This study was supported as basic research by CREST from the Japan Science and Technology Agency (JST) and by grants from JSPS/MEXT KAKENHI (Number 26291083) and the Canon Foundation to M.M. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

注:此协议不涉及水样采集和metabarcoding方法。水可以以不同的方式取决于研究的目的16进行采样，看看宫等 12使用MiFish引物metabarcoding方法的细节。注意，采样水应保持非常冷，并在数小时内过滤，以避免的eDNA的降解。另请注意，该协议涉及使用旋转振荡器和培养箱的，而后者必须足够大，以容纳前者。此外，离心分离机，可容纳均为15和50ml锥形管是不可缺少的，从过滤后的过滤器除去残留的液体和分别暗盒内以收集提取的DNA。 1.处理螺帽和1升的塑料袋注:如果过滤量&gt; 4 L.跳过此步骤<ol><li>通过一个螺丝c的中心钻一个孔AP（附连到一次性1升塑料袋）具有相同的直径（4.8 MM）作为管从阳路厄锁连接器突出。使用斜口钳，缩短阳路厄锁连接器的管以适当的长度（ 约 3毫米），以避免水的过滤后的帽内有少量的堵塞。 </li><li>申请专门用于聚乙烯（PE）和聚丙烯（PP）的粘合剂胶水的阳路厄锁连接器的底部和表面都和螺旋盖，分别。等待好的粘接几分钟（参照制造商的说明）。 </li><li>插入阳鲁尔锁连接器插入螺丝帽的孔。等到两部分（通常&gt; 24小时）的完整的粘接。用消毒使用前用10％的市售漂白剂（ 约 0.6％次氯酸钠）阳路厄锁连接器上的螺丝帽。 </li><li>冲在1升塑料袋的两个底部角落的两个孔，以从一个MES挂ħ面板（步骤3）。确保该孔的直径比所述网格板上的齿尖的尺寸。 </li></ol> 2.过滤系统的装配<ol><li>附加在高真空油管抽吸泵的输入连接器和管的另一端连接到一个歧管。可以肯定的歧管的三个红色叔阀处于"关闭位置"（ 即 ，横向）。 </li><li>戴着干净组手套，插入一个注射器阴配件，并在真空橡胶管用于过滤的顶端和底端的真空连接器，分别。 </li><li>仔细附加注射器阴配件的滤筒的出口和真空连接器连接到一个硅胶塞。 </li></ol> 3.使用滤芯水样（≤4L）的过滤注:如果过滤量&gt; 4 L.该过滤系统跳过此步骤需要自立F组或悬挂塑料袋装满1升的水。网状面板，多叉，而对于面板的立场，都可以从网上商店，将是组装这个单位非常有用。高压釜的入口和出口的路厄氏帽在使用前过滤筒。 <ol><li>倒入1升采样水入塑料袋中并与阳鲁尔锁连接器关闭螺旋盖。 </li><li>小心地用塑料袋的阳路厄锁定连接器连接的组装过滤器滤芯的流入口。不要过度拧紧鲁尔锁连接器;否则一旦开始抽水设备将泄漏。确保所有的连接都从网状面板挂塑料袋之前的安全。 </li><li>小心地用滤筒从两个叉悬挂网格面板上的塑料袋中，并插入一个硅胶塞进入歧管的入口。 </li><li>打开抽吸器。打开红色T阀过滤。运行歧管，直到滤芯干燥，T母鸡将红色叔阀到关闭位置。 </li><li>重复3.1-3.4步骤，直到水的所需量的过滤。 注:为每升从亚热带珊瑚礁取水，它需要的过滤约三分钟。 </li><li>小心地从塑料袋和真空连接器的滤芯。 </li><li>帽与入口和出口的路厄氏帽上的卡盒的两端。 </li><li>标签盒和入口路厄帽适当地使用溶剂型笔快干和非涂抹标记。 </li><li> DNA提取之前储存在-20℃的滤筒。 </li></ol> 4.水过滤（&gt; 4 L）采用滤芯样品注意:跳过这一步，如果水过滤体积是≤4L。该过滤系统需要配备一个阀门和一次性10毫升移液管尖端的10升书瓶中。