We gratefully acknowledge funding for this project from the USDA National Institute of Food and Agriculture grants 2009-01208 and 2009-01984.

细菌1。从农业用水的使用MMS大体积浓度<ol><li>彩信准备<ol><li>剪下的4层纱布测量40×12厘米的矩形截面。 </li><li>折沿两个轴的干酪，得到20×6厘米的矩形。 </li><li>滚动纱布沿其长轴紧密，形成大约6公分，直径3厘米的圆柱形棒。 </li><li>在高压灭菌铝箔粗棉布擦拭，不要高压磁带。净化盒通过浸泡在10％家用漂白剂30分钟，浸泡在盒15分钟，在一个五水合硫代硫酸钠（的Na 2 S 2 O 3·5H 2 O）溶液（50 mg / L的失活的残留氯在无菌水中制备）。 </li><li>将拭子插入彩信磁带。 注意：如果MMS盒的非一次性使用的版本，从粗棉布的4 - 层片我asuring 80×22厘米，把它折叠40×11厘米，把它卷成圆筒状约11公分，直径6厘米。 </li><li>组装彩信。 </li><li>连接乙烯管到轴颈上的彩信的两侧，保证了管材完全粘在轴颈，并修复管道进入蠕动泵。确保该蠕动泵的管道上游有足够的长度用于采样。 </li></ol></li><li>细菌浓度<ol><li>样品至少10个通过运行蠕动泵在高速升农业用水。 </li><li>取样结束后，从水样品撤回蠕动泵的进，继续运行泵，直到没有出水观察退出MMS。 </li><li>如果歧管是用来在同一时间执行多个样品，收集每个MMS的流出物，并且当10 L被通过一个单一的MMS过滤关闭该阀用于该特定MMS和继续运行泵。 <ol><li>当采样我已落成，从农业用水撤回蠕动泵的入口，打开歧管的所有阀门，并继续运行泵，直到没有出水观察退出彩信。 </li></ol></li></ol></li><li>当浓度已经完成所有彩信，小心地从彩信轴颈除去乙烯管和盖在轴颈上的彩信的两侧。盖紧彩信可以添加丰富的媒体之前存储长达12小时。 </li></ol> 2，彩信富集和样品制备<ol><li>丰富<ol><li>拧开彩信的盖子，并在无菌条件下加入20毫升无菌适当浓缩肉汤。使用一个彩信每个选择性富集。另外，执行非选择性富集传播这三个目标细菌在同一样品中。 注意：如果MMS盒的非一次性使用的版本，在无菌条件下从纸盒中取出粗棉布过滤，并放置在无菌袋广告之前鼎225毫升适当浓缩介质。 <ol><li>对于E的浓缩大肠杆菌 ，加入20毫升缓冲蛋白胨水（BPW）补充有盐酸万古霉素的8毫克/升。 </li><li>对于沙门氏菌的富集。，添加20毫升BPW的补充与补充沙门氏菌 （4毫升/升）。 </li><li>对于L的浓缩菌 ，加入20毫升VIDAS UP李斯特菌（LPT）肉汤。 </li><li>同时对于非选择性富集使用单一MMS的全部三种微生物，使用通用preenrichment肉汤（UPB）。 </li></ol></li><li>用螺丝将彩信的后盖盖上紧。确保轴颈盖紧，以免富集过程中泄漏和可能的污染。 </li><li>孵育MMS长达18-24小时，在摇动培养箱中于200转。用42°C为E。大肠杆菌和沙门氏菌 。选择性富集。用30℃L。菌选择性富集，并用37＆＃176，C为非选择性富集。 </li></ol></li><li>样品制备<ol><li>当孵化完成后，从孵化器中删除彩信，开盖的轴颈之一，并轻轻免除约0.5ml至1.5 ml离心管中。另外，拧开MMS和吸管出0.5毫升一夜致富。 注意：如果MMS磁带的未一次性使用的版本，吸取0.5毫升从包里富集。 </li><li>对于E的比色分析大肠杆菌选择性富集，或用于非选择性富集的分析，超声处理0.5毫升富集于5瓦，22千赫，持续20秒使用探针超声波仪。 </li></ol></li></ol> 3，准备和比​​色法基质嵌入在μPADs <ol><li>准备足够的μPADs每个样品和目标进行测试。为了检测大肠杆菌大肠杆菌准备CPRG和X-GLUCμPADs，为沙门氏杆菌 。检测准备管委会μPADs，和L。 monocytogenes检测准备的X磷化铟μPADs。 <ol><li>准备HEPES缓冲液[0.1M的HEPES和0.1％牛血清白蛋白（BSA）]的比色底物并根据基板，是要被用于调节缓冲液的pH：pH为7.5制备CPRG或X-GLUC的， pH值为9为MC，和pH为7的X的InP。 </li><li>制备储备溶液的pH值在基板中的3毫米（CPRG），62.5毫米（X-GLUC），15毫米（MC），100毫米（X-InP）材料的浓度调节HEPES缓冲液。避光股票的解决方案。 </li><li>将μPADs无菌培养皿内，然后嵌入的μPAD微点与24微升底物各原液。 </li></ol></li></ol> 4，检测和数据分析<ol><li>发现<ol><li>加入6微升的富集样品中所含的培养皿中各适当μPAD微点。 </li><li>将外盖放回培养皿中，并用封口膜密封。 </li><li>孵育样品与μPAD在37℃达3小时，以允许发色微光点​​和干燥。 </li></ol></li><li>结果的目视检查<ol><li>通过的μPADs的颜色变化的简单视觉检查进行检测。 注：通常情况下，产生最终的颜色变化的强烈反应，预计下18-24小时富集，应避免需要进一步澄清，应视为阳性。 </li><li>确定大肠杆菌的存在通过观察微光嵌有从黄色CPRG变化到红紫色和微光嵌有由无色的X GLUC变化至蓝绿色的大肠杆菌阳性样品。 </li><li>确定L的存在通过观察嵌入微光使用X-InP的变化由无色变成靛蓝菌阳性样品。 </li><li>确定沙门氏菌的存在。由嵌有从山坳MC变化观测微光阳性样品orless到紫色紫红色。 </li></ol></li><li>软件辅助数据分析 注：传导，以明确判断弱反应是正还是负，以使结果的半定量分析。 <ol><li>让μPADs充分干燥。 </li><li>使用平板扫描器扫描μPAD。 </li><li>使用ImageJ的软件来处理图像。 <ol><li> “打开”扫描图像中的ImageJ位于主菜单（或按“CLT + O”）上的“文件”选项卡下进行分析。 </li><li>反转图像选择“反转”位于主菜单上的“编辑”选项卡下（或按“CLT + SHIFT + I”）。 </li><li>使用主菜单上的“分析”选项卡，并选择“设置测量...”，选择分析报告的测量。 </li><li>确保“平均灰度强度”，“限阈值”和“显示标签”框勾选，然后按“确定”，这是如何设置的软件分析所选择的每个领域，以及它如何报告它。 </li><li>使用“椭圆”工具，绘制一个圆，列出您希望在测量区域。 </li><li>在“分析”选项卡中选择“测量”（或按“CLT + M”）。该软件将测量的平均灰度值定义的区域内，并在可以复制并粘贴到Excel作进一步分析一个弹出式屏幕举报。 注：为了更准确的分析，每个样品至少两个技术复制应该被执行。 </li></ol></li></ol></li></ol>

