The authors thank Dr. Mark Borchardt, U.S. Department of Agriculture, Marshfield, WI, for supplying the Sabin poliovirus serotype 3 used in this study; Mary Jean See, Nancy Schable, and Jenifer Jones of Dynamac Corporation for preparation of BGM cultures; Nichole E. Brinkman, Shannon M. Griffin, Brian R. McMinn, Eric R. Rhodes, Eunice A. Varughese, Ann C. Grimm, Sandhya U. Parshionikar, and Larry Wymer for contributions in the overall evaluation of USEPA Method 1615; Gretchen Sullivan for technical assistance; and local private well owners and utilities for allowing us to collect water samples. Although this work was reviewed by USEPA and approved for publication, it may not necessarily reflect official Agency policy. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

注:请参阅补充材料S1节为定义列表。与USEPA方法1615相关的质量保证程序的补充资料部分S2描述。 1.过滤程序洗脱<ol><li>首先洗脱<ol><li>放置500毫升缓冲1.5％牛肉提取物，pH为9.0的，温热至室温下，在量筒。打开过滤器滤芯外壳和添加牛肉提取物的足够量的完全覆盖正电的过滤器。更换过滤器外壳盖子，倒入剩余的牛肉膏到无菌烧杯中。 </li><li>的接触时间1分钟后，通过在壳体中的牛肉提取液用缓慢通过过滤器使用压力容器或蠕动泵残留在无菌烧杯一起。收集洗脱液成2升玻璃烧杯中。 </li></ol></li><li>第二次洗脱<ol><li>重复使用附加500毫升缓冲的1.5％，牛肉膏1.1步的，但增加在步骤1.1.2接触时间为15分钟。 </li><li>从第二次洗脱添加牛肉提取物，以含有从第一洗脱2L烧杯中。无菌搅拌棒加到烧杯中。 </li></ol></li></ol> 2.有机絮凝浓缩过程<ol><li>消毒用0.525％次氯酸钠的组合型pH电极至少5分钟。冲洗用无菌的dh 2 O电极，然后用0.05M硫代硫酸钠脱氯。校准使用pH值为4和7的标准pH计。 </li><li>将装有小的轰动板的洗脱液中的烧杯中。打开板和增加搅拌速度，直到形成涡流。 </li><li>逐滴加入1.2M的HCl与洗脱液缓慢调节洗脱物至3.5±0.1的pH值。添加盐酸滴，因为快速另外将可灭活病毒。在此期间，作为沉淀物开始形成洗脱液将变得浑浊。 </li><li>降低混合SPE编一个缓慢的搅拌，然后继续监控和维护洗脱液在3.5±0.1在室温下的pH 30分钟。 </li><li>倾析出牛肉膏悬浮液到一个或多个离心瓶，离心15分钟，在4℃下2,500 xg离心。从离心机和任一抽吸取出瓶子或慢慢倒出上清液，以防止沉淀沉淀物的损失。弃去上清液。 注意:可以有牛肉提取大量的沉淀物的数量和质量之间的相当大的变化。从有的地段产生的沉淀物会迅速溶解，而从其他许多难以溶解。 </li><li> 30毫升的0.15M磷酸钠添加到含有沉淀的离心瓶中。 <ol><li>使用的0.15M磷酸钠，用于从牛肉提取大量该5分钟内溶解的沉淀物的pH 9.0。搅拌10分钟后的沉淀物完全溶解，然后转到立即步骤2.7。 </li><li>使用的0.15M磷酸钠，pH 7.0-7.5所有其他沉淀。搅拌10-15分钟以溶解，或者用于更难以析出物，用移液管搅拌期间它们分开用无菌刮刀，通过反复吸取溶液上下摇动，在160转的沉淀定轨振荡器上，或通过这些方法的组合。 注意:当使用一个以上的离心瓶中，结合使用小于30毫升磷酸盐钠的沉淀，然后用30毫升的其余部分上的析出物合并成一个瓶或烧杯后冲洗瓶。 </li><li>如果组合的沉淀物是在一个平底离心瓶中，搅拌棒添加到瓶中。