作者感谢众多EPA的人员，他们的贡献作出ICR和期间进行监控UCMR3可能，其他EPA研究以下铅研究者报告：丹尼尔Dahling，阿尔弗雷德·杜福尔，安德烈·叶戈罗夫，苏珊Glassmeyer，ASJA Korajkic，理查德·利伯曼，罗伯特Safferman，和蒂姆·韦德;和Shannon格里芬和迈克尔·洁具的严格审查本手稿。作者感谢印度山自来水厂对使用其泵房之一展示样品采集。虽然这项工作是由美国环保署审查和批准发布，也未必反映了官方机构的政策。商品名称或商品的提及并不构成认可或推荐使用。

1.初步程序<ol><li>装配一个标准滤波器装置如图1选自由摄取模块，盒外壳模块，和一个放电模块的如补充材料所述。 </li><li>校准流量计/累加器在第一次使用之前，用于取样的流速。检查校准第一次使用后，再一个月后使用。如果流速校准后或者在第一次使用或在第一个月的使用后发生了变化，确定所述频率校准检查如补充材料所述。 <ol><li>排出模块连接到一个吸气模块，然后进模块连接到一个抽头。设置流量计/累加器来读取流量。打开水龙头完全，然后调整用青铜截止阀的流率，以10升/分。一旦流量是稳定的，在10升/分钟，重置流量计/累加器读总体积。 </li><li>将出让放电模块的成4升或更大的量筒并且同时复位累加器归零。测量累加器读数在4升的标记，并达到在缸的4个L标记所需的时间。改变流量计/累加器设置回流量。 注意：如果流量下降到低于9.95 L或完成步骤1.2.2之后增加至超过10.05 L，等到流量稳定并重复步骤1.2.2。一个正确校准仪将达到4升大关量筒在24±1秒，4.0±0.04 L.累加器阅读</li><li>如果累加器读数小于3.96或大于4.04 L时，计算需要调整为所观察到的差异的校正因子。例如，如果流量计/累加器读3.9 l位于上量筒4 L标记，校正因子是1.026（4÷3.9）。因此，如果一个场的样品的所需体积分别为300升的容积根据第r收集eading上流量计/累加器应为300÷1.026 = 292.4 L。 </li></ol></li><li>消毒标准过滤设备并准备样品采集。 <ol><li>由任一完全浸入所述模块中的溶液或通过虽然使用泵模块循环次氯酸钠每次使用前消毒的摄入和仓外壳模块0.525％次氯酸钠至少30分钟。冲洗模块用无菌的dh 2 O和然后脱氯在含有5分钟加入50ml每升无菌试剂级水的1M硫代硫酸钠的溶液中。覆盖用无菌铝箔滤波器采样装置模块结束。 </li><li>添加一个正电筒式过滤器到盒外壳模块无菌。 注：请注意，以确保滤芯过滤器在外壳正确就位。正确安装过滤器不会泄露和动摇，当过滤器将不会在壳体内移动。在AP上的垫片roperly坐在滤波器将有明显的凹痕表示在过滤器接触他们。垫片应当始终被检查之后立即将过滤器从壳体移除凹陷。 </li><li>记录在样本数据表独特的样本号（见补充材料的一个例子），然后采取或运送过滤器的采样设备和采样数据表到谁将会收集现场采样的个人，以及关于样品的任何必要的说明集合（ 例如 ，样品体积，取样位置等 ）。 </li></ol></li></ol> 2.准备样品采集在采样现场<ol><li>抵达后洗手，在收集的网站，不要手术手套，以尽量减少样品污染的可能性。