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The authors have nothing to disclose.

本协议描述了用于检测大肠杆菌集成方法大肠杆菌 ， 沙门氏菌和L。菌在农业用水。这里，细菌从大量的农业用水MMS浓度（10升），被加上细菌富集，并使用μPADs细菌，指示比色检测。彩信程序可以应付水样中高颗粒含量而集中的细菌10倍，是强大和现场应用通过微创培训的人员足够简单，并且可以以最小的成本来完成。通过将细菌富集步骤，活细菌进行检测，该方法的...

重要的是，食源性疾病代理人，以减少食源性疾病的负担迅速，最好在检测场为基础的环境。共同战略，以检测食源性致病菌包括生化分析，选择性和鉴别培养，免疫隔离和检测和分子检测。然而，这些方法是由零星污染的阻碍，测试样本量小，则往往低浓度的食源性致病菌，需要很长的处理时间，和/或不适用于字段设置。此外，在许多食物基质的化合物是抑制到检测和诊断应用。为了提高微生物检测的可能性，美国食品和药物管理局已提出，测试农业用水（如洗涤用水和灌溉用水），其中任一接触到新鲜产品有很大的表面积接触或用作车辆对produce污染是一个可行的替代食品1的直接测试。即便如此，往往低自然病原体负担再加上代表性的农业水样的稀释作用使样品制备方法的病原体浓度至关重要。这种方法将需要采样大量的水（≥10 L），适当的病原体浓度，并与下游检测策略的兼容性。 修改摩尔拭子（MMS）是用于从浓缩水2-4大容量（≥10L）细菌便宜，简单，坚固耐用的设备。彩信由一个塑料盒填充用纱布，它作为一个粗过滤器，大体积的水，用蠕动泵通过盒泵入的。彩信是细菌浓度（≥10倍浓缩）的非歧视性的方法，捕捉有机和无机颗粒材料，包括在处理里的微生物镑样品。它很可能是由MMS靶微生物的浓度的优良功效可通过一个事实，即微生物预期将附着在粉砂质粘土组分或有机微聚集的悬浮物3进行说明。坚固耐用的设计，彩信允许克服与其它过滤方法捕获从水中细菌，如堵塞的过滤器，无法处理大量，过滤器样品浊度高，而且成本高和浓度有关的大多数缺点。由于这些原因，美国食品药品管理局的建议将彩信的纳入环境和生产相关的样品收集程序5官方程序。 这里，描述了一种方法用于浓缩，富集和检测大肠杆菌 ， 沙门氏菌 ， 单增李斯特菌来自农业的水域。彩信用于BACT的浓度ERIA，并且还用作用于选择性或非选择性富集细菌的容器。使用的纸张为基础的分析设备（μPADs）6达到细菌检测生化。 μPADs可以被制造为流体网络，或使用了多种方法，包括光刻法，喷墨印刷，压印，和蜡印7-11斑点试验。的流体设计的例子可以是其中样品被沉积在中间，并随后流入其中样品或基底被从通道的外储层被拉通过毛细作用进入中心12前端水库或单声道模式的树突状通道模式。对于这个协议，我们选择采用的嵌有显色底物7毫米直径的腊纸点阵列，可以通过指示在这里测试的微生物的酶进行处理：氯酚红-β-D-吡喃半乳糖苷（CPRG）和5 - 溴-4 - 氯-3 - 吲哚基β-D-葡糖苷酸（X-GLUC）用于检测β-半乳糖苷酶和β-葡糖苷酸酶由大肠杆菌产生大肠杆菌 ; 5 -溴-6 -氯-3 -吲哚辛酸盐（品红辛酸盐），用于由沙门菌属生产的C8-酯酶的检测;和5 -溴-4 -氯-3 -吲哚基- 肌醇 -磷酸（X-磷化铟），用于检测磷脂酰肌醇特异性磷脂酶C（PI-PLC）制作的L。菌等 6。因此，一个特定的细菌的存在下，可以目视观察，而不需要复杂的设备或数据的解释。的基于酶的比色μPAD检测这些特定的目标细菌的特异性和灵敏度先前已探索6。此外，对于这些靶细菌的集成浓度检测方法的灵敏度由尖峰的大体积的水的微生物（未发表数据和比沙等人 13）的预先确定的水平进行评价。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Agricultural water</td>
			<td></td>
			<td></td>
			<td>Irrigation water, produce wash water, well water, etc.</td>
		</tr>
		<tr>
			<td>Vinyl tubing</td>
			<td>Wilmar</td>
			<td>BN-CVT1005</td>
			<td>&#160;1/4&#34; inner diameter,&#160; 3/8&#34; outer diameter, available at:&#160; http://www.wilmar.com</td>
		</tr>
		<tr>
			<td>Modified Moore Swab cartridge&#160;</td>
			<td>Lumiere Diagnostics</td>
			<td></td>
			<td>11 &#189; cm in length and 4 &#189; cm in width, available at:&#160; http://www.lumierediagnostics.com.&#160; Alternativelly, a non-disposable version of the cartridge can be used (refer to the text)</td>
		</tr>
		<tr>
			<td>Cheesecloth</td>
			<td>Chesapeake Wiper &#38; Supply, Inc.</td>
			<td>CC90</td>
			<td>Grade #90, 44 &#215; 36 weave, available at:&#160; www.raglady.com</td>
		</tr>
		<tr>
			<td>Household Bleach</td>
			<td>Various</td>
			<td></td>
			<td>Sodium hypochlorite concentration approx. 6%</td>
		</tr>
		<tr>
			<td>Sodium thiosulphate 5-hydrate</td>
			<td>Mallinckrodt Baker Inc</td>
			<td>8100-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Manifold</td>
			<td>Built in-house</td>
			<td></td>
			<td>Optional, device can be constructed from PVC pipes and appropriate fittings</td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Micron Meters</td>