转到步骤2.6.5。 </li><li>如果组合的沉淀物是在离心机瓶具有圆锥形底部，将其转移到一个小玻璃烧杯中，并搅拌棒添加到烧杯中。 </li><li>放置瓶或烧杯上磁力搅拌器，搅拌直至公关ecipitate完全溶解。 </li><li>重新消毒用0.525％的次氯酸钠组合型pH电极和步骤2.1说明用硫代0.05酸钠脱氯。校准用pH 7和10的标准的pH计。缓慢调节完全溶解沉淀至9.0用1M NaOH的pH值，然后搅拌10分钟。 </li></ol></li><li>取出搅拌棒，并在4,000-10,000 XG，4℃离心沉淀溶解10分钟。 </li><li>小心倾上清液到玻璃烧杯中，而不会干扰沉淀。搅拌棒添加到烧杯中，并弃沉淀。 </li><li>放置烧杯上磁力搅拌器，并搅拌该溶液。添加1.2 M盐酸逐滴以调节pH至7.0-7.5。 </li><li>滤波器通过包含已预处理用15毫升的1.5％牛肉膏，0.05M的甘氨酸，pH值7.0-7.5预滤器灭菌过滤消毒由通道上清液。 </li><li>分析样品VolumE（S）计算: <ol><li>使用公式1来计算S表示，除了实验室筑堡垒空白，实验室试剂空白的所有测试样本， <img alt="式（1）" src="/files/ftp_upload/52437/52437eq1.jpg" width="100" />式（1） 其中D（检测大鼠原水样量）是500升的地下水，TSV（总样本量）是通过实验室收到的5英寸阳电滤芯过滤器通过现场样品的体积和FCSV（最终浓缩样品体积）是以下步骤2.10过滤的体积。一个例子示于补充材料节S4.1。 </li><li>计算S为实验室空白加标（LFB; 即使用种子试剂级水阳性质控）和实验室试剂空白（LRB; 即使用试剂级水阴性质量控制）由0.3 FCSV相乘。 </li></ol></li><li>划分成FCSV THREê子样本。 <ol><li>准备副样1和2等于1.04倍的测定样品体积的体积。冻结这些子样本在或低于-70℃，如果他们不能使用的总可培养病毒试验（次采样1，步骤4）进行分析，或24小时内的分子检测（未示出）进行处理;否则，保持在4℃。 </li><li>在等于或低于冻结剩余体积（子样本3）-70℃。 </li></ol></li><li>通过除以10在S计算接种量 。 </li></ol> 3.总可培养病毒量子含量注:对于所有的步骤，始终仔细添加解决方案，以避免干扰细胞单层。 <ol><li>滗析或从含有BGM细胞单层在3-6天后分裂试验容器抽吸介质，然后添加平衡盐溶液的等于除去培养基的体积。 </li><li>倒出或抽吸从细胞培养德的平衡盐溶液使用圣血管再接种细胞培养物测试容器<ol><li> 10接种试验容器与子样本1等于接种量 ，总可培养病毒检测量子控制（补充材料部分S2.4）沿的体积每个测试样本。 </li><li>为LFB和实验室强化样品基质（LFSM; 也就是 ，接种水基质样品），准备用子样本3和0.15M的磷酸钠，pH 7.0-7.5作为稀释剂5-，25-，和125倍稀释液。用于使稀释的过程的一个例子中的补充材料部分S3中给出。接种使用上除了在步骤3.2.1未稀释子样本1接种容器各试验容器中的接种物体积各稀释系列10洗涤的细胞培养试验容器。 </li><li>从步骤3.2.1（高于3.2.2步接种除外）的任何测试样品有CPE在孵化后14天的所有10个重复（见步骤3.3），预削子样本3的5-，25-，和125-和625倍稀释液接种10洗涤的细胞培养试验容器使用上的每个测试容器中的接种物体积与一组新的总可培养病毒量子沿着各稀释系列实验对照组（补充资料部分S2.4）。 </li><li>通过倾斜容器来回分发细胞单层表面上的接种物。在室温下孵育试验容器为80-120分钟在机械摇动平台在1-5振荡/分或与血管的摆动每15-20分钟，以允许任何存在的病毒吸附到细胞。 </li><li>添加预热的维持培养基，然后在36.5±1℃下孵育试验容器。 </li></ol></li><li>用显微镜寻找的CPE中的每个测试容器外观每日为第3天，然后检查它们每2-3天至第14天转移任何试验容器，显示≥75％的CPE到冷冻机设定在或低于-70℃。冻结所有剩余的文化，总培养的病毒检测量子控制在或低于-70℃考察的最后一天血管后。 </li><li>解冻所有培养物，并通过0.2μm的无菌过滤器过滤从每个CPE阳性测试容器中的介质的≥15％。如果指定的卷不能穿过过滤器在1,500-18,000 XG传递由于堵塞，离心培养基10分钟，4℃之前过滤。 </li><li>执行使用洗BGM测试所有的船只1 日通过测定容器的第二通道。 <ol><li>接种与表示从所有阴性试验容器和从阳性血管的过滤介质的解冻介质的10％的接种量的新的试验容器。 </li><li>重复步骤3.2.4-3.4.3，但冻结任何试验容器如步骤3.3描述的是在1 日通过的消极和积极的第二位 。执行第三通作为仅使用阴性测定对照和分别在第二通道中的1 次通过期间的阴性和阳性细胞培养物中的第 2 次通道描述的年龄。 </li></ol></li><li>确定各个测试容器如病毒阳性时，显示出在第1 次和第2 次传代或两者的CPE，在CPE不发生，直到第二通道，同时在第 2 次和第3 次传代的情况下。 </li><li>使用美国环保局的最大可能数计算器与见表S2中计算所有测试样品的病毒滴度设置的默认程 ​​序设置。 <ol><li>输入来自步骤3.6的每个测试样品的病毒阳性复制试验容器的数量到计算器确定MPN / ml的值（M 毫升 ）和上部（CL UML）和下（CL 为1mL）95％置信限/ ml的值。 </li><li> Øbtain使用等式2相应的测试样品的MPN / L值（M L）。 <img alt="公式（2）" src="/files/ftp_upload/52437/52437eq2.jpg" width="100" />公式（2） 中号毫升是在步骤3.7 MPN / ml的值，S是测定样品体积 ，D是检测大鼠原水样品的体积 。 </li><li>通过替换用于M 毫升值为CL值UML计算置信上限/ L。通过替换用于M 毫升值CL 为1mL值计算置信下限/ L。一个例子计算显示在补充材料科S4.2。 </li><li> 0为≤-1 / D报告中号 毫升值。例如，≤0.002 MPN / L（≤1/500 L）地下水样本。 </li><li>计算MPN和95％可信限值为每个实验室强化空白和实验室的重复剂通过第一乘以用S计算器获得的值/毫升，然后通过0.3除以结果空白。 </li></ol></li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+method+using+a+positively+charged+microporous+filter+and+ultrafiltration+for+concentration+of+viruses+from+tap+water.">New method using a positively charged microporous filter and ultrafiltration for concentration of viruses from tap water.</a> Appl. Environ. Microbiol. 77 (10), 3500-3506 (2011).</li><li>Karim, M. R., Rhodes, E. R., Brinkman, N., Wymer, L., Fout, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+electropositive+filter+for+concentrating+enteroviruses+and+noroviruses+from+large+volumes+of+water.">New electropositive filter for concentrating enteroviruses and noroviruses from large volumes of water.</a> Appl. Environ. Microbiol. 75 (8), 2393-2399 (2009).</li><li>Cashdollar, J. L., Wymer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+primary+concentration+of+viruses+from+water+samples:+a+review+and+meta-analysis+of+recent+studies.">Methods for primary concentration of viruses from water samples: a review and meta-analysis of recent studies.</a> J Appl Microbiol. 115 (1), 1-11 (2013).</li></ol>