不收集样品，如果遇到肠道或呼吸道症状。更换手套频繁，尤其是触摸水龙头及其他项目后，可能会续胺化。 </li><li>记录相关样本跟踪一个样本数据表的信息。相关信息包括网站和样品标识，采样的名字，设备型号和序列号。 </li><li>放水水龙头2-3分钟以清除在该行已经解决任何碎屑。如有必要，可使用一个花园软管或油管在这一步，达到排水。 </li><li>如果取样装置模块连接，断开它们和保护用无菌箔盒外壳模块的开口端。排出模块连接到进气模块然后两者连接到水龙头。连接一个花园软管在放电模块的端部，并放置在1升聚丙烯容器中的自由端。 </li><li>打开水龙头，并通过至少加入75μL的水通过该装置进行采样。 <ol><li>测量并在水冲洗周期流入聚丙烯容器氯渣油记录水质参数UAL，pH，温度，和浊度。 </li><li>关闭水水龙头后，断开从吸气模块的放电模块，然后暗盒壳体模块连接到进气模块的出口与排出模块到盒外壳模块的出口。重置累计读数为零。 </li></ol></li></ol> 3.现场样品采集<ol><li>记录初始累加器读数连同日期和时间，然后缓慢打开水龙头。 <ol><li>确保该盒式外壳处于直立位置，它完全与水填充。一些外壳有可压在填充过程，以排出空气从壳体的排气按钮。 </li><li>完全打开水龙头。使用青铜截止阀调节流量，以10L /分钟，然后记录的初始流速。 </li></ol></li><li>经过300升的地表水或使用1500至1800 L的地下水通过设备累加器读数（如调节，如果需要的话）。如果所需的体积之前，过滤器堵塞达到和超过一半的体积已经被收集起来，用第二过滤器壳体模块以收集剩余的样本，如果有一个可用。 </li><li>观察的流速为样品体积接近取样周期结束，然后关闭水流在样品抽头。记录最终的流速，日期，当天的时间，最后累加器读数，总样品体积。 </li><li>断开水龙头过滤器取样装置。断开其它模块盒外壳模块。 <ol><li>转动过滤器外壳（S）上下颠倒，并允许过量水流出，直到没有更多的水会流失从两个进口和出口。 </li><li>打开外壳直立覆盖每端用无菌铝箔的快速连接。放置在外壳成一个或多个可密封的塑料袋。 </li><li>从进气以及d沥干水ischarge模块。放置模块为一个或多个可密封的塑料袋。 </li></ol></li></ol> 4.装运现场样品<ol><li>将过滤器取样装置模块到隔热存储和运输凉爽。 </li><li>加冷冻冰袋或双袋装冰块到存储和运输冷却器，以确保样品在运输过程中保持之间的1-10℃。两个大或小的6-8冰袋应该足够了。 </li><li>放置在保温储存和运输的胸部在一个位置的温度记录装置不接触冰袋或冰袋。 注：温度记录装置是可选的，如果分析实验室将使用一个视觉红外线温度计测量仓外壳到达后立即与容器的开口部的温度。 </li><li>填补空隙与包装材料，然后将样本数据表，在一个密封的塑料袋保护，在T运算。关闭隔热存储和运输的胸部，然后用胶带防止水泄漏。 </li><li>交通运输或运送样品的分析实验室。保证样品字段样本采集开始后到达分析实验室不迟于24小时（ 即 ，记录在步骤3.1的时间）。 </li></ol>