			<td>RPP1300</td>
			<td>Available at:&#160; http://www.micronmeters.com</td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Various</td>
			<td></td>
			<td>Disposable, 10ml</td>
		</tr>
		<tr>
			<td>Universal preenrichment broth</td>
			<td>Difco</td>
			<td>223510</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffered peptone water</td>
			<td>Difco</td>
			<td>218105</td>
			<td></td>
		</tr>
		<tr>
			<td>Salmonella supplement</td>
			<td>Biom&#233;rieux Industry</td>
			<td>42650</td>
			<td>http://www.biomerieux-usa.com</td>
		</tr>
		<tr>
			<td>VIDAS UP Listeria (LPT) Broth</td>
			<td>Biom&#233;rieux Industry</td>
			<td>410848</td>
			<td>http://www.biomerieux-usa.com</td>
		</tr>
		<tr>
			<td>Vancomycin</td>
			<td>Sigma-Aldrich</td>
			<td>861987</td>
			<td>http://www.sigmaaldrich.com</td>
		</tr>
		<tr>
			<td>Pipet-Aid</td>
			<td>Various</td>
			<td></td>
			<td>Drummond DP-110 used here</td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Various</td>
			<td></td>
			<td>Excella E25, New Brunswick Scientific used here</td>
		</tr>
		<tr>
			<td>Micropipette&#160;</td>
			<td>Various</td>
			<td></td>
			<td>10 &#956;l, 1 ml</td>
		</tr>
		<tr>
			<td>Micropipette tips</td>
			<td>Various</td>
			<td></td>
			<td>Barrier, 10 &#956;l, 1 ml</td>
		</tr>
		<tr>
			<td>1.5 microcentrifuge tubes</td>
			<td>Various</td>
			<td></td>
			<td>RNase- and DNase- free</td>
		</tr>
		<tr>
			<td>Probe sonicator</td>
			<td>Q Sonica LLC</td>
			<td>XL-2000 series</td>
			<td></td>
		</tr>
		<tr>
			<td>&#181;PADs</td>
			<td>Avant&#160;</td>
			<td></td>
			<td>Wax printed 7 mm diameter circles, with 4 pt line thickness. Contact Dr. Charles Henry for additional information</td>
		</tr>
		<tr>
			<td>HEPES [N-(2-Hydroxyethyl)piperazine-N&#8242;-2-ethanesulfonic acid]</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A8022</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorophenol red-galactopyranoside (CPRG)</td>
			<td>Sigma-Aldrich</td>
			<td>59767</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-glucuronide (X-Gluc)</td>
			<td>Sigma-Aldrich</td>
			<td>B8174</td>
			<td></td>
		</tr>
		<tr>
			<td>5-bromo-6-chloro-3 indolylcaprylate (magenta caprylate)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>53451</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-myo-inositol phosphate (X-InP)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>38896</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, polystyrene 100mm by 15 mm</td>
			<td>Various</td>
			<td></td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Flat bed scanner</td>
			<td>Various</td>
			<td></td>
			<td>Xerox USB scanner</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>http://rsb.info.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

