用阴离子交换树脂检测 Bioaerosols 病毒
1High Plains Intermountain Center for Agricultural Health and Safety, Department of Environmental and Radiological Health Sciences,Colorado State University, 2National Wildlife Research Center, Wildlife Services, Animal and Plant Health Inspection Service,United States Department of Agriculture, 3Western Sydney University, 4Leprino Foods, Inc, 5Department of Food Science and Agricultural Chemistry,McGill University, 6Department of Mechanical Engineering,Colorado State University, 7Department of Animal Science,University of Wyoming
Summary
证明了一种基于液体冲击的气溶胶病毒取样方法。当与下游分子检测相结合时, 该方法允许对 bioaerosols 的病毒进行简便、灵敏的检测。
Abstract
该协议演示了一种针对病毒的自定义气溶胶采样方法。在该系统中, 负离子交换树脂与液体撞击式空气取样装置相结合, 有效地集中了 bioaerosols 的负电荷病毒。因此, 树脂在气溶胶取样工作流中充当额外的浓度步骤。然后直接从阴离子交换树脂中提取出病毒微粒的核酸, 得到的样品适用于分子分析。此外, 本议定书还描述了一个定制的气溶胶室, 能够在各种环境条件下产生带有病毒的 bioaerosols, 并允许对环境变量 (如温度、湿度) 进行连续监测,风速和气溶胶质量浓度。使用此协议的主要优点是增强了病毒检测的灵敏度, 这是通过与未修改的常规液体冲击器进行直接比较来评估的。其他优势包括可能集中各种负电荷病毒, 低成本的阴离子交换树脂 (每样0.14 美元), 并易于使用。缺点包括本议定书无法评估树脂吸附的病毒微粒的传染性, 并可能需要优化冲击器中使用的液体取样缓冲器。
Introduction
该方法的目的是提供一个高度敏感的气溶胶取样平台, 以促进分子检测的负电荷病毒从 bioaerosols。微生物, 包括病毒微粒, 可以在 bioaerosols 中存活1长时间。Bioaerosols 可以旅行较远的距离和保持生存能力和传染性, 这证明了军团的疾病爆发的起源于距离6公里的工业冷却塔从受影响的人和导致18人死亡2。通过 bioaerosols 介导的病毒向人类间接传播可能发生在多种环境中, 并已证明诺如病毒在学校和餐馆3、4爆发。同样, 气溶胶传播病毒可能发生在农业环境中, 如在猪和家禽养殖场, 这一传输路线被认为是一个主要因素在病毒的移动之间的生产设施5,6,7,8,9。
对病毒携带的 bioaerosols 的有效取样可以改善快速诊断和预防疫情的准备, 如在中国活动物市场 bioaerosols 检测到 H5 流感病毒的示威活动中所示, 以及美国10,11。目前的气溶胶取样技术涉及许多不同的粒子捕获原理, 可大致分为 impingers、旋风、撞击和过滤器12。在本议定书范围之外, 详尽地涵盖了这些平台对 bioaerosols 病毒取样的所有利弊;但是, 可以说, 大多数这些取样装置尚未针对病毒和噬菌体13的收集进行优化。此外, 病毒微粒的传染性往往受到负面影响, 液体 impingers 认为更有效地保持病毒传染性比取样设备, 如固体撞击或过滤器14。然而, 液体撞击的一个缺点是靶稀释效应, 这是因为病毒在收集容器中相对较大的体积 (通常是≥20毫升) 收集的。另一个重要的缺点是液体 impingers 的次优效率, 浓缩颗粒 < 0.5 µM 大小15。然而, 这些装置的捕获效率可以通过固定在固体基质上来改善, 因为固定化可以增强病毒核酸的保存和病毒传染性16,17。
我们以前已经证明, 阴离子交换树脂是一种有效的工具, 从液体基质中捕获和浓缩病毒, 包括 F RNA 噬菌体, 甲型肝炎病毒, 人腺病毒, 和轮状病毒18,19 ,20。根据制造商的定义, 在这项工作中使用的阴离子交换树脂是一种吸附聚苯乙烯强碱性阴离子交换树脂, 功能性季胺基团介导在液体介质中对阴离子的吸引和捕获21.因此, 阴离子交换树脂有望捕获病毒的净负表面电荷, 包括许多肠道病毒, 流感病毒和其他与公共和动物健康有关的病毒。
目前的协议涉及增加负离子交换树脂的液体冲击器。在该系统中, 树脂作为冲击器液中捕获的病毒微粒的二次浓缩步骤。核酸可以直接洗脱在小体积, 提供了一个浓缩样本的分子分析。因此, 该方法的主要优点是提高了病毒检测灵敏度, 主要是通过减少样本量。另外, 由于对负电荷病毒的固有非特定捕获, 该方法可能适用于检测大量感兴趣的病毒。对 A 型和 B 型流感病毒的疫苗株和 FRNA 噬菌体 MS2 (MS2) 进行了验证。这些病毒随后使用标准的 qRT PCR 方法检测, 如前所述22。终端用户不应期望在执行此方法时遇到困难, 因为对当前现有设备的修改不构成对常规气溶胶取样和分析流程的重大干扰。
Protocol
1. 气溶胶室的设置 (见图 2) 预加载液体 impingers 与20毫升0.01 米磷酸盐缓冲盐水, pH 值 7.5 (PBS)。 加入0.5 克阴离子交换树脂, 悬浮在其中一个液体 impingers 的 PBS 内, 另一种液体冲击器用作控制。 在气溶胶腔内平行放置液体 impingers, 使用夹持器, 面对喷雾器的气溶胶入口。注: 有关其他详细信息, 请参见图 2 。…
Representative Results