的10毫升吸管尖端（15.0毫米）内径和尖端的锥形端应该适合的阀（15.0毫米）的外直径和过滤器滤芯的流入口，分别。这两个连接在一摩擦配合在过滤过程中牢固地保持。用消毒使用前10％市售漂白剂（ 约 0.6％次氯酸钠）枪头。 <ol><li>在书瓶准备的采样适量水。紧紧插入10毫升吸管尖端放入10升本书瓶的阀门。 </li><li>紧紧插入滤筒插入吸移管前端的出口并插入硅胶塞进入歧管的进气口。打开抽吸器。打开红色T阀过滤。运行所述歧管，直到滤筒是干的，然后打开红色叔阀到关闭位置。 注:对于10升从亚热带外珊瑚礁取海水，它需要的过滤约30-40分钟。 </li><li>小心地从塑料袋和真空连接器的滤芯。这两个帽与入口和出口的路厄氏帽上的卡盒的结束。标签盒和入口路厄帽适当地使用溶剂型笔快干和非涂抹标记。在DNA提取之前-20℃保存滤芯。 </li></ol>从过滤器埃德娜5.提取注:在步骤4和5，我们使用商业套件，主要针对以下"核血"的套件中提供的协议。为简单起见，我们将描述用于处理单个盒的过程。在实践中，我们建议在一个时间处理8（或离心机的最大数目）或更少的过滤器。 <ol><li>预热的孵化器56℃。 </li><li>通过从管切割铰链式盖制备2.0毫升管。丢弃上限。 注:此管被用作用于在过滤器中剩余的液体的收集管。 </li><li>从过滤器滤芯的入口（未出口）鲁尔盖，将进气口我n要收集管。紧密地密封采用自密封膜盒和收集管之间的连接。 </li><li>将合并的单元成15毫升锥形管离心适配器。离心盒在5000 xg离心1分钟，以去除剩余的液体在过滤器。 </li><li>从滤筒移除收集管并弃去管。重新盖盒的入口。 </li><li>制备的20微升蛋白酶K溶液的混合物中，220微升PBS（磷酸盐缓冲盐水;不是由试剂盒提供）和200微升缓冲液AL。 注:滤芯可容纳该混合物的四倍体积（ 加利福尼亚州 1.8ml）中。 </li><li>卸下入口盖和该混合物（440微升）加入到用枪头滤筒。插入吸移管完全插入进气口，使得枪头是盒刚好在膜上方内可见。 </li><li>重新盖上的进气口，然后将过滤器的车盒在旋转摇床上。 </li><li>放置在预热的培养箱的旋转振荡器在56℃和20 rpm的速度打开摇动20分钟。 </li><li>在温育后，准备一个新的2.0毫升管，标号管子适当地使用溶剂型笔，并将其放置在50ml的锥形管中。 注:前者（2.0毫升）和后者管（50毫升）被用于提取的DNA的收集和用于保持2.0毫升管，分别。 </li><li>培养后，除去入口盖和盒的端口插入入口（未出口）进50毫升的锥形管内2.0毫升管。用螺丝帽关闭50ml锥形管中。 </li><li>离心50毫升锥形管中，在5000 xg离心1分钟，以收集从盒中提取的DNA。移除滤筒并使用无菌镊子2.0毫升管和帽的管中。丢弃滤筒。 </li></ol> 6.纯化提取的DNA 注意:我们洗脱的eDNA用100微升缓冲AE，而不是商业套件手册中规定的200微升。 <ol><li>加入200μl乙醇（96-100％），以所提取的DNA在2.0毫升试管从上述步骤（ 约 440微升），并通过涡旋调匀。 </li><li>吸管将混合物成放置在2 ml收集管旋转柱。离心在5000 xg离心1分钟。丢弃流通和收集管。 </li><li>放置旋转柱在新的2ml收集管中，在5000×g下加入500μl缓冲液AW1，离心1分钟。丢弃流通和收集管。 </li><li>放置旋转柱在新的2ml收集管中，在20,000×加入500微升缓冲液AW2，与离心3分钟g。丢弃流通和收集管。 </li><li>准备一个新的1.5毫升管（未由试剂盒提供），并适当地使用快干和不涂抹标记防水笔标记管。转移自旋T列Ø1.5 ml管。 </li><li>通过加入100μl的缓冲液AE到离心柱膜的中央洗脱DNA。孵育在室温下1分钟，然后在6000 xg离心离心1分钟。 </li><li>丢弃旋转柱和帽的管中。储存在-20℃的纯化的DNA。 </li></ol>