colorimetric paper-based detection of escherichia coli, salmonella spp., and listeria monocytogenes from large volumes of agricultural water

本协议描述快速比色检测农产品水域的大肠杆菌 ， 沙门氏菌和李斯特菌的大量（10升）。这里，水通过无菌改性摩尔棉签（MMS），它由装在一个塑料盒的简单纱布过滤器的过滤，浓缩的细菌。过滤后，非选择性或选择性富集的靶细菌在MMS中进行。对于比色法检测目标细菌的富集，然后用纸张为基础的嵌入式细菌指示基板分析设备（μPADs）测定。每个基片会与目标指示细菌的酶，产生可以目视（定性检测）上μPAD来检测有色产物。可替换地，可以使用公共扫描或摄影装置生成并使用ImageJ软件，人分析了反应μPADs的数字图像仰角调整器的结果更加客观和标准化的解释。尽管生化筛查程序的设计，以确定上述的细菌病原体，在某些情况下背景微生物或比色底物的降解​​酶的产生可能会产生假阳性。因此，需要确认使用的是更多的歧视诊断。然而，这种细菌浓度和检测平台价格低廉，敏感（0.1 CFU / ml的检测限），易于操作，快速的（是在大约24小时进行浓缩，富集和检测），其理由作为初步筛选的使用方法对农业用水的微生物质量。