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Microbiol. 75 (8), 2393-2399 (2009).</li><li>Ko, G., 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+electropositive+filtration+for+recovering+norovirus+in+water.">Evaluation of electropositive filtration for recovering norovirus in water.</a> J Water Health. 9 (1), 27-36 (2011).</li><li>Liu, P., 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=Laboratory+Evidence+of+Norwalk+Virus+Contamination+Levels+on+the+Hands+of+Infected+Individuals.">Laboratory Evidence of Norwalk Virus Contamination Levels on the Hands of Infected Individuals.</a> Appl. Environ. Microbiol. 79 (24), 7875-7881 (2013).</li></ol>

The authors have nothing to disclose.

浓缩的环境水域病毒不同的过滤器类型已经使用多年26。目前的方法采用超滤27，负电的过滤器13,28,29，玻璃棉过滤器23和正电的过滤器30。负电的过滤器被广泛地使用了许多年，但对于添加盐和前或取样期间水的pH值在现场的调整的要求限制了它们的效用13。最实用的过滤器选择现场采样正电的过滤器。利用这些过滤器的大体积的水的采样在高流速和没有水的任何设施。玻璃棉过滤器是最便宜的选项，但具有较慢的流速比正电的过滤器和市场上不能买到。超滤提供最高的病毒回收率在大范围的水的质量，但需要sampli设备纳克不易字段便携式和收集样品所需要的时间要长得多27。方法最近已开发了利用预处理阴电过滤器以避免需要调整的领域，但这些可能不适用，用于收集大体积样品28,29。 EPA方法1615使用阳电滤芯其中获得无论从氧化铝纳米纤维或季胺的正电荷。前者超过后者的优点是，它是便宜的，并有效地收集病毒从水域在更宽的范围内的天然pH值的值30,31;然而，每个盒，以及使用博尔夏特和他的同事的玻璃棉过滤得到肠病毒和诺沃克病毒从水23,31,32（Cashdollar，未发表的数据）的类似的回收率。筒式过滤器被放置成，其被设计来简化样品的集合的简单采样装置和取样过程中减少污染。 标准方法是非常宝贵的，当大型研究使用多个分析实验室进行。 EPA方法1615提供了标准的程序和指导，以尽量减少可能会影响在这些收集的数据的两大样本采集问题的研究，假阳性结果所产生的污染过程中样本采集或充分消毒的设备部件和过滤毛孔由组件堵塞水进行采样。 正如肠道病毒可波及人对人由于卫生不足，病毒可以被引入到从洗涤不好的手或双手的样品与被污染的手套33。至关重要的是，采样理解污染的潜在途径和采样过程中使用无菌技术。采样应该明白，手套主要用于保护免于暴露采样，而不是为protect从污染的装置。手要洗净前的取样和关怀的开始必须手套的穿戴过程中注意防止手手套污染。取样与胃肠炎或呼吸道症状不能收集样本，因为它们可能被脱落的肠道病毒或诺如病毒在较高的水平。 其次，必须小心，以防止病毒交叉污染，从上次采样事件。为了尽量减少这种潜在的污染的来源，在方法1615取样装置是从该ICR的由不包括进口和仓外壳模块之间的压力调节器和压力计修改。这些组件被删除，因为采样期间观察到的事件的压力总是低于最高住房等级（ 例如 ，125磅的5英寸盒式磁带的外壳），因为他们难以消毒。通过ST设备的使用空白对照后一个问题被证明udies到ICR 6,20以后。在何种程度上影响了它的ICR数据是未知的，但很可能很小;在研究过程中，只有两个假阳性负面绩效考核的样本（数据未显示）。为了进一步减少遗留污染的可能性，也建议每个采样事件之后所述入口模块管代替。它是特别重要的，以确保有足够的消毒，如果该装置被用于被接种病毒一质量或性能的控制。之前进行消毒，游离氯的浓度应测量储存过程中的任何损失。除改变上述的装置的结构中，很重要的是正规的设备空白运行，以表明消毒是有效的。方法1615的设备使用的空白已经被用于病毒种子的控制后，经过消毒设备执行任务，从而简化由于不需要的程序之前，通过该装置中的病毒种子溶液来消毒。消毒剂的浓度增加至0.525％，次氯酸钠为方法1615，因为这个浓度是必需既以灭活设备上的任何可行的病毒和降解的核酸。因此，设备的空白，必须使用两个细胞培养和qPCR测定来进行分析。 这两种类型的过滤器正电的是主体在报告表1中的研究堵塞。未知的样本数量与体积的减少可能是由于抽样误差，如累加器的误读或抽样的有意尽早停止，以满足另一最后期限，特别是对水浊度读数低于20 NTU。堵塞的程度取决于两个经滤波器类型和水的质量的参数。预过滤器提供一些改善的措施，但如果使用的话，应进行处理和分析分别从正电的过滤器。为UCMR3当前的建议是，将样品在不使用氧化二铝纳米纤维基过滤器，如果至少有一半的体积可以使用第一过滤器来收集预过滤器收集。

