我们要感谢John Carra博士和Rowena Schokman博士提供纯化的埃博拉纳维VLP，Rajini Mudhasani博士提供基孔肯雅病毒，以及Charles（Jason）Shoemaker博士提供表达埃博拉病毒糖蛋白的小鼠白血病VLP。我们还要感谢MAJ Carl Soffler为夏季实习计划（SIP）和科学与工程学徒计划（SEAP）和凯瑟琳·威廉森博士提供实验室安全培训。

1.在使用病毒样本之前，在BSL-2环境中进行实验准备<ol><li>准备或购买Formvar和碳涂层的TEM铜网格，通常为200-400目。 </li><li> 将涂层的TEM网格插入胶囊。 <ol><li>使用放大镜可以使此过程容易执行。一个或两个网格可以插入每个胶囊。如果需要，可以购买预装胶囊以消除此步骤。 </li></ol></li><li>将带有TEM涂层网格的胶囊与其他用品和试剂一起转移到生物体内，其中病毒将被染色。 </li></ol> 2.使用水性戊二醛和1％四氧化锇蒸气失活的生物达标中的阴性染色的胶囊方法<ol><li>在生物驯化BSC内，将40μL病毒悬浮液吸入附着在移液管上的胶囊。 注意:移液器仍然连接到e胶囊，直到过程完成。病毒准备是根据我们以前的出版物14 。 </li><li>将移液管放在其侧面10分钟，网格水平定向。这是为了促进病毒颗粒在涂层网格上的均匀分布。 </li><li> 在Biocontainment BSC内的胶囊内灭活病毒。 <ol><li>拿起移液管并按压柱塞将病毒溶液分配到废物容器中。 </li><li>将40μL2％戊二醛固定液吸入胶囊。 注意:戊二醛是一种危险化学品，需要适当的保护。戊二醛可以在正常BSC中短暂使用，但延长的开放试剂需要在管道BSC或化学通风橱中工作。 </li><li>将移液器放在其侧面20分钟。这是为了确保样品固定好。 </li><li>推出固定剂并将40μL的dI水吸入胶囊中灰掉了固定剂。重复此洗涤步骤总共3次漂洗循环。 </li></ol></li><li>吸入40μL的1％乙酸铀酰（UA）或1％磷酸钨酸（PTA）到胶囊中，并使其静置30秒。 注意:基于病毒样本，染色时间可能在10秒至1分钟之间变化。 注意:UA是α发射体，是一种累积的毒素。处理适当的保护。 </li><li>从移液管中取出胶囊，然后通过在网格保留在胶囊内的同时将一块滤纸接触网格边缘，来吸干网格。 </li><li> 氧化锇蒸汽灭活程序。 <ol><li>将胶囊盖子打开放入含有浸泡在1％四氧化锇溶液中的滤纸的50mL离心管中。 注意:四氧化锇在极低的蒸气压下毒性极大。必须在管道BSC或化学通风橱中使用。处理适当的保护。发布警告在工作区域的信息。 </li><li>将50 mL离心管密封1 h，以使四氧化锇蒸气完全渗透。然后，随后对BSL-2 EM设施进行净化和转移出生物处理。 </li></ol></li><li> 从胶囊中取出EM网格。 <ol><li>在BSL-2 EM设备中，从离心管中取出胶囊并将其放在移液管上。 </li><li>将40μL的dI水吸入胶囊中，并将水分配到废物容器中三次。 </li><li>从移液器上取下胶囊，用滤纸吸干网格以接触网格边缘。 </li><li>空气干燥后，存放电网用于随后的TEM成像。 </li></ol></li></ol> 3.用2％戊二醛生物达标灭活的胶囊方法，随后在BSL-2实验室中进行阴性染色<ol><li> 病毒灭活程序。 <ol><li&gt;在生物驯化BSC内，将病毒悬浮液与相同体积的4％戊二醛混合，达到终浓度为2％的戊二醛。 注意:戊二醛是一种危险化学品，需要适当的保护。戊二醛可以在正常BSC中短暂使用，但延长的开放试剂需要在管道BSC或化学通风橱中工作。 </li><li>在包装，去污和将其转移到BSL-2 EM设施之前，先将固定剂灭活至少24小时。 </li></ol></li><li>在BSL-2 EM设备中，将病毒和固定剂混合物吸入胶囊，包含两个连接到移液管上的TEM网格。 </li><li>将移液器水平放置10分钟，网格水平定向。 注意:这是为了促进病毒颗粒均匀分布到TEM网格上。 </li><li>拿起移液管并按压柱塞将病毒排出到废物容器。吸出40μL的dI水进入胶囊并将其排入废物容器中3次漂洗循环。 </li><li>将40μL的1％UA或1％PTA吸入胶囊中30 s。 注意:基于病毒样本，染色时间从10秒变化到1分钟。 注意:UA是α发射体，是一种累积的毒素。处理适当的保护。 </li><li>从移液器上取下胶囊，并通过将网格边缘接触到一张滤纸来吸干网格。空气干燥网格并存放它们用于随后的TEM成像。 </li></ol>