如本方案所述，可以在约15-20分钟来进行浓度使用MMS细菌（ 图1）。在MMS从丙烯腈 - 丁二烯 - 苯乙烯（ABS）在两个分开的部件构成;一个盖和盒都带有集成轴颈组件被插入其中的圆筒形干酪拭子（ 图1A）。然后这两个组件被拧在一起形成MMS（ 图1B）。基于MMS的处理是由一个电池供电的蠕动泵驱动的，允许样品被集中在字段设置。通过耦合MMS浓度，选择性富集（18...

Watch this Scientific Journal Video about Colorimetric Paper-based Detection of Escherichia coli, Salmonella spp., and Listeria monocytogenes from Large Volumes of Agricultural Water at JoVE.com

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 ...

colorimetric-paper-based-detection-escherichia-coli-salmonella-spp

Research

JoVE Journal

Environment

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

14.3K 次观看.  University of Wyoming. 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 (µPAD) imbedded with bacterial-indicative colorimetric substrates.

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

Laboratory-determined Phosphorus Flux from Lake Sediments as a Measure of Internal Phosphorus Loading

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) loading from lake sediments can account for a substantial portion of the total P load in eutrophic, and some mesotrophic, lakes. Laboratory determination of P release rates from sediment cores is one approach for determining the role of internal P loading and guiding management decisions. Two principal alternatives to experimental determination of sediment P release exist for estimating internal load: in situ measurements of changes in hypolimnetic P over time and P mass balance. The experimental approach using laboratory-based sediment incubations to quantify internal P load is a direct method, making it a valuable tool for lake management and restoration.
Laboratory incubations of sediment cores can help determine the relative importance of internal vs. external P loads, as well as be used to answer a variety of lake management and research questions. We illustrate the use of sediment core incubations to assess the effectiveness of an aluminum sulfate (alum) treatment for reducing sediment P release. Other research questions that can be investigated using this approach include the effects of sediment resuspension and bioturbation on P release.
The approach also has limitations. Assumptions must be made with respect to: extrapolating results from sediment cores to the entire lake; deciding over what time periods to measure nutrient release; and addressing possible core tube artifacts. A comprehensive dissolved oxygen monitoring strategy to assess temporal and spatial redox status in the lake provides greater confidence in annual P loads estimated from sediment core incubations.

Lake eutrophication is a water quality issue worldwide, making the need to identify and control nutrient sources critical. Laboratory determination of phosphorus release rates from sediment cores is a valuable approach for determining the role of internal phosphorus loading and guiding management decisions.

实验室确定的磷通量湖泊沉积物作为内部磷载入一个测量

Eutrophication is a water quality issue in lakes worldwide, and there is a critical need to identify and control nutrient sources. Internal phosphorus (P) ...

科研

环境科学

Measurement of Greenhouse Gas Flux from Agricultural Soils Using Static Chambers

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the biogeochemical drivers of climate change and to the development and evaluation of GHG mitigation strategies based on modulation of landscape management practices. The static chamber-based method described here is based on trapping gases emitted from the soil surface within a chamber and collecting samples from the chamber headspace at regular intervals for analysis by gas chromatography. Change in gas concentration over time is used to calculate flux. This method can be utilized to measure landscape-based flux of carbon dioxide, nitrous oxide, and methane, and to estimate differences between treatments or explore system dynamics over seasons or years. Infrastructure requirements are modest, but a comprehensive experimental design is essential. This method is easily deployed in the field, conforms to established guidelines, and produces data suitable to large-scale GHG emissions studies.

This article showcases the static chamber-based method for measurement of greenhouse gas flux from soil systems. With relatively modest infrastructure investments, measurements may be obtained from multiple treatments/locations and over timeframes ranging from hours to years.

从农业土壤使用静态钱伯斯温室气体流量测量

Measurement of greenhouse gas (GHG) fluxes between the soil and the atmosphere, in both managed and unmanaged ecosystems, is critical to understanding the ...

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 ...