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Genet. 17, 1-17 (2016).</li><li>Sorokulova, I., Olsen, E., Vodyanoy, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biopolymers+for+sample+collection,+protection,+and+preservation.">Biopolymers for sample collection, protection, and preservation.</a> Appl. Microbiol. Biotechnol. 99, 5397-5406 (2015).</li><li>Renshaw, M. A., Olds, B. P., Jerde, C. L., McVeigh, M. M., Lodge, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+room+temperature+preservation+of+filtered+environmental+DNA+samples+and+assimilation+into+a+Phenol-Chloroform-Isoamyl+alcohol+DNA+extraction.">The room temperature preservation of filtered environmental DNA samples and assimilation into a Phenol-Chloroform-Isoamyl alcohol DNA extraction.</a> Mol. Ecol. Res. 2014, (2014).</li></ol>

The authors have nothing to disclose.

在使用环境样品，例如水和土壤许多metabarcoding研究中，过滤器滤芯的过滤后处理通常如下24,25:1）切开或开裂用手动工具（管切割器或钳子）的外壳; 2）清除墨盒上的过滤器;和3）切割所述滤波器小块用刀片用于DNA提取。为了避免过滤器滤芯的外壳内的这种麻烦的和费时的，它们很容易在实验室污染的程序，我们尝试几种DNA提取方法使用一种广泛使用的，价格低廉市售的试剂盒，并成功地开发了本协议具有有利从的eDNA metabarcoding分析结果（ 图1）。 一个在本协议的更重要的步骤是在预热的培养箱使用旋转振荡器，即能够提供后续文库prepar好的DNA产量的通货膨胀在的eDNA metabarcoding分析。在最佳温度在外壳内的蛋白酶K溶液中不断搅拌下可能有利于从截留在过滤器上的颗粒的DNA提取。但是请注意，这个协议包括使用两个旋转振荡器和培养箱的，而后者必须能容纳前者。此外，离心分离机，可容纳均为15和50ml锥形管是用于分别从过滤后过滤去除残留的液体和盒中收集提取的DNA，是必不可少的。 还有另一种选择，以实现这样的DNA提取，而不必切开盒（参见材料/试剂的表）。所述试剂盒不使用DNA提取任何酶;相反，它使用与珠击附加的机械裂解组合的特殊裂解缓冲液。在基于天然海水从海岸温带太平洋初步实验中，我们学尝试泰德DNA提取与使用一个原型使用商品化试剂盒在本议定书沿着这个套件。随着使用后者的原型协议，我们一贯承认不同的扩增产物凝胶电泳为第1轮PCR。与此相反，我们未能通过使用不同的试剂盒，以获得任何的PCR产物（见材料/试剂表 ）尽管按照制造商提供的两个备选方案。考虑到的PCR抑制剂去除步骤包括在第二试剂盒的两个协议（见材料/试剂表 ），这一观察表明缺乏足够量的模板DNA。从环境样品使用第二试剂，例如相对低DNA产量列于最近的研究26,27。 在本协议中，我们选择了过滤器滤芯大标称孔径（0.45微米），而水生微生物的研究传统领LLY使用通过较大的孔尺寸的过滤器（1至3微米）28预过滤后的较小的一个（0.22微米）。我们选择的原因是简单和直接的，而不是基于理论参数:1）孔尺寸更接近，该玻璃纤维过滤器（0.70微米），我们在先前的eDNA已经常规使用的研究12,14， 22,23; 2）它可望允许比中较小的一个被过滤更大体积的水。后者的特点是我们目前的研究项目稀缺鱼丰度和生物量公海和深海生态系统发生尤为显著。事实上，我们证实了从没有明显的堵塞黑潮罐海水的独立采样10-L的成功过滤（未示出结果）。然而，最近，Turner 等人29表明，0.2微米的过滤最大化鲤鱼的eDNA捕获（85％±6％），同时最小化总（ 即非靶）的eDNA捕获（48％±3％），但滤波器此孔径堵塞限于小于250毫升的样品体积。需要进一步研究以确定滤筒为的eDNA各类鱼数量和生物量的不同水平的水生环境中metabarcoding的最优滤波器孔径和水过滤体积。 一个这种协议的局限性是，水过滤应该在一个电源是可用的实验室中进行。由于DNA的有限的化学稳定性的，的eDNA开始尽快降低，因为它是从生物体棚和是可检测的只为在水生环境时间（几小时到几天）短时间30。因此它是理想的采集后立即过滤水样品，以避免的eDNA的显著降解。在这方面，使用的过滤器滤芯内的过滤方法的发展将是一个有希望的下一个步骤。便携性和sterilit滤芯的Ÿ启用现场过滤方法，这将提供更完整的eDNA可以埃德娜metabarcoding更准确地反映鱼类群落的物种组成发展。在过滤器上的墨盒的收集的eDNA可以通过商业试剂31的喷射被稳定（参见材料/试剂表 ）或LONGMIRE把它回实验室前缓冲器32到盒。