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文章中提出的意见，解释，结论和建议是作者的意见，并不一定得到美国陆军或国防部的认可。

负染是用于评估和筛选病毒，蛋白质复合物和纳米颗粒的有价值的TEM技术。通过手动将网格从试剂转移到阴性污渍，这些样品的液滴制备已经是半个多世纪以来的典型方案。这是一个简单的过程，但需要通过培训获得成功完成的专业知识。优秀的阴性染色仍被认为是最先进的技术，并且在许多TEM实验室中非常期望。胶囊方法相对于手动液滴方法具有几个明显的优点，特别是在生物实验室中。首先，手动液滴方法需要大量的培训和经验才可以成功执行，而使用简单的胶囊方法可以通过简单的移液技术进入入门级技术人员。手动液滴法最大的挑战是使用BSL-3和BSL-4 PPE中的镊子成功处理TEM网格o大大降低了触觉和灵巧度。脆性涂层TEM网格容易被镊子损伤或刺穿。涂层TEM网格的损伤通常不会透露，直到用TEM观察。胶囊的第二个优点是TEM格栅只有当它们插入胶囊中并且当它们被移除以插入电子显微镜时才被直接处理。无论是在生物实验室还是BSL-2 EM设施工作，都不会直接进行电网处理。胶囊方法的第三个优点是网格的两侧被病毒和污渍覆盖。当样品浓度低时，这是有用的;然而，如果浓度高，可能导致过多的样品，或者在使用前导致样品稀释。 使用手动液滴技术的阴性染色多个网格是耗时的，因为每个样品需要单独制备和染色。这导致了困难ning一致，可重复染色。此外，跟踪许多个别网格总是一个挑战。每个胶囊容纳两个网格，并且多个胶囊可以连接到多通道移液管，从而简化了该过程。因此，胶囊确保在一致的样品条件下和同时使用非常相似的方法对网格进行染色。胶囊可以标记以帮助组织，从而减少网格混合或错误识别的可能性。使用手动液滴方法时面临的另一个常见问题是网格污染。当网格留在露天太久或掉落时，可能会发生这种情况。胶囊可保护TEM格栅免受露天影响，并将其牢牢固定，使其不会掉落。当制备和阴性染色时，胶囊提供更大的实验控制和重复性，因为胶囊是更受控制的环境。 胶囊可以预购，如果de首创。将网格手动装入胶囊需要一些做法才能确保网格不被损坏。胶囊方法还需要比手动液滴方法稍微更多的样品和污点。与手动液滴法相比，胶囊方法需要至少40μL的样品，可以用少至8μL进行。 病毒灭活是BSL-3和BSL-4阴性染色的重要组成部分，因为它允许灭活的病毒从生物实验室中取出，用于EM设施中成像。使用固定剂灭活具有修复病毒的附加益处，防止降解。灭活病毒样本有两种不同的方法，这两种方法都在本研究中进行了研究。首先将病毒粘附到涂覆的TEM网格上，将病毒在2％戊二醛溶液中固定20分钟，然后将栅格暴露于四氧化锇蒸气1小时（ 图1C ）。这个灭活离子法是有利的，因为通过使样品在1小时20分钟后能够从生物实验室中取出来节省时间。它也可以防止病毒稀释到涂覆的TEM网格之前，因此可能需要怀疑接近TEM检测限的样品。然而，1％的四氧化锇蒸气会降低TEM图像的质量，因为锇可以进一步污染样品（ 图3 ）。以前的报告显示，四氧化锇蒸气暴露5分钟，当阴性染色时可以获得很好的效果;然而，绝对病毒灭活所需的更长的暴露产生正和负染色17的混合物。第二种灭活方法在2％戊二醛溶液中至少需要24小时，不需要四氧化锇蒸气处理（ 图1D ）。第二种方法需要更长时间才能完成，因为样品没有从生物修复实验室中移除但是可以有利于在BSL-2环境中进行阴性染色，而不需要有害的四氧化锇。我们的研究结果表明，当用四氧化锇蒸气处理样品时，24小时的戊二醛灭活产生更高质量的TEM图像（ 图3 ）。 在本实验中使用了两种阴性污染物:UA和PTA。两种污渍均用作1％溶液。 UA，铀的醋酸盐，作为一个负面的污点，因为浓铀原子散射电子5 。 PTA，一种杂多酸，类似地作为由于致密的钨原子而产生的负污染5 。 PTA有时比UA优选，因为它毒性低得多，如果吸入或接触只有轻微的刺激物。当阴性染色未固定样品时，必须考虑UA的较低pH，因为它可能导致损伤o样品。在该实验中使用的病毒需要固定以进行灭活，因此UA和PTA都实现了类似的结果，并且对于两种染色都没有检测到明显的优点。两种污渍易于使用，并且它们都产生类似质量的TEM图像（ 图4 ）。 总之，胶囊方法在生物环境中更容易使用，可用作手动液滴法的替代方法。使用胶囊可以减轻样品操作缺陷，并产生良好质量的TEM图像。使用胶囊方法产生的图像与使用手动液滴方法产生的图像相当。用四氧化锇进行短病毒灭活可提高速度，但只有降低图像质量可接受才能使用，否则疑似稀释病毒会使其低于手术的TEM检测限。 UA和PTA都与本研究中使用的固定病毒样品提供了类似的结果;浩wever，当染色未固定病毒时，结果可能不同。