图 1展示了基于电荷的从 bioaerosols 捕获病毒的原理, 通过将树脂加入液体基 impingers。图 2显示了自定义构建的气溶胶室的设置。图 3描述了设置 aerosolization 实验的步骤和确保质量控制的措施。图 4显示了从 bioaerosols 捕获的负电荷病毒 qRT PCR 检测的放大曲线。表 1显示了用?…
Discussion
本协议概述了一种使用改进的液体 impingers 对 bioaerosols 进行敏感病毒捕获的方法。该方法对 bioaerosols 中病毒载量的检测和定量进行了优化。这里所展示的具体修改包括在普通液体冲击器中加入负离子交换树脂。该方法是为简化下游样品加工而开发的, 而其他样品处理技术, 如离心、过滤和沉淀法等, 并没有提供这样的优势。核酸的提取是直接从树脂, 不需要高容量 elutions 和多样品准备步骤, 最终有?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了来自 CDC/NIOSH 高平原山间农业健康与安全中心 (5U54OH008085) 和科罗拉多生物科学发现评估补助金计划 (14 改编-16) 的资助。
Materials
| Escherichia coli bacteriophage MS2 (ATCC 15597-B1) | American Type Culture Collection | ATCC 15597-B1 | |
| FluMist Quadrivalent | AstraZeneca | Contact manufacturer | Viral constitutents of this vaccine are subject to change on an annual basis |
| CFX96 Touch Real-Time PCR Detection System | Bio-Rad | 1855195 | |
| Primers and probes | Integrated DNA Technologies | NA | |
| 0.2 µM sterile filter | NA | NA | |
| 1 L pyrex bottles or equivalent | NA | NA | |
| 1 mL pipet tips | NA | NA | |
| 1 mL pipettor | NA | NA | |
| 50 mL serological pipet | NA | NA | |
| PCR tubes | NA | NA | |
| Pipet-aid or equivalent | NA | NA | |
| QIAamp Viral RNA Mini Kit | Qiagen | 52904 | |
| QuantiTect Probe RT-PCR Kit | Qiagen | 204443 | |
| Amberlite IRA-900 chloride form | Sigma-Aldrich | 216585-500G | |
| Phosphate buffered saline | Sigma-Aldrich | P5368-10PAK | |
| Water (molecular biology grade) | Sigma-Aldrich | W4502-1L | |
| Eppendorf DNA LoBind Microcentrifuge Tubes | ThermoFisher | 13-698-791 | |
| Falcon 50 mL Conical Centrifuge Tubes | ThermoFisher | 14-432-22 | |
| Falcon Polypropylene Centrifuge Tubes | ThermoFisher | 05-538-62 | |
| SuperScript III Platinum One-Step qRT-PCR Kit w/ROX | ThermoFisher | 11745100 | |
| SKC Biosampler 20 mL, 3-piece glass set | SKC Inc. | 225-9593 | |
| Vac-u-Go sample pumps | SKC Inc. | 228-9695 | |
| Collison nebulizer (6-jet) | BGI Inc. | NA | |
| HEPA capsule | PALL | 12144 | |
| Q-TRAK indoor air quality monitor 8554 | TSI Inc. | NA | |
| Alnor velometer thermal anemometer AVM440-A | TSI Inc. | NA | |
| SidePak AM510 personal aerosol monitor | TSI Inc. | NA | |
| Bioaerosol chamber | NA | NA |
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Cite This Article
Schaeffer, J. W., Chandler, J. C., Davidson, M., Magzamen, S. L., Pérez-Méndez, A., Reynolds, S. J., Goodridge, L. D., Volckens, J., Franklin, A. B., Shriner, S. A., Bisha, B. Detection of Viruses from Bioaerosols Using Anion Exchange Resin. J. Vis. Exp. (138), e58111, doi:10.3791/58111 (2018).