在水生环境环境DNA（的eDNA）是指水体中发现的遗传物质。最近的研究表明的eDNA的效用从各种水生环境，包括池塘1-3，4-8江河鱼类检测，溪流9，和海水10-14。这些研究大多集中在检测的单个或几个侵入1,4-6,8,14和珍稀或濒危物种3,9，而最近的一些研究试图在当地鱼类群落7,9多个物种的同时检测， 12,13,15和生物群落11,12。 后一种方法被称为"metabarcoding"和的eDNA metabarcoding使用的PCR引物的一个或多个集合横跨分类学上不同的样品coamplify的基因区域。其次是与索引和适配器此外文库制备和索引库由一个高通量平行测序分析平台。最近宫等人 12开发的通用PCR引物用于从鱼类（称为"MiFish"）metabarcoding的eDNA。所述MiFish引物靶向线粒体12S rRNA基因（163-185碱基），它包含足够的信息，以确定鱼生物分类科，属和除了一些密切相关的同源物种的高变区。随着使用中的eDNA metabarcoding的引物，美哉等 12检测到水族箱230余属亚热带海洋物种，水族馆附近的已知物种组成和珊瑚礁。 同时优化metabarcoding协议容纳天然海水以改变来自鱼类的eDNA浓度的水平，我们已经注意到，MiFish引物偶尔未能扩增后续文库制备目标区域。一个为这个不成功的PCR扩增更可能的原因是缺乏足够数量的TE的中所含的水的小体积的过滤mplate的DNA（ 即 1-2升）。虽然从特定分类群的eDNA浓度放大之前是不可知的大型水量（&gt; 1-2升），过滤是一个简单而有效的手段，从稀缺的鱼类数量和生物量，比如水环境收集更多的eDNA公海和深海生态系统。 相对于磁盘纤维过滤器在许多鱼的eDNA研究16的常规使用的，滤芯具有堵塞17之前容纳较大水量的优点。事实上，最近的一项研究表明，大体积（&gt; 20 L）采用滤芯18沿海海水样品的过滤。此外，它们被单独包装和无菌，实验工作流的若干步骤可以在过滤器外壳中进行，从而降低了从实验室19污染的可能性。后者特征为的eDNA metabarcoding关键，其中污染的风险仍然之间的最大实验挑战20,21。尽管滤芯这些技术优势，它尚未在鱼类的eDNA研究中使用的有两个例外8,15。 在这里，我们提供了从它的过滤器的eDNA的滤筒和提取水样的过滤的协议，而不必切开壳体。我们还提供了根据不同的水量（≤4L或&gt; 4升）两种可供选择的水过滤系统。为了比较新开发协议的性能，在我们的研究小组使用12,14,22,23玻璃纤维过滤先前使用的协议，我们执行的eDNA metabarcoding海水分析从一个巨大的鱼缸（7 500 立方米 ）与已知的物种组成，并示出了从两个协议作为代表性结果得出检测物种的数目。该协议ħ作为已经开发了用于从鱼类metabarcoding的eDNA，但也适用于的eDNA从其他生物体。