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

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

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

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

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 ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

表征和被动采样的应用在农药水监测

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

Continuous Instream Monitoring of Nutrients and Sediment in Agricultural Watersheds

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in water resources is a prerequisite for understanding the drivers of the pollutant loads and for making informed water resource management decisions. The commonly used &#34;grab sampling&#34; method provides the concentrations of pollutants at the time of sampling (i.e., a snapshot concentration) and may under- or overpredict the pollutant concentrations and loads. Continuous monitoring of nutrients and sediment has recently received more attention due to advances in computing, sensing technology, and storage devices. This protocol demonstrates the use of sensors, sondes, and instrumentation to continuously monitor in situ nitrate, ammonium, turbidity, pH, conductivity, temperature, and dissolved oxygen (DO) and to calculate the loads from two streams (ditches) in two agricultural watersheds. With the proper calibration, maintenance, and operation of sensors and sondes, good water quality data can be obtained by overcoming challenging conditions such as fouling and debris buildup. The method can also be used in watersheds of various sizes and characterized by agricultural, forested, and/or urban land.

With the advancement of technology and the rise in end-user expectations, the need and use of higher temporal resolution data for pollutant load estimation has increased. This protocol describes a method for continuous in situ water quality monitoring to obtain higher temporal resolution data for informed water resource management decisions.

农业流域营养盐和泥沙的连续河道监测

Pollutant concentrations and loads in watersheds vary considerably with time and space. Accurate and timely information on the magnitude of pollutants in ...

Measuring and Mapping Patterns of Soil Erosion and Deposition Related to Soil Carbonate Concentrations Under Agricultural Management

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonate (CaCO3) profiles. The objective is to describe a simple conceptual model and detailed protocol for repeatable field and laboratory measurements of these quantities. Here, accurate elevation is measured using a ground-based differential global positioning system (GPS); other data acquisition methods could be applied to the same basic method. Soil samples are collected from prescribed depth intervals and analyzed in the lab using an efficient and precise modified pressure-calcimeter method for quantitative analysis of inorganic carbon concentration. Standard statistical methods are applied to point data, and representative results show significant correlations between changes in soil surface layer CaCO3 and changes in elevation consistent with the conceptual model; CaCO3 generally decreased in depositional areas and increased in erosional areas. Maps are derived from point measurements of elevation and soil CaCO3 to aid analyses. A map of erosional and depositional patterns at the study site, a rain-fed winter wheat field cropped in alternating wheat-fallow strips, shows the interacting effects of water and wind erosion affected by management and topography. Alternative sampling methods and depth intervals are discussed and recommended for future work relating soil erosion and deposition to soil CaCO3.

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes in elevation are related to changes in near-surface soil carbonates. Repeatable methods for field and laboratory measurements of these quantities and data analysis methods are described here.

农业经营条件下土壤侵蚀和沉积与土壤碳酸盐浓度关系的测量与制图

Spatial patterns of soil erosion and deposition can be inferred from differences in ground elevation mapped at appropriate time increments. Such changes ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

在排水过程中捕获农用运河中的流动加权水和悬浮颗粒

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

Continuous Hydrologic and Water Quality Monitoring of Vernal Ponds

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. However, they are vulnerable parts of the landscapes that are often poorly understood and understudied. Land use and management practices, as well as climate change are thought to be a contribution to the global amphibian decline. However, more research is needed to understand the extent of these impacts. Here, we present methodology for characterizing a vernal pond's morphology and detail a monitoring station that can be used to collect water quantity and quality data over the duration of a vernal pond's hydroperiod. We provide methodology for how to conduct field surveys to characterize the morphology and develop stage-storage curves for a vernal pond. Additionally, we provide methodology for monitoring the water level, temperature, pH, oxidation-reduction potential, dissolved oxygen, and electrical conductivity of water in a vernal pond, as well as monitoring rainfall data. This information can be used to better quantify the ecosystem services that vernal ponds provide and the impacts of anthropogenic activities on their ability to provide these services.

Understanding the ecosystem services and processes provided by vernal ponds and the impacts of anthropogenic activities on their ability to provide these services requires intensive hydrologic monitoring. This sampling protocol using in-situ monitoring equipment was developed to understand the impact of anthropogenic activities on water levels and quality.

春季池塘连续水文水质监测

Vernal ponds, also referred to as vernal pools, provide critical ecosystem services and habitat for a variety of threatened and endangered species. ...

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 ...