The authors have nothing to disclose.

USEPA方法1615是为无管制的污染物监测法规的第三监视周期（UCMR3）17中使用而开发的，并主要用于测量地下水发生病毒设计。这股数与信息收集规则（ICR）方法，15,18常见的步骤，但有两个细微的差别。两者都使用量子实验来测量被基于最大可能数（MPN）的计算产生CPE的BGM细胞单层与定量病毒。方法1615允许从各种水基体集中的病毒采用了较新的正电过滤器，并减少细胞培养试验容器的数量每稀释重复20至10这两个微小的变化降低整体成本的方法。在重复的数量的减少降低了劳动，但导致略微较低的检测极限。虽然地下水预期具有较低浓度的病毒比表层水，19,20第测定样品电子量是五倍地表水，在用于不同部分补偿。使用更少的重复的将是足以满足大多数地表水，但有些需要稀释样品。 方法1615有几个关键的步骤和限制。牛肉提取物有所不同批次。每批应进行病毒洗脱效率和通过二级浓缩步骤的能力，病毒浓度测试所描述的是补充材料部分S2.3。该方法使用精确的公式，用于计算样品的量接种到BGM的细胞培养物，并确定病毒滴度。如果这些式不严格遵循不准确的结果将生成。必须用于维持细胞培养物适当无菌技术。该显示在14天的潜伏期窘迫未感染的BGM细胞培养控件可能表明与细胞培养维护问题。大保养也必须TA肯中吹打涉及的接种和培养基除了细胞培养瓶中，以避免交叉污染的步骤。在补充材料S2节中描述的质量控制必须严格的执行。第S2还提供故障排除建议的质量问题。 病毒吸附的主要机制，以正电的过滤器是与过滤器上的正电荷的强度和病毒的有关它的等电点负电荷的强度和被测试21的水的pH值的电荷相互作用。从过滤洗脱也由这些相互作​​用的强度的影响。因为它们之间病毒类型和甚至在相同类型的内菌株而变化，从过滤器的洗脱不统一。这意味着，任何结果可能低估病毒存在于环境水的实际水平。使用单一的BGM细胞系也低估了病毒的发生。范围的，可以在该细胞系主要被限制为脊髓灰质炎病毒和肠病毒乙物种的血清型，以及一些呼肠孤病毒14,15,22产生的CPE肠溶病毒。其他传染性病毒类型将不被检测到。 从地面和试剂级水脊髓灰质炎病毒回收率满足USEPA方法1615性能验收标准为性能评价（PE; 也就是 ，接种试剂级水样与用于评估分析之前的起动性能滴度未知分析师一项研究）和LFB样品（补充材料表S1）的。从使用培养过程地下水58％的回收率是类似于由其他人使用自来水23,24的报道。从111％的LFB样本变异的100％（CV）的系数，平均回收率也达到了性能的方法验收标准，即使他们比，对于PE样品观察到更高DURING的ICR。 ICR意味着实验室间回收率为56％与92％的变化（CV）的一个系数而平均实验室内回收率从36变化到85％（的CV 58至131％;从所述ICR体育数据库未公布的数据）。在这项研究中用于低种子LFB样品比更高的种子样品（122对比42％）观察到更高的回收率。在该ICR正在计划的时候，有人预计，接受较低的种子值PE样品将具有更低的复苏，那些接受高种子。类似于这里观察到LFB样品，ICR PE样品脊髓灰质炎病毒回收率为71％（CV 100％），54％（CV 69％），和44％（CV 71％）为种子值≤300MPN，300- 1500 MPN和&gt; 1500 MPN，分别为。 有水样25测量传染性病毒许多方法。这种方法是在相对于在标准化程度的其它方法显著。标准化不仅包括质量和性能的控制，而且还使用定义的卷和公式，以确保所有的分析实验室进行相同的方法。没有标准化，所以很难在整个实验室比较的结果，并且因此导通在多个分析实验室规模的研究，当标准化是必不可少的。与内置在标准化此方法可以在将来进行扩展以包括额外的病毒类型和细胞系。研究正在进行中，以纳入腺病毒的进入方法提供数据。