<table border="0" cellpadding="0" cellspacing="0" width="927">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Mesh panel</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MPP-3060-BE</td>
			<td style="width:411px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Metal prong</td>
			<td style="width:167px;">Iris Ohyama</td>
			<td style="width:119px;">MR12F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stand for the mesh panel</td>
			<td style="width:167px;">No brand</td>
			<td>4184-9507</td>
			<td>available from Amazon Japan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1-L plastic bag with screw cap</td>
			<td style="width:167px;">Yanagi</td>
			<td style="width:119px;">DP16-TN1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer-lock connector</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">11620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-mL pipette tip</td>
			<td style="width:167px;">Eppendorf</td>
			<td style="width:119px;">0030 000.765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">10-L book bottle with valve</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-2169-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sterivex-HV filter</td>
			<td style="width:167px;">Millipore</td>
			<td style="width:119px;">SVHVL10RC</td>
			<td>denoted as &#34;filter cartridge&#34; throughout the ms and used in the protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Male luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7379-04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Female luer fitting</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">5-1043-14&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Inlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRMP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Outlet luer cap</td>
			<td style="width:167px;">ISIS</td>
			<td style="width:119px;">VRFP6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">High vacuum tubing</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-590-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Vacuum connector</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">6-663-02</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicone stopper</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7650-07</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Manifold</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">2-258-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Aspirator-GAS-1</td>
			<td style="width:167px;">As One</td>
			<td style="width:119px;">1-7483-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">DNeasy Blood &#38; Tissue Kit (250)</td>
			<td style="width:167px;">Qiagen</td>
			<td style="width:119px;">69506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">PowerWater Sterivex DNA Isolation Kit</td>
			<td style="width:167px;">MO BIO</td>
			<td style="width:119px;">14600-50-NF</td>
			<td>denoted as &#34;optional kit&#34; in the ms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Tabletop Centrifuge</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">Model 4000</td>
			<td>Maximum speed 6,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Fixed-angle rotor</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">AT-508C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adaptor for a 15 mL conical tube</td>
			<td style="width:167px;">Kubota</td>
			<td style="width:119px;">055-1280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">RNAlater Stabilization Solution</td>
			<td style="width:167px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM7020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;"></td>
			<td style="width:167px;">Parafilm</td>
			<td style="width:119px;">PM992</td>
			<td>denoted as &#34;self-sealing film&#34;</td>
		</tr>
	</tbody>
</table>

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.