人类肠道病毒在胃肠道内复制并通过粪 - 口途径分布。这些病毒经常发现在污水中高浓度的1-3。他们能坚持生活污水4,5，并在表面6,7，地面8-10和处理饮用水11水域。当存在时，病毒在美国环境水的浓度典型地过低，可直接测量12,13。这需要的病毒被浓缩从大量的水。在信息收集规则（ICR）监测由美国环境保护署（USEPA）14进行，病毒浓度大事业，从0.009全国性范围的水源水阳性标本的19.7最可能的传染性单位数（MPN）/ L.中位数和平均浓度阳性标本分别为0.03和0.17 MPN / L的水源流入溪流，0.010.07 MPN / L为那些来自湖泊和水库，以及0.04〜0.74 MPN / L为那些使用地下水11（从2000年4月25日日的ICR辅助118个月访问数据库中的数据）。阳性标本来自全国地下水一个USEPA研究的病毒浓度从0.009到2.12传染性单位/ L用中位数和平均浓度为0.13和0.29传染性单位/ L范围8。病毒阳性地下水样品中的浓度高于溪流更高。大多数在这些研究中使用地下水的设施获得的水从位于岩溶地区地下蓄水层。这些连同那些位于石灰石和结晶（断裂基岩）设置有可能有更高的病毒浓度高于在其他设置8,15,16。 USEPA病毒方法指定的200升的地表水（ICR）300 L（方法1615年）和1000升的地下水17,18（ICR）至1500 L（方法1615年）的采样量。然而，即使使用大体积的样品，大多数地表水和地下水的样品是阴性病毒8,11,19,20。 目前在地表水中的病毒构成潜在的健康风险对消费者饮用水。在地表水处理规则要求采用地表水由至少4日志，以减少病毒浓度的所有污水处理厂。即使使用4-log减少，感染性病毒的浓度在源水小至0.0044 MPN / L时有可能导致每天1感染假设这样平均曝光和处理条件和剂量反应参数为轮状病毒11,21。从病毒中未处理地下水的风险可能更大，由于缺乏治疗和病毒的发生。博查特和他的同事估计，多达急性胃肠炎的22％的成年人和63％的儿童不到五年的时间可能是由于病毒使用未经处理的地下水19饮用水的社区。 USEPA Method 1615开发非常规污染物监测法规的第三监测周期（UCMR3）22作为国家过程中检测肠道病毒和诺瓦克病毒后续博查特的调查结果和同事19,23。美国环保局方法主要设计用于使用未经处理的地下水检测病毒系统，但被写得更普遍，包括其它水矩阵类型。新方法是一种混合型保持从ICR 17期间所使用的先前的病毒的方法，许多组件，加入分子程序基于博尔夏特等人 19,23和额外的引物组的诺如病毒24的方法。本文的目的是描述了采样过程，并收集和运输过程中保持样品的完整性所需的步骤。总体方法进行评估，Cashdollar 等人 25进行说明。该协议涵盖了简单的现场收藏品要采样的n地表水和地下水，其中一个泵和预滤器是不必要的，在那里不需要用于pH或在水中的消毒剂的存在的调整。更复杂的采样要求中的F out 等人 17,18进行说明。

<table border="0" cellpadding="0" cellspacing="0" width="711">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:215px;">Name of the reagent/Equipment</td>
			<td style="width:99px;">Company</td>
			<td style="width:129px;">Catalogue number</td>
			<td style="width:271px;">Comments/Description</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">1-L polypropylene bottle</td>
			<td style="width:99px;">Nalgene</td>
			<td style="width:129px;">2104-0032</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Aluminum foil squares</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">06275-40</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Autoclave</td>
			<td style="width:99px;">Steris</td>
			<td style="width:129px;">Amsco Lab Series</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Bubble wrap</td>
			<td style="width:99px;">U.S. Plastics</td>
			<td style="width:129px;">50776</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Closable bag</td>
			<td style="width:99px;">Uline</td>
			<td style="width:129px;">S-12283</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Closable bag</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">S31798C</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Commercial ice packs</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">06345-20</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Cool safe box</td>
			<td style="width:99px;">Diversified Biotech</td>
			<td style="width:129px;">CSF-BOX</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Gauze sponge</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">22-415-469</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Graduated cylinder</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">06135-90</td>
			<td style="width:271px;">4-L or larger</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Hype Wipe</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">14-412-56</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">iButtons temperature data logger</td>
			<td style="width:99px;">Maxim</td>
			<td style="width:129px;">DS1921G</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Insulated storage and transport chest</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">11-676-12</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Packing tape</td>
			<td style="width:99px;">U.S. Plastics</td>
			<td style="width:129px;">50083</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Portable chlorine colorimeter II test kit</td>
			<td style="width:99px;">Hach</td>
			<td style="width:129px;">5870062</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Portable pH and temperature probe</td>
			<td style="width:99px;">Omega</td>
			<td style="width:129px;">PHH-830</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Portable turbidity meter</td>
			<td style="width:99px;">Omega</td>
			<td style="width:129px;">TRB-2020-E</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">PTFE thread tape</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">08270-34</td>
			<td style="width:271px;">Use on all threaded connections</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Pump, Centrifugal Magnetic Drive</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">72010-20</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Reduction nipple</td>
			<td style="width:99px;">Cole-Parmer</td>
			<td style="width:129px;">06349-87</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Sodium hypochlorite (NaClO)</td>
			<td style="width:99px;"></td>
			<td style="width:129px;"></td>
			<td style="width:271px;">Use locally available household bleach</td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Sodium thiosulfate (Na2S2O3)</td>
			<td style="width:99px;">Sigma Aldrich</td>
			<td style="width:129px;">217247</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Surgical gloves</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">19-058-800</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;">Waterproof marker</td>
			<td style="width:99px;">Fisher Scientific</td>
			<td style="width:129px;">22-290546</td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:215px;"></td>
			<td style="width:99px;"></td>
			<td style="width:129px;"></td>
			<td style="width:271px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:215px;">Media</td>
			<td style="width:99px;">Composition</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="299">
			<td height="299" style="height:299px;width:215px;">0.525% sodium hypochlorite (NaClO)</td>
			<td style="width:99px;">Prepare a 0.525% NaClO solution by diluting household bleach 1:10 in dH2O.&#160; Store 0.525% NaClO solutions for up to 1 week at room temperature.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="240">
			<td height="240" style="height:240px;width:215px;">1 M sodium thiosulfate (Na2S2O3) pentahydrate</td>
			<td style="width:99px;">Prepare a 1 M solution by dissolving 248.2 g of Na2S2O3 in 1 L of dH2O.&#160; Store sodium thiosulfate for up to 6 months at room temperature.</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