透射电子显微镜（TEM）可以观察生物样本太小的形态和超微结构的有效工具与传统的光学显微镜1，2，3，4中看到的。 TEM通过非常薄的样品射出电子，产生更高分辨率的图像，因为电子的波长比光短得多。弯曲或阻挡电子的样品的区域看起来很暗，而电子透明的区域看起来是白色的。 缺少电子致密物质使得病毒难以在TEM下观察，因为它们不能散射电子。阴性染色是用于通过TEM创建对比度和观察病毒的最常用方法。 Brenner和Horne在1959年提出了第一个负染色程序，基于Hall（1955）和Huxley（1957）观察到的实验当浸入电子密度物质5时，反向对比生物结构的外观。阴离子染色过程在过去半个世纪几乎没有改变。阴性染色涉及在TEM网格上简单地将重金属盐溶液施用于样品，以试图用致密材料包围病毒而不渗透病毒6 。这产生了一个黑色的边框，并显示了颗粒的形状5 。本研究使用两种阴性染色试剂，乙酸双氧铀（UA）和磷钨酸钾（PTA）。这两个污渍通常用于小的生物样品，如病毒，蛋白质复合物和纳米颗粒7，8，9负染色。 常规的阴性染色技术是手动液滴阴性染色技术nique 7 。该方法需要用镊子精确处理小而脆弱的TEM网格，以应用少量病毒样品，染色和漂洗。典型的制备方法包括将一滴样品悬浮液施加到膜涂布的TEM网格的表面上（ 图1A ）。在将样品连接到膜表面之后，漂洗网格以除去非粘附病毒，并且根据样品的类型用UA或PTA染色数秒至一分钟。通过将一块滤纸接触到网格的边缘，将过量的液体从网格中除去。 手动液滴方法要求每个格子单独制作。如果不小心处理，涂层的TEM网格容易被刺穿，弯曲或污染。处理多个样品可能导致跟踪网格的困难，并确保每个样品的染色一致。本手册染色程序更多d由于这些环境所需的个人防护装备（PPE），在生物安全水平（BSL）-3和-4生物实验室进行培训时很困难。 PPE是繁琐的，与常规实验室相比，生物环境的变化更为动荡。在BSL-3生物实验室工作的人员必须佩戴2双手套，并在生物安全柜（BSC）工作。这双手套可以降低触感，并限制电动机运动。保护用户并有助于防止样品污染的BSC的气流可导致样品和污渍太快地干燥，从而影响染色质量。 BSC中强烈的湍流气流也可以快速吹走不太牢固的电网。在BSL-4生物实验室中，还有其他安全要求。人员需要穿正压服，这进一步限制身体运动和清晰看见和操纵的能力网格。在BSL-4工作的技术人员还穿着至少2双手套，外套是厚厚的手套，大大降低了灵巧和触感。最后，用于处理TEM网格的镊子是锋利的，因此由于其穿刺手套的能力而对技术人员造成风险。使用含有网格的胶囊，镊子不是必需的，因此提供了一种安全的无镊子的替代方案，用于操纵生物体的网格。最后，胶囊还提供了在加工过程中储存网格的有效方式，锇蒸气去污和储存期间;从而保持网格的组织和安全免受损害。 在本报告中，我们介绍负染色TEM网格在生物防护实验室利用mPrep / g的胶囊，网格处理和染色10，11，12基于胶囊的装置的新方法。胶囊accom改变两个TEM网格，最大限度地减少直接处理，并减少电网损坏的可能性。胶囊以与移液管尖端相同的方式直接附接到单通道或多通道移液管，允许将各种液体施加到包含在其中的网格上。这使得能够同时准备具有重复格栅的多个样品（ 图1B ）。用胶囊阴性染色，将病毒样品吸入胶囊中并保持10分钟，使病毒吸附在网格表面上。随后用去离子水（dI）洗涤带有吸附病毒的网格，并用UA或PTA染色数秒至1分钟。该方法使用与手动液滴法相同的方案步骤和试剂;不同之处在于，所有的工作都发生在胶囊内，没有物理处理网格。 （ 图1C ，1D ）。 本研究的目的是评估胶囊一种用于生物环境中病毒样品阴性染色的新方法。本研究还检测了由两种不同病毒灭活程序产生的TEM图像的质量:1）快速灭活，1％四氧化锇蒸气，2）用2％戊二醛灭活24小时。这些都是使用胶囊进行的。最后，我们评估了两种常用的阴性污染物UA和PTA，用于胶囊。 13