A correction was made to: <a href="www.jove.com/video/52437">EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay</a>. The values for Reagent Grade Water shown in Figure 2 were changed from a mean value of 111% with a standard error of 8% to a mean value of 82% with a standard error of 26%.
The penultimate sentence of the last paragraph of the Representative Results section was changed from:
 These controls performed similarly with a mean recovery of 111% and a coefficient of variation of 100% (Figure 2), 
 
 to: 
 
 These controls had a mean recovery of 82% and a coefficient of variation of 110% (Figure 2).
 
The third sentence of the fourth paragraph of the Discussion section was changed from:
 The mean recovery from the LFB samples of 111% with a coefficient of variation (CV) of 100% also met the method performance acceptance criteria even though they are higher than that observed for PE samples during the ICR, 
 
 to: 
 
 The mean recovery from the LFB samples of 82% with a coefficient of variation (CV) of 110% also met the method performance acceptance criteria even though they are higher than that observed for PE samples during the ICR.

A correction was made to: <a href="www.jove.com/video/52437">EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay</a>. The values for Reagent Grade Water shown in Figure 2 were changed from a mean value of 111% with a standard error of 8% to a mean value of 82% with a standard error of 26%.

Erratum: EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

肠道病毒是可感染人体肠道系统并且经由粪 - 口途径被发送的病毒不同群体。这些病毒通过污水处理厂和化粪池污水，设计不当或破损化粪池，下水道坏了线条，结合下水道溢出，等点和非点源1-4进入地表水和地下水。从水性病毒人类感染和疾病经由受污染的或未经消毒的水耗，或通过休闲水接触发生。疾病症状可能涉及轻微到严重胃肠炎;结膜炎;发热;上呼吸道窘迫;手足口病;心肌炎;无菌性脑膜炎;脑炎;麻痹; 5-8败血症，甚至死亡9,10。 USEPA方法1615提供了一个方法来衡量环境和水饮用肠道传染病毒颗粒。这些水域可含有感染性和非感染性病毒体的混合物，但只有感染性颗粒构成潜在的健康危害。感染性病毒颗粒由于可能存在的11-13任何消毒剂丧失感染性超过在从蛋白质衣壳的完整性，核酸由于来自太阳光的紫外线辐射，以及损坏损坏损失环境和饮用水的时间。在该方法中提供的总可培养病毒程序是基于生产的布法罗绿猴肾（BGM）细胞系细胞病变效应（CPE）。该细胞系的选择是因为它在环境病毒学领域14,15的广泛使用，即使检测到的感染性病毒类型的范围主要限于某些肠道病毒15。本文的目的是描述为五英寸的阳电筒式过滤洗脱，仲浓度和总可培养病毒测量方法1615的程序。的评估整个方法中Cashdollar 等人 16进行说明。