过滤器堵塞是可以与方法1615可能遇到堵塞减小的水通过过滤器的流率的一个主要的潜在问题。在某些情况下，这可以通过打开截止阀，以允许更大的流动来克服。在其他情况下，该过滤器将变成完全堵塞之前所需的体积已经通过它。 表1示出样品具有减少量作为过滤型，水型，和浊度的函数的百分比。堵塞发生了地下水和地表水样，与氧化铝纳米纤维为基础的过滤器跑赢季胺类过滤地下水样品，而相反的是地表水样品的情况下。总体而言，使用的季胺系滤波器收集的样本6％是缺乏量，同时与氧化铝纳米纤维为基础的过滤器收集的样品的10％没有达到所要求的最小规定体积由于堵塞。在属升，堵塞随浊度，但是许多不同的组件有助于浊度和其中的一些不会导致堵塞。例如，所述的ICR研究期间发生的所有事件的堵塞的43％被限制到2水系（数据未示出）。在ICR的最低目标卷的地表水为200 L.在研究过程中收集的平均量为217±32升用208升的容积中位数（日期为2000年4月25日从ICR辅助118个月访问数据库中的数据） 。因此，全卷可以从许多水域收集即使在高浊度。 该ICR要求采样使用预过滤器时，浊度均超过75 NTU，但即使有一个预过滤器，收集水域浊度超过75样本的34％NTU堵塞。方法1615建议使用预过滤器与水超过50 NTU;但是，由于方法1615的出版物中，除了在该方法中列出的几个不同的预过滤器的选择已经测试。所有这些导致了样品体积显著改善（数据未显示）。 <img alt="图1" src="/files/ftp_upload/52067/52067fig1.jpg" /> 图1.标准过滤设备的标准过滤器装置由进气，过滤器外壳，放电模块（见补充材料的每个模块的介绍）。 <a href="https://www.jove.com/files/ftp_upload/52067/52067fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 总样本 一 </td><td> 对缺乏样品 B 号码 </td><td> 百分比缺乏样品 </td><td> 浊度（NTU） </td></tr><tr><td colspan="4" fo:padding-left="200px" style="text-align:center">使用NanoCeram地下水<html>过滤器Ç </td></tr><tr><td> 113 </td><td> 1 </td><td> 1 </td><td> &lt;20 </td></tr><tr><td colspan="4" fo:padding-left="200px" style="text-align:center">使用地表水NanoCeram过滤器Ç </td></tr><tr><td> 83 </td><td> 12 </td><td> 14 </td><td> &lt;20天</td></tr><tr><td> 8 </td><td> 4 </td><td> 50 </td><td> 20 - &lt;50 </td></tr><tr><td> 6 </td><td>五</td><td> 83 </td><td> ≥50ð </td></tr><tr><td colspan="4" fo:padding-left="230px" style="text-align:center">总NanoCeramÇ </td></tr><tr><td> 210 </td><td> 22 </td><td> 10 </td><td> ≥0 </td></tr><tr><td colspan="4" fo:padding-left="200px" style="text-align:center">使用1MDS过滤地下水Ç </td></tr><tr><td> 374 </td><td> 75 </td><td> 20 </td><td> &lt;20 </td></tr><tr>分机对齐：中心“&gt;地表水使用1MDS过滤器Ç </td></tr><tr><td> 269​​3 </td><td> 27 </td><td> 1 C </td><td> &lt;20个电子</td></tr><tr><td> 505 </td><td> 36 </td><td> 7 F </td><td> 20 - &lt;50架F </td></tr><tr><td> 122 </td><td> 19 </td><td> 16ê </td><td> 50 - &lt;75克 </td></tr><tr><td> 175 </td><td> 60 </td><td> 34 E，F </td><td> ≥75 E，F，G </td></tr><tr><td colspan="4" fo:padding-left="240px" style="text-align:center">总1MDSÇ </td></tr><tr><td> 3869 </td><td> 217 </td><td> 6 </td><td> ≥0 </td></tr></tbody></table> 表1.过滤能力 一个样品是从下面的研究从体积和浊度的数据是可用的： 从 4/2日的ICR辅助118个月access数据库1）美国环保局的ICR研究（数据5/2000），2）美国环保局地下水研究8,3）USEPA劳伦斯和洛厄尔，MA研究（未发表资料），4）USEPA密西西比河研究（未发表资料），5）USEPA饮用水处理厂的研究（未发表资料） 6）USEPA方法1615评价研究25和7）的前三个月的UCMR3监测（未公布数据）。B瓶尿样 ，其中的每个研究规定的水体积最小才堵塞过滤器收集。这是200升的地表水和1500 L代表地下水，但200升的水源，正在接受美国环保局ICR地下水和1893 L代表美国环保局地下水研究。C到收集指定数量的最低全面的能力是不同的显著为使用样品收集NanoCeram和1MDS过滤器（P = 0.002）之间，地表水和地下水均整体（P &lt;0.001），并为每个过滤器类型（P &lt;0.001）采用Mann-Whitney秩和检验。D＆＃8211; G组相同的标值是显著差异（P &lt;0.05），根据方差的秩和检验单向分析秩和检验和邓恩的成对多重比较程序。对于所有的统计检验的因变量是通过过滤器作为最小指定体积的一小部分通过了体积，但具有1.0的最大值。

