analytical devices (&#181;PAD) imbedded with bacterial-indicative colorimetric substrates.

比色纸为基础的检测<em&gt;大肠杆菌</em&gt;<em&gt;沙门氏菌</em&gt;属，和<em&gt;单核细胞增生李斯特氏菌</em&gt;从农业用水大容量

This protocol describes rapid colorimetric detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from large volumes (10 L) of ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Clean Sampling and Analysis of River and Estuarine Waters for Trace Metal Studies

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination control. Developments of some techniques aiming to reduce trace metal contamination in the last couple of decades have resulted in concentrations reported now being orders of magnitude lower than those in the past. These low concentrations often necessitate preconcentration of water samples prior to instrumental analysis of samples. Since contamination can appear in all phases of trace metal analyses, including sample collection (and during preparation of sampling containers), storage and handling, pretreatments, and instrumental analysis, specific care needs to be taken in order to reduce contamination levels at all steps. The effort to develop and utilize "clean techniques" in trace metal studies allows scientists to investigate trace metal distributions and chemical and biological behavior in greater details. This advancement also provides the required accuracy and precision of trace metal data allowing for environmental conditions to be related to trace metal concentrations in aquatic environments.
This protocol that is presented here details needed materials for sample preparation, sample collection, sample pretreatment including preconcentration, and instrumental analysis. By reducing contamination throughout all phases mentioned above for trace metal analysis, much lower detection limits and thus accuracy can be achieved. The effectiveness of "clean techniques" is further demonstrated using low field blanks and good recoveries for standard reference material. The data quality that can be obtained thus enables the assessment of trace metal distributions and their relationships to environmental parameters.

Special care using "clean techniques" is required to properly collect and process water samples for trace metal studies in aquatic environments. A protocol for sampling, processing, and analytical procedures with the aim of obtaining reliable environmental monitoring data and results with high sensitivity for detailed trace metal studies is presented.

清洁取样和痕量金属研究河和河口水域分析

Most of the trace metal concentrations in ambient waters obtained a few decades ago have been considered unreliable owing to the lack of contamination ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of scientific publications address microplastic pollution of the sea surface. The protocol below describes the methodology for sampling, sample preparation, separation and chemical identification of microplastic particles. A manta net fixed on an &#187;A frame&#171; attached to the side of the vessel was used for sampling. Microplastic particles caught in the cod end of the net were separated from samples by visual identification and use of stereomicroscopes. Particles were analyzed for their size using an image analysis program and for their chemical structure using ATR-FTIR and micro FTIR spectroscopy. The described protocol is in line with recommendations for microplastics monitoring published by the Marine Strategy Framework Directive (MSFD) Technical Subgroup on Marine Litter. This written protocol with video guide will support the work of researchers that deal with microplastics monitoring all over the world.

The protocol below describes the methodology for: microplastics sampling on the sea surface, separation of microplastic and chemical identification of particles. This protocol is in line with the recommendations for microplastics monitoring published by the MSFD Technical Subgroup on Marine Litter.

协议塑料微粒采样的海面和样品分析

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment during manufacturing and transport. Because of their environmental persistence, they are widely distributed in the oceans and on beaches all over the world. They can act as a vector of potentially toxic organic compounds (e.g., polychlorinated biphenyls) and might consequently negatively affect marine organisms. Their possible impacts along the food chain are not yet well understood. In order to assess the hazards associated with the occurrence of plastic pellets in the marine environment, it is necessary to develop methodologies that allow for rapid determination of associated organic contaminant levels. The present protocol describes the different steps required for sampling resin pellets, analyzing adsorbed organochlorine pesticides (OCPs) and identifying the plastic type. The focus is on the extraction of OCPs from plastic pellets by means of a pressurized fluid extractor (PFE) and on the polymer chemical analysis applying Fourier Transform-InfraRed (FT-IR) spectroscopy. The developed methodology focuses on 11 OCPs and related compounds, including dichlorodiphenyltrichloroethane (DDT) and its two main metabolites, lindane and two production isomers, as well as the two biologically active isomers of technical endosulfan. This protocol constitutes a simple and rapid alternative to existing methodology for evaluating the concentration of organic contaminants adsorbed on plastic pieces.