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Formvar/carbon coated TEM grids</td>
			<td style="width:71px;">SPI</td>
			<td style="width:119px;">3420C-MB</td>
			<td style="width:167px;">200 mesh Cu Pk/100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">mPrep/g capsules</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">85010-01</td>
			<td>box</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">mPrep/g couplers</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">85010-11</td>
			<td>standard 16/Pk</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">glutaraldehdyde</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">16320</td>
			<td>50% solution, EM grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Osmium Tetroxide</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">19190</td>
			<td>4% aqueous solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Uranyl Acetate</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">22400</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Potassium phosphotungstic acid</td>
			<td style="width:71px;">EMS</td>
			<td style="width:119px;">19500</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1450-090</td>
			<td>size 50</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:233px;">Tranmission Electron Microscope</td>
			<td style="width:71px;">JEOL</td>
			<td style="width:119px;">JEM-1011</td>
			<td>TEM</td>
		</tr>
	</tbody>
</table>

utilization of capsules for negative staining of viral samples within biocontainment

透射电子显微镜（TEM）用于观察病毒和其他微生物病原体超微结构的纳米分辨率。大多数生物材料不含能够散射电子的致密元件以产生图像;因此，需要在样品周围放置稠密的重金属盐的负污渍。为了在TEM下观察悬浮液中的病毒，它们必须应用于涂覆有仅纳米厚的透明表面的小网格。由于其尺寸小巧易碎，这些网格难以处理，容易被气流移动。薄表面容易损坏，使样品难以或不可能成像。传染病毒必须在生物安全柜（BSC）中进行处理，有些需要生物化学实验室环境。生物安全水平（BSL）-3和-4中的染色病毒特别具有挑战性，因为这些环境更为动荡，技术人员需要to佩戴个人防护装备（PPE），降低灵活性。 在这项研究中，我们评估了一种新的装置，以帮助负染色的病毒生物获得。该设备是一种胶囊，可用作专用移液器吸头。一旦将网格装入胶囊中，用户只需将试剂吸入胶囊中，将病毒和污渍传递到封装的网格上，从而消除用户对网格的处理。虽然这种技术专门设计用于BSL-3或-4生物测定，但它可以通过实现病毒容易的阴性染色来缓解任何实验室环境中的样品制备。同样的方法也可用于制备纳米颗粒，大分子和类似标本的负染色TEM样品。