这在技术上是难以分离和量化从所提取的散装的eDNA仅鱼的eDNA，因为MiFish引物从一些非鱼脊椎动物，如鸟类和哺乳动物coamplify目标区域，与同样大小的PCR产物（ 约 170 bp的）12。相反，量化鱼埃德娜，我们执行MiFish从使用过滤和DNA提取的两种不同的方法已知的物种组成的水族箱metabarcoding埃德娜分析，并从两个协议比较每个库检测物种的数量。这个简单的实验旨在显示本议定书的可行性的"代表性的结果"，而不是严格地证明其对他人的优势。 我们在冲绳美丽海水族馆，日本冲绳（26˚41&#39;39"N共30升海水取样从黑潮罐，127˚52&#39;41"E）。黑潮水箱35米×27米×10μm且7500米3的总水量的设计展示海洋巨型动物，具有尺寸（长x宽x厚），设有约60大-sized海水鱼类围绕黑潮，沿日本的整个长度东北中流动的西边界电流中的一个区域的特性。在以前的一个MiFish metabarcoding研究，宫等人 12检测出的63种的61（97％）中含有在从五个2升海水样品罐。 对于海水的过滤，我们使用滤芯（孔径= 0.45微米）和玻璃纤维盘过滤器（孔径= 0.70微米）。我们同时使用两种协议为四水体积（;总八个滤波器1，2，3，4升）上过滤同一歧管的海水。海水的过滤后，我们还过滤的1升的PCR级水与两个滤波器（六ILTER盒和玻璃纤维过滤器）作为阴性对照（两个过滤空白）。从这些类型的过滤器，我们提取的eDNA使用新的和先前使用的协议12，这两者都使用相同的商业试剂盒。总共10个的eDNA样品（包括两个阴性对照）进行MiFish metabarcoding分析如在宫等人所述。12 简要地说，我们进行了第1轮PCR扩增靶区域（线粒体12S rRNA基因; CA 170碱基对）和追加用于配对末端测序适配器。八的eDNA样品（不含两个阴性对照），我们进行了10×PCR复制以查看在每个样品（总共80的PCR）内检测到的种类的数目的变化。我们还添加了过滤器和PCR空白，以每组8个样品的eDNA（共16个项目完成报告）。我们总共获得96 PCR产物从第一轮PCR，他们被用来作为第二轮P模板CR双索引和附加的文库制备以下宫等额外的适配器。12 96库的配对末端测序（2×150碱基对），得到4127546读取，每个文库的42995的平均读取（40539读取滤筒和43121中的玻璃纤维过滤器阅读）。解复用和原始数据的后续预处理后，1068266读取被保留用于分类分配的BLAST搜索。这些读，830788读取被确定为包含在黑潮罐（ 表1）的物种。我们检测60罐物种从830788读取和10的PCR检测物种的平均数目从八个的eDNA样品从29范围为1升（玻璃纤维滤器），以55复制为4升（滤筒）（ 表1） 。从八个的eDNA样品两个空白的读数分别为次要的，范围从012为油箱物种。 我们发现，检测物种通过过滤器滤芯的数量明显高于玻璃纤维过滤器（ANCOVA，F = 381.8，p &lt;0.001; 图1）的显著更高横跨1-4大号海水卷。检测物种通过过滤器滤芯的数量分别比使用玻璃纤维过滤器的那些和在这两个方法高约1.5倍，检测物种的过滤（ANCOVA，F = 164.2，p &lt;0.001）的水的体积增加的数量。 检测物种通过过滤器滤芯的数字较高，可能会导致从一个或多个在实验流程如下步骤:通过（1）不同的材料所采样的水的过滤（亲水性PVDF [聚偏二氟乙烯] 对玻璃微纤维）和/或（2）不同的公称孔尺寸（0.4581，在滤芯和玻璃纤维过滤器与米0.70微米），可从原过滤才对保留更多的eDNA; （3）在预热的培养箱使用旋转振荡器的产生更多的DNA产量从滤筒比在放置一动不动的热块上的旋转柱折叠玻璃纤维过滤器; （4）从滤筒提取的DNA通过离心收集比从由于不同的过滤材料和/或不同的离心分离的方法在玻璃纤维过滤器更有效。显然，一个更严格设计的研究是需要查明负责通过在本研究的滤筒的一贯高数检测物种的因素。 <img alt="图1" src="/files/ftp_upload/54741/54741fig1.jpg" /> 图1: 检测种类的标绘对过滤水卷MiFish Metabarcoding安娜的数<html>从黑潮坦克，冲绳美丽海水族馆。检测物种的数量的eDNA的裂解被限制容纳在罐中的物种。从30升海水的，我们使用的过滤筒过滤的1，2，3和4 L个采样和使用本和先前描述的方案12中 ，分别玻璃纤维过滤器和提取的eDNA从这些过滤器。我们对10 PCR使用两个协议的四水卷复制进行MiFish metabarcoding分析（多物种的同时检测）。盒子里的单杠代表的中位数;盒子的上下边缘对应跨四分位数，竖条对应1.5倍四分位数，而圆点代表异常值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54741/54741fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> /files/ftp_upload/54741/54741tbl1.jpg"/&gt; 表1: 分类组成，并从黑潮坦克的eDNA样品MiSeq分析检测的60种阅读仅限于数字包含在与自定义数据库的参考序列坦克的物种显示<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54741/54741tbl1large.jpg" target="_blank">，请点击这里查看大图此表。</a>

Watch this Scientific Journal Video about Use of a Filter Cartridge for Filtration of Water Samples and Extraction of Environmental DNA at JoVE.com

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

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 ...

use-filter-cartridge-for-filtration-water-samples-extraction

Nanyang Technological University

Research

JoVE Journal

Environment

24.8K 次观看.  Natural History Museum and Institute, Chiba. 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.

