使用脱脂牛奶絮凝和超滤法浓缩环境水和废水样品中的病毒颗粒
Summary
环境水和废水样品中的病毒浓度是一项具有挑战性的任务，主要用于病毒的鉴定和定量。虽然已经开发和测试了几种病毒浓缩方法，但我们在这里展示了超滤和脱脂牛奶絮凝对不同样品类型的RNA病毒的有效性。
Abstract
基于水和废水的流行病学已成为监测和预测社区疫情进程的替代方法。从废水和环境水样中回收微生物组分，包括病毒、细菌和微真核生物，是这些方法中具有挑战性的步骤之一。在这项研究中，我们专注于使用ArmorRNA作为测试病毒的顺序超滤和脱脂牛奶絮凝（SMF）方法的回收效率，该方法也被其他一些研究用作对照。超滤前采用0.45 μm和0.2 μm膜盘式过滤器进行预过滤，去除固体颗粒，防止超滤装置堵塞。用顺序超滤方法处理的测试样品以两种不同的速度离心。速度的提高导致铠甲RNA的恢复率和阳性率降低。另一方面，SMF导致装甲RNA的恢复率和阳性率相对一致。对环境水样进行的其他测试表明，SMF可用于浓缩其他微生物组分。考虑到废水样品超滤之前应用的预过滤步骤，将病毒分成固体颗粒可能会影响整体回收率。由于样品中的固体浓度较低，因此具有预过滤功能的SMF在应用于环境水样品时表现更好，因此降低了对固体的分配率。在本研究中，使用顺序超滤方法的想法源于在COVID-19大流行期间减少病毒浓缩物最终体积的必要性，当时常用的超滤设备的供应有限，并且需要开发替代病毒浓度方法。
Introduction
确定地表和废水样品中微生物的有效浓度以进行微生物群落分析和流行病学研究，是监测和预测社区暴发过程的重要步骤之一1，2。COVID-19大流行揭示了改进集中方法的重要性。COVID-19 于 2019 年底出现，截至 2023 年 3 月，仍对人类健康、社会生活和经济构成威胁。有效的监测和控制策略，以减轻COVID-19疫情对社区的影响已成为一个重要的研究课题，因为除了病毒的快速传播和传播以及未报告和未确诊的无症状病例外，还出现了COVID-19的新浪潮和变种3，4，5.民间社会组织、政府机构以及公共或私营公用事业使用基于废水的COVID-19流行病学有助于提供快速暴发相关信息并减轻COVID-19疫情的影响6、7、8、9。然而，废水样品中SARS-CoV-2（一种包膜RNA病毒）的浓度仍然构成挑战10。例如，这些挑战之一是废水固体中SARS-CoV-2的分配，当固体在浓度11期间被消除时，这可能会影响回收。如果是这种情况，定量/评估的重点应放在环境水样的固相和水相上，而不仅仅是水相。此外，可以根据下游测试和分析修改浓缩方法的选择。随着测序和微生物组领域的发展，环境样本中病毒颗粒和病原体的浓度已成为一个紧迫的研究课题。
在环境水和废水样品的病毒浓度领域已经应用了各种病毒浓缩方法。一些常用的方法有过滤、脱脂牛奶絮凝（SMF）、吸附/洗脱和聚乙二醇沉淀12-17。其中，SMF被认为是一种廉价有效的方法，已成功测试，并应用于从废水和地表水中回收病毒，包括SARS-CoV-212，15，16，18。SMF 程序是一种相对较新的方法，在许多环境研究中得到了越来越多的认可，是一种合适的方法，可以同时从所有类型的水样（即污泥、原始污水、废水和污水样本）中回收各种微生物，例如病毒、细菌和原生动物19.与其他已知的从环境样品中回收病毒的方法（例如超滤和甘氨酸碱洗脱、基于冻干的方法或超速离心和甘氨酸-碱洗脱）相比，SMF 已被报道为最有效的方法，具有更高的病毒回收率和检出率18，20.在本研究中，我们使用ArmorRNA作为测试病毒来评估病毒浓缩方法的回收效率，包括评估SARS-CoV-2回收率的测试21，22。
在这里，我们测试了废水和环境水样，以证明SMF和顺序超滤方法在浓缩微生物级分以进行定量聚合酶链反应（qPCR），基于序列的宏基因组学和深度扩增子测序的实用性。与超滤方法相比，SMF 是一种相对便宜的方法，最适合大量样品。使用顺序超滤方法的想法源于在 COVID-19 大流行期间减少病毒浓缩物最终体积的必要性，当时常用超滤设备的供应有限，并且需要开发替代病毒浓缩方法。
Protocol
1. 连续超滤与脱脂牛奶絮凝浓缩废水样品中病毒的比较 样品制备收集2 L的24小时流量比例复合原始（进料）废水样品。2020年夏季和秋季，从加拿大温尼伯的三个主要废水处理厂（WWTP）收集了样品（表1）。 将样品装在保温箱中的防光瓶中运送到实验室，并在24小时内进行处理。收集废水理化和生物特性数据。 病毒浓度测定注意：?…
Representative Results
病毒RNA浓度方法的评估用UF-3k x g处理的所有六个样品均为阳性，回收率为13.38%±8.14%（图1）。当样品用UF-7.5k x g处理时，只有一个样品呈阳性。用SMF处理的所有样品均为阳性，回收率为15.27%±2.65%（图1）。UF-3K x g和SMF的平均回收率显著且一致（p < 0.0001）高于UF-7.5K x g。另一方面，UF-3K x g和SMF的回收?…
Discussion