<table border="0" cellpadding="0" cellspacing="0" width="624">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Name of the reagent</td>
			<td style="width:120px;">Company</td>
			<td style="width:143px;">Catalogue number</td>
			<td style="width:149px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Beef extract, desiccated powder</td>
			<td style="width:120px;">BD Bacto</td>
			<td style="width:143px;">211520</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Cryogenic tubes</td>
			<td style="width:120px;">Thermo Fisher</td>
			<td style="width:143px;">3775/945373</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Hank&#8217;s balanced salt solution</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:143px;">14170-112</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Mechanical rocking platform</td>
			<td style="width:120px;">Daigger</td>
			<td style="width:143px;">EF4907G</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Orbital shaker</td>
			<td style="width:120px;">Thermo Fisher</td>
			<td style="width:143px;">14-285-729</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Sterilizing filter with prefilter</td>
			<td style="width:120px;">VWR</td>
			<td style="width:143px;">28143-295</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Sterilizing syringe filter</td>
			<td style="width:120px;">Corning</td>
			<td style="width:143px;">431219</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">pH Standards</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:143px;">33643, 33646, 33648</td>
			<td style="width:149px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">MEM</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:143px;">M1018 or M4642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Leibovitz L-15</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:143px;">L4386</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Sodium bicarbonate, 7.5%</td>
			<td style="width:120px;">Sigma-Aldrich</td>
			<td style="width:143px;">S8761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Fetal bovine serum</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:143px;">10082-139</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Antibiotic-Antimycotic</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:143px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:212px;">Trypsin, EDTA</td>
			<td style="width:120px;">Invitrogen</td>
			<td>25200072</td>
			<td></td>
		</tr>
	</tbody>
</table>

epa method 1615. measurement of enterovirus and norovirus occurrence in water by culture and rt-qpcr. ii. total culturable virus assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

病毒从三个饮用水处理厂和私人以及使用正电的过滤器的源地下水浓缩。两个样本集，它由一个场样品和LFSM控制的，从该处理厂收集在不同的场合，以及一个样品组从私人井收集。使用LFSM样品测定从两个植物和私人井（从一种植物和从另一个样品两个样品的整体病毒回收被排除在计算，因为与各样品中使用的种子的MPN值不能被准确地，由于确定复制烧瓶中异常CPE结果）。从地下水样品意味着脊髓灰质炎病毒回收率平均为79％变异系数58％（ 图2）16。在任何重复的未接种的地下水字段样品中没有检测到可培养病毒。 方法表现也使用测量LFB小号amples通过使用两个不同的种子水平修改。脊髓灰质炎病毒的300 MPN的"低"价被用来评估在低于"标准"的500 MPN LFB在USEPA方法1615年"高"价平使用性能级别的脊髓灰质炎病毒1000 MPN被用来在测试性能在LFSM控制水平。这些控制用的111％的平均回收率为100％的变化系数（ 图2）执行类似。所有样品LRB均为阴性和接受范围（补充材料表S1）内执行的所有LFB样本。 <img alt="图1" src="/files/ftp_upload/52437/52437fig1.jpg" /> 图1.过滤洗脱。一种使用的滤芯过滤器洗脱的压力容器原理图。正空气压力用于通过暗盒壳体CON推在压力容器牛肉提取液泰宁阳电筒式过滤器。蠕动泵可以取代的压力容器，与泵的入口被置于容器中保持的牛肉提取液。 <img alt="图2" src="/files/ftp_upload/52437/52437fig2.jpg" /> 图2.脊髓灰质炎病毒的平均回收率（％）从地面和水面试剂级，平均回收率显示从地面脊髓灰质炎病毒（ <img alt="图3" src="/files/ftp_upload/52437/52437fig3.jpg" /> ; N = 4）和试剂级水（ <img alt="图4" src="/files/ftp_upload/52437/52437fig4.jpg" /> ; N = 12）的样品。十二试剂级水样包括6种入300 MPN和六脊髓灰质炎病毒的1000 MPN接种。误差条代表标准误差。

Watch this Scientific Journal Video about EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay at JoVE.com

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

EPA方法1615年测量和肠道病毒水中诺如病毒发生由文化部和RT-qPCR的的。 II。总可培养病毒试验

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

epa-method-1615-measurement-enterovirus-norovirus-occurrence-water

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JoVE Journal

Environment

8.7K 次观看.  U.S. Environmental Protection Agency. Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

视频: EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A Small Volume Procedure for Viral Concentration from Water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

科研

环境科学

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

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。病毒样本的收集I.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking 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 ...