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

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. ...

科研

环境科学

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is not an appropriate measure for its availability; bioavailability rather depends on the dynamic interplay of potential mass transfer (flux) of a compound to a microbial cell and the capacity of the latter to degrade the compound. In water-unsaturated parts of the soil, mycelia have been shown to overcome bioavailability limitations by actively transporting and mobilizing organic compounds over the range of centimeters. Whereas the extent of mycelia-based transport can be quantified easily by chemical means, verification of the contaminant-bioavailability to bacterial cells requires a biological method. Addressing this constraint, we chose the PAH fluorene (FLU) as a model compound and developed a water unsaturated model microcosm linking a spatially separated FLU point source and the FLU degrading bioreporter bacterium Burkholderia sartisoli RP037-mChe by a mycelial network of Pythium ultimum. Since the bioreporter expresses eGFP in response of the PAH flux to the cell, bacterial FLU exposure and degradation could be monitored directly in the microcosms via confocal laser scanning microscopy (CLSM). CLSM and image analyses revealed a significant increase of the eGFP expression in the presence of P. ultimum compared to controls without mycelia or FLU thus indicating FLU bioavailability to bacteria after mycelia-mediated transport. CLSM results were supported by chemical analyses in identical microcosms. The developed microcosm proved suitable to investigate contaminant bioavailability and to concomitantly visualize the involved bacteria-mycelial interactions.

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

全细胞Bioreporter方法​​评估交通有机污染物的生物利用度和水饱和系统

Bioavailability of contaminants is a prerequisite for their effective biodegradation in soil. The average bulk concentration of a contaminant, however, is ...

Use of Chironomidae (Diptera) Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. Unfortunately, traditional benthic larval sampling methods, such as the dip-net, can be time-consuming and expensive. An alternative protocol involves collection of Chironomidae surface-floating pupal exuviae (SFPE). Chironomidae is a species-rich family of flies (Diptera) whose immature stages typically occur in aquatic habitats. Adult chironomids emerge from the water, leaving their pupal skins, or exuviae, floating on the water&#8217;s surface. Exuviae often accumulate along banks or behind obstructions by action of the wind or water current, where they can be collected to assess chironomid diversity and richness. Chironomids can be used as important biological indicators, since some species are more tolerant to pollution than others. Therefore, the relative abundance and species composition of collected SFPE reflect changes in water quality. Here, methods associated with field collection, laboratory processing, slide mounting, and identification of chironomid SFPE are described in detail. Advantages of the SFPE method include minimal disturbance at a sampling area, efficient and economical sample collection and laboratory processing, ease of identification, applicability in nearly all aquatic environments, and a potentially more sensitive measure of ecosystem stress. Limitations include the inability to determine larval microhabitat use and inability to identify pupal exuviae to species if they have not been associated with adult males.

Use of Chironomidae Diptera Surface-Floating Pupal Exuviae as a Rapid Bioassessment Protocol for Water Bodies

Rapid bioassessment protocols using benthic macroinvertebrates are often used to monitor and assess water quality. An efficient protocol involves collections of Chironomidae surface-floating pupal exuviae (SFPE). Here, techniques for field collection, laboratory processing, slide mounting, and identification of Chironomidae SFPE are described.

使用摇蚊科（双翅目）的表面浮蛹蜕为快速生物议定书水体

Rapid bioassessment protocols using benthic macroinvertebrate assemblages have been successfully used to assess human impacts on water quality. ...

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 ...

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

内毒素和DNA含量的空气采样滤波器分析

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

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 ...

Extraction of Organochlorine Pesticides from Plastic Pellets and Plastic Type Analysis

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

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

从塑料颗粒中提取有机氯农药和塑料类型分析

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

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.