Microplastics act as vector of potentially toxic organic contaminants with unpredictable effects. This protocol describes an alternative methodology for assessing the levels of organochlorine pesticides adsorbed on plastic pellets and identifying the polymer chemical structure. The focus is on pressurized fluid extraction and attenuated total reflectance Fourier transform infrared spectroscopy.

从塑料颗粒中提取有机氯农药和塑料类型分析

Plastic resin pellets, categorized as microplastics (&#8804;5 mm in diameter), are small granules that can be unintentionally released to the environment ...

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf mottling, vein yellowing, defoliation, root decay, and ultimately, the death of the citrus plant. When infected, the citrus plants have stunted growth and produce flowers out of season. These flowers rarely yield fruit, and those that do yield small, bitter, irregularly shaped citrus fruit that are not desirable. This disease is spread by the Asian citrus psyllid, Diaphorina citri, and by the grafting of infected citrus tissue. The pathogen has a long and variable incubation period within the citrus plant&#8212;sometimes years, before symptoms appear. Attempts to culture this pathogen in vitro have been unsuccessful, possibly due to the low and uneven concentration of the pathogen within infected citrus tissue, or because it is difficult to replicate the environmental conditions conducive to growth of the pathogen. It is very difficult to identify the disease before it has spread, due to its long incubation period and researchers&#39; inability to culture the pathogen. As a result, the disease only becomes apparent after suddenly destroying a citrus farmer&#39;s entire yield. Presented here is a method for the accurate and specific detection of the citrus greening pathogen, Candidatus liberibacter spp. using a genomic DNA extraction kit and PCR. This method is simple, efficient, cost effective, and adaptable for quantitative analysis. This method can be adapted for use on any citrus tissue; however, it is potentially limited by the amount of pathogen present in the tissue. Nevertheless, this method will allow citrus farmers to identify infected citrus plants earlier, and curb the spread of this destructive disease before it can further spread.

Specific and Accurate Detection of the Citrus Greening Pathogen Candidatus liberibacter  spp. Using Conventional PCR on Citrus Leaf Tissue Samples

Citrus Greening is a particularly destructive disease affecting citrus crops globally. Presented here is a simple method using PCR and genomic DNA extraction of citrus leaf tissue for the accurate and precise identification of the citrus greening pathogen, Candidatus liberibacter spp.

利用常规 PCR 技术对柑桔叶片组织样品Candidatus liberibacter的特异和准确检测

Citrus greening, also known as huanglongbing, is a destructive citrus disease ravaging citrus farms globally. This disease causes asymmetrical yellow leaf ...

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus (Murayama), Using a Low-Cost, Small, and Easily Constructed Flight Mill

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). Therefore, knowing the dispersal capacity may help inform trapping/tree removal efforts to prevent this disease more effectively. In this study, we measured the flight velocity and duration and estimated the flight distance of the beetle using a newly developed flight mill. The flight mill is low cost, small, and constructed using commonly available items. Both the flight mill arm and its vertical axis comprise a thin needle. A beetle specimen is glued to one tip of the arm using instant glue. The other tip is thick due to being covered with plastic, thus it facilitates the detection of rotations of the arm. The revolution of the arm is detected by a photo sensor mounted on an infrared LED, and is indicated by a change in the output voltage when the arm passed above the LED. The photo sensor is connected to a personal computer and the output voltage data are stored at a sampling rate of 1 kHz. By conducting experiments using this flight mill, we found that P. quercivorus can fly at least 27 km. Because our flight mill comprises cheap and small ordinary items, many flight mills can be prepared and used simultaneously in a small laboratory space. This enables experimenters to obtain a sufficient amount of data within a short period.

Measuring the Flight Ability of the Ambrosia Beetle, Platypus Quercivorus Murayama, Using a Low-Cost, Small, and Easily Constructed Flight Mill

We developed a low cost and small flight mill, constructed with commonly available items and easily used in experimentation. Using this apparatus, we measured the flight ability of an ambrosia beetle, Platypus quercivorus.

利用低成本、小型、易于建造的Quercivorus测量豚草甲虫、鸭嘴兽的飞行能力 (村山富市)

The ambrosia beetle, Platypus quercivorus (Murayama), is the vector of a fungal pathogen that causes mass mortality of Fagaceae trees (Japanese oak wilt). ...

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

从刺客臭虫和其他 Heteropteran 昆虫身上采集毒液毒素

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

一种用自动表型分析预测线虫化学毒性的高通量测定

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...