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 ...

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and detritus, and excreting particulate and dissolved compounds, they serve as important agents for benthic-pelagic coupling. Accurately measuring the compounds removed and excreted by suspension feeders (such as sponges, ascidians, polychaetes, bivalves) is crucial for the study of their physiology, metabolism, and feeding ecology, and is fundamental to determine the ecological relevance of the nutrient fluxes mediated by these organisms. However, the assessment of the rate by which suspension feeders process particulate and dissolved compounds in nature is restricted by the limitations of the currently available methodologies. Our goal was to develop a simple, reliable, and non-intrusive method that would allow clean and controlled water sampling from a specific point, such as the excurrent aperture of benthic suspension feeders, in situ. Our method allows simultaneous sampling of inhaled and exhaled water of the studied organism by using minute tubes installed on a custom-built manipulator device and carefully positioned inside the exhalant orifice of the sampled organism. Piercing a septum on the collecting vessel with a syringe needle attached to the distal end of each tube allows the external pressure to slowly force the sampled water into the vessel through the sampling tube. The slow and controlled sampling rate allows integrating the inherent patchiness in the water while ensuring contamination free sampling. We provide recommendations for the most suitable filtering devices, collection vessel, and storing procedures for the analyses of different particulate and dissolved compounds. The VacuSIP system offers a reliable method for the quantification of undisturbed suspension feeder metabolism in natural conditions that is cheap and easy to learn and apply to assess the physiology and functional role of filter feeders in different ecosystems.

VacuSIP, an Improved InEx Method for In Situ Measurement of Particulate and Dissolved Compounds Processed by Active Suspension Feeders

We introduce the VacuSIP, a simple, non-intrusive, and reliable method for clean and accurate point sampling of water. The system was developed and evaluated for the simultaneous collection of the water inhaled and exhaled by benthic suspension feeders in situ, to cleanly measure removal and excretion of particulate and dissolved compounds.

VacuSIP，对改进的方法INEX<em&gt;原位</em&gt;颗粒物与测量溶解的化合物加工后主动悬挂给料机

Benthic suspension feeders play essential roles in the functioning of marine ecosystems. By filtering large volumes of water, removing plankton and ...

A Lipid Extraction and Analysis Method for Characterizing Soil Microbes in Experiments with Many Samples

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial communities, a relatively precise but effort-intensive technique to assay microbial community composition is phospholipid fatty acid (PLFA) analysis. PLFA was developed to analyze phospholipid biomarkers, which can be used as indicators of microbial biomass and the composition of broad functional groups of fungi and bacteria. It has commonly been used to compare soils under alternative plant communities, ecology, and management regimes. The PLFA method has been shown to be sensitive to detecting shifts in microbial community composition.
An alternative method, fatty acid methyl ester extraction and analysis (MIDI-FA) was developed for rapid extraction of total lipids, without separation of the phospholipid fraction, from pure cultures as a microbial identification technique. This method is rapid but is less suited for soil samples because it lacks an initial step separating soil particles and begins instead with a saponification reaction that likely produces artifacts from the background organic matter in the soil. 
This article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples. The method combines the accuracy achieved through PLFA profiling by extracting and concentrating soil lipids as a first step, and a reduction in effort by saponifying the organic material extracted and processing with the MIDI-FA method as a second step.

The article describes a method that increases throughput while balancing effort and accuracy for extraction of lipids from the cell membranes of microorganisms for use in characterizing both total lipids and the relative abundance of indicator lipids to determine soil microbial community structure in studies with many samples.