Watch this Scientific Journal Video about EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples at JoVE.com

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

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. ...

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12.0K 次观看.  U.S Environmental Protection Agency. 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.

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

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. 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.

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 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 ...

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

对水样的自动固相萃取小污染物的免疫学分析的简单方法

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

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 ...

Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies employed disk fiber filters to collect eDNA from water samples, although a number of microbial studies in aquatic environments have employed filter cartridges, because the cartridge has the advantage of accommodating large water volumes and of overall ease of use. Here we provide a protocol for filtration of water samples using the filter cartridge and extraction of eDNA from the filter without having to cut open the housing. The main portions of this protocol consists of 1) filtration of water samples (water volumes &#8804;4 L or &gt;4 L); (2) extraction of DNA on the filter using a roller shaker placed in a preheated incubator; and (3) purification of DNA using a commercial kit. With the use of this and previously-used protocols, we perform metabarcoding analysis of eDNA taken from a huge aquarium tank (7,500 m3) with known species composition, and show the number of detected species per library from the two protocols as the representative results. This protocol has been developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

We describe a protocol for filtration of water samples with a filter cartridge and extraction of environmental DNA (eDNA) without having to cut open the housing to remove the filter. This protocol is developed for metabarcoding eDNA from fishes, but is also applicable to eDNA from other organisms.

滤芯水过滤样品和环境DNA的提取使用

Recent studies demonstrated the use of environmental DNA (eDNA) from fishes to be appropriate as a non-invasive monitoring tool. Most of these studies ...

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 ...

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

真菌 DNA 分析用职业空气标本的收集与提取

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

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 ...