本研究的关键步骤之一是通过使用0.2μm和0.45μm膜过滤器进行预过滤步骤来消除固体颗粒。考虑到病毒被分割成固体颗粒，特别是包膜病毒，预过滤会导致病毒回收的显着损失30。虽然对于环境和废水样品几乎总是需要超滤方法的预过滤步骤以防止超滤设备堵塞，但根据下游分析的类型，可能需要对SMF进行预过滤。对于靶向PCR研究，可能不需要预过滤。另一方面，预过滤可能会显?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NSERC联盟Covid-19资助（奖项编号431401363，2020-2021，袁博士和乌亚瓜里-迪亚斯的支持）。MUD要感谢大学研究资助计划（奖项编号325201）。JF和JZA都得到了视觉和自动化疾病分析（VADA）研究生培训计划的支持。KY和JF都获得了Mitacs Accelerate计划的奖学金。MUD及其实验室成员（KY，JF，JZA）得到了NSERC-DG（RGPIN-2022-04508）和马尼托巴省研究新研究者运营补助金（第5385号）的支持。特别感谢曼尼托巴省温尼伯市。这项研究是在曼尼托巴大学进行的。我们要承认，曼尼托巴大学校园位于Anishinaabeg，Cree，Oji-Cree，Dakota和Dene人的原始土地上以及梅蒂斯民族的家园。
Materials
| 0.2 M sodium phosphate buffer with a pH 7.5 | Alfa Aesar | J62041AP | Fisher Scientific, Fair Lawn, NJ, USA |
| 0.2 μm 47-mm Supor-200 membrane disc filters | VWR | 66234 | Pall Corporation, Ann Arbor, MI |
| 0.45 μm 47-mm Supor-200 membrane disc filters | VWR | 60043 | Pall Corporation, Ann Arbor, MI |
| 4X TaqMan Fast Virus 1-Step Master Mix | Thermo Fisher Scientific | 4444432 | Life Technologies, Carlsbad, CA, USA |
| Armored RNA Quant IPC-1 Processing Control | Asuragen | 49650 | Asuragen, Austin, TX, USA |
| Brand A, Jumbosep Centrifugal Device, 30-kDa | Pall | OD030C65 | Pall Corporation, Ann Arbor, MI |
| Brand B, Microsep Advance Centrifugal Device, 30-kDa | Pall | MCP010C46 | Pall Corporation, Ann Arbor, MI |
| Centrifuge tubes (50 ml) | Nalgene | 3119-0050PK | Thermo Fisher Scientific |
| DNAse I | Invitrogen | 18047019 | Thermo Fisher Scientific |
| Dyna Mag-2 | Invitrogen | 12027 | Thermo Fisher Scientific |
| GWV High Capacity Groundwater Sampling Capsules – 0.45 µm | Pall | 12179 | Pall Corporation, Ann Arbor, MI |
| Hydrochloric acid, 1N standard solution | Thermo Fisher Scientific | AC124210025 | Fisher Scientific, Fair Lawn, NJ, USA |
| MagMAX Microbiome Ultra Nucleic Acid Isolation Kit | Applied biosystems | A42358 | Thermo Fisher Scientific |
| Nuclease free water | Promega | P1197 | Promega Corporation, Fitchburg, WI, USA |
| Peristaltic pump | Masterflex, Cole-Parmer instrument | 7553-20 | Thermo Fisher Scientific |
| pH meter | Denver instrument | RK-59503-25 | Cole-Parmer. This product has been discontinued |
| Phenol:chloroform:isoamyl alcohol 25:24:1 | Invitrogen | 15593031 | Fisher Scientific, Fair Lawn, NJ, USA |
| Primers and probe sets | IDT | Integrated DNA Technologies, Inc., Coralville, IA, USA | |
| Qiagen All-prep DNA/RNA