用于表征土壤微生物的脂质提取和分析方法在许多样品的实验中

Microbial communities are important drivers and regulators of ecosystem processes. To understand how management of ecosystems may affect microbial ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

用小型化磷测定法测定膦酸在粒状氢氧化铁上吸附的优化方法

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Detection of Tilapia Lake Virus Using Conventional RT-PCR and SYBR Green RT-qPCR

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be used as part of surveillance programs, biosecurity measures and in TiLV basic research laboratories. The gold standard of virus diagnostics typically involves virus isolation followed by complementary techniques such as reverse-transcription polymerase chain reaction (RT-PCR) for further verification. This can be cumbersome, time-consuming and typically requires tissue samples heavily infected with virus. The use of RT-quantitative (q)PCR in the detection of viruses is advantageous because of its quantitative nature, high sensitivity, specificity, scalability and its rapid time to result. Here, the entire method of PCR based approaches for TiLV detection is described, from tilapia organ sectioning, total ribonucleic acid (RNA) extraction using a guanidium thiocyanate-phenol-chloroform solution, RNA quantification, followed by a two-step PCR protocol entailing, complementary deoxyribonucleic acid (cDNA) synthesis and detection of TiLV by either conventional PCR or quantitative identification via qPCR using SYBR green I dye. Conventional PCR requires post-PCR steps and will simply inform about the presence of the virus. The latter approach will allow for absolute quantification of TiLV down to as little as 2 copies and thus is exceptionally useful for TiLV diagnosis in sub-clinical cases. A detailed description of the two PCR approaches, representative results from two laboratories and a thorough discussion of the critical parameters of both have been included to ensure that researchers and diagnosticians find their most suitable and applicable method of TiLV detection.

This protocol diagnoses Tilapia Lake Virus (TiLV) in tilapia tissues using RT-PCR methodologies. The entire method is described from tissue dissection to total RNA extraction, followed by cDNA synthesis and detection of TiLV by either conventional PCR or quantitative PCR using dsDNA binding a fluorescent binding dye.

常规 rt-pcr 和 sybr 绿色 rt-qpcr 检测罗非鱼

The aim of this method is to facilitate the fast, sensitive and specific detection of Tilapia Lake Virus (TiLV) in tilapia tissues. This protocol can be ...

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research&#160;design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N&#8729;ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.

Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis

A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.

微图设计及植物和土壤样品制备，进行15次氮气分析

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

Electrostatic Method to Remove Particulate Organic Matter from Soil

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of roots and plant material from soil can result in highly variable carbon measurements. Methods to remove the plant material are often limited to the largest, most visible plant materials. In this manuscript we describe how electrostatic attraction can be used to remove plant material from a soil sample. An electrostatically charged surface passed close to dry soil naturally attracts both undecomposed and partially decomposed plant particles, along with a small quantity of mineral and aggregated soil. The soil sample is spread in a thin layer on a flat surface or a soil sieve. A plastic or glass Petri dish is electrostatically charged by rubbing with polystyrene foam or nylon or cotton cloth. The charged dish is passed repeatedly over the soil. The dish is then brushed clean and recharged. Re-spreading the soil and repeating the procedure eventually results in a diminishing yield of particulates. The process removes about 1 to 5% of the soil sample, and about 2 to 3 times that proportion in organic carbon. Like other particulate removal methods, the endpoint is arbitrary and not all free particulates are removed. The process takes approximately 5 min and does not require a chemical process as do density flotation methods. Electrostatic attraction consistently removes material with higher than average C concentration and C:N ratio, and much of the material can be visually identified as plant or faunal material under a microscope.

Removing recently deposited and incompletely decomposed plant material from soil samples reduces the influence of temporary seasonal inputs on soil organic carbon measurements. Attraction to an electrostatically charged surface can be used to quickly remove a substantial amount of particulate organic matter.

从土壤中去除颗粒有机物的静电方法

Estimations of soil organic carbon are dependent on soil processing methods including removal of undecomposed plant material. Inadequate separation of ...