胶囊方法产生优质的阴性染色用于TEM成像: 首先，我们通过使用手动液滴法和用于阴性染色扎伊尔埃博拉病毒的胶囊方法来评估产生的图像的质量。伏博病毒属于Filoviridae家族，还有Marburg病毒。埃博拉病毒的直径通常为80至100纳米，长度可超过1000纳米。埃博病毒必须在BSL-4生物环境中处理。 图2示出了使用手动液滴法和用于阴性染色的胶囊方法产生的图像。 图2A （手动液滴法）和图2B （胶囊法）显示在病毒粒子中心具有明确定义细胞核结构的微卫星病毒样品，可见的埃博拉病毒糖蛋白在表面上。因此，胶囊和液滴方法都具有产生类似质量的TEM图像的能力。 使用胶囊法，用戊二醛水溶液和1％四氧化锇蒸气快速灭活，并用2％含水戊二醛进行24小时灭活，比较和评估EM图像质量: 我们评估了使用基孔肯雅病毒从两种不同的样品灭活方法产生的TEM图像的质量。基孔肯雅病毒是披膜 病毒科中的甲病毒属的成员。它是直径为60-70nm的球形。病毒粒子含有丰富的糖蛋白的信封，其在病毒表面上形成三聚体峰。基孔肯雅病毒必须在BSL-3中处理。使用2％戊二醛20分钟后快速灭活，然后1小时暴露于1％四氧化锇蒸气，整个阴离子在染色过程中发生在BSC内的胶囊中的生物实验室（ 图1C ）。然而，当使用2％戊二醛24小时来灭活病毒时，灭活发生在生物环境中，但是在BSL-2实验室中使用胶囊法进行1％UA阴性染色程序（ 图1D ）。这些灭活程序不产生相同质量的图像（ 图3 ）。从图3可以看出，戊二醛中没有四氧化锇存在下的固定（ 图3A ，3B ）显示出比用戊二醛和四氧化锇制备的样品更多的超微结构细节（ 图3C ，3D ）。 胶囊法使用醋酸铀（UA）和磷酸盐戊酸（PTA）作为醛固定样品的阴性污渍。 图4中示出使用胶囊方法的UA和PTA阴性染色的实例在醛固定病毒样颗粒（VLP）上。 VLP是组装成病毒样结构的蛋白质，但不含有任何病毒遗传物质。它们通常用于疫苗开发和基础病毒研究。负染色是评估VLP装配和形态的有价值的工具。我们使用UA和PTA用胶囊法对VLP进行阴性染色。两种污渍都显示出高质量的结果，糖蛋白可见和明确界定埃博拉纳维VLP 15 （ 图4A ，4B ）和鼠白血病VLPs 16 （ 图4C ，4D ）。 图1:负染方法概述。 （ A ）通过将TEM网格从液滴逐步移动到样品液滴，冲洗和污渍来进行手动液滴法。 （ B ）用网格设置胶囊方法并应用样品悬浮液。 （ C ）使用胶囊方法在1％四氧化锇蒸气短时间灭活的典型程序。 （ D ）使用胶囊法在2％戊二醛长期灭活的生物培养中的推荐方法。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56122/56122fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 图2:1％PTA阴性埃博病毒颗粒的比较。 （ A ）用手动液滴法进行阴性染色。 （ B ）使用胶囊法进行阴性染色。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56122/56122fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/56122/56122fig3.jpg" /> 图3:使用不同灭活程序的基孔肯雅病毒胶囊方法1％UA阴性染色。 （ A ， B ）仅用2％戊二醛灭活至少24小时。 （ C ，D）用2％戊二醛和1％四氧化锇蒸气快速灭活。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56122/56122fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/56122/56122fig4.jpg" /> 图4:使用胶囊方法的磷钨酸（PTA）和乙酸铀酯（UA）阴性病毒样颗粒（VLP）的实例。 （ A ）1％PTA染色的埃博拉纳维VLP的低放大概率。 （ B ）显示PTA染色的埃博拉纳米VLP的结构细节的高倍放射TEM图像。 （ C ）1％UA染色的小鼠白血病VLP的低放大概率。 （ D ）高倍放大TEM图像显示结构detUA染色的小鼠白血病VLP在其表面具有禽流感病毒糖蛋白。图先前已发布，并重新使用参考13的权限。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56122/56122fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment at JoVE.com

Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

该协议为负染色病毒样本提供了指导，可以很容易地用于BSL-2，-3或-4实验室。它包括使用创新的加工胶囊，其保护透射电子显微镜网格并且在生物达到的更湍流环境中为使用者提供更容易的处理。

利用胶囊对生物达标中病毒样品的阴性染色

Transmission electron microscopy (TEM) is used to observe the ultrastructure of viruses and other microbial pathogens with nanometer resolution. Most ...

utilization-capsules-for-negative-staining-viral-samples-within

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.1K 次观看.  United States Army Medical Research Institute of Infectious Diseases (USAMRIID). 该协议为负染色病毒样本提供了指导，可以很容易地用于BSL-2，-3或-4实验室。它包括使用创新的加工胶囊，其保护透射电子显微镜网格并且在生物达到的更湍流环境中为使用者提供更容易的处理。 

视频: Utilization of Capsules for Negative Staining of Viral Samples within Biocontainment

Development of a Negative Selectable Marker for Entamoeba histolytica

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up- and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain.

Development of a Negative Selectable Marker for Entamoeba histolytica

We report development of a negative selection system in E. histolytica based upon transgenic expression of a chimeric protein (FCU1) and selection with the prodrug 5-fluorocytosine. The FCU1 protein is a fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase. Expression of FCU1 resulted in increased E. histolytica sensitivity towards 5-fluorocytosine.

一个负选择标记的发展阿米巴</em

Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the ...

科研

免疫与感染

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Flow Cytometry Analysis of Immune Cells Within Murine Aortas

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam cells, immune cells, vascular endothelial and smooth muscle cells, platelets, extracellular matrix, and a lipid-rich core with extensive necrosis and fibrosis of surrounding tissues.1 The innate and adaptive arms of the immune response are involved in the initiation, development and persistence of atherosclerosis.2, 3 There is a significant body of evidence that different subsets of the immune cells, such as macrophages, dendritic cells, T and B lymphocytes, are present within the aortas of healthy and atherosclerosis-prone mice4. Additionally, immune cells are found in the surrounding aortic adventitia which suggests an important role of this tissue in atherogenesis.2
For some time, the quantitative detection of different types of immune cells, their activation status, and the cellular composition within the aortic wall was limited by RT-PCR and immunohistochemical methods for the study of atherosclerosis. Few attempts were made to perform flow cytometry using human aortas, and a number of problems, such as a high autofluorescence, have been reported5,6. Human atherosclerotic plaques were digested with collagenase 1, and free cells were collected and stained for CD14+/CD11c+ to highlight macrophage-derived foam cells. In this study, a "mock" channel was used to avoid false-positive staining.6 Necrotic materials accumulating during the digestion process give rise in a large amount of debris that generates a high autofluorescence in aortic samples. To resolve this problem, a panel of negative and positive controls has been proposed, but only double staining could be applied in these samples. We have developed a new flow cytometry-based method7 to analyze the immune cell composition and characterize the activation, proliferation, differentiation of immune cells in healthy and atherosclerosis-prone aorta. This method allows the investigation of the immune cell composition of the aortic wall and opens possibilities to use a broad spectrum of immunological methods for investigations of immune aspects of this disease.

This paper presents a flow cytometry-based method to investigate the immune composition of aortas. The paper also illustrates an additional technique that allows examining surrounding adventitia and vessel wall separately. This method opens possibilities to perform phenotypical analyses of aortic leukocytes and apply several immunological assays for atherosclerosis studies.

流式细胞仪分析小鼠主动脉内流的免疫细胞

Atherosclerosis is a chronic inflammatory process of medium and large size vessels that is characterized by the formation of plaques consisting of foam ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

富达的RNA病毒和病毒的突变频率的表征的变种的分离

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin&#160;contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.

The application of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) directly to blood culture broth expedites the identification of bacteria. The presented method is a rapid and reliable method for identification of Gram negative bacteria directly from blood culture broth.

革兰从血培养液使用MALDI-TOF质谱阴性细菌快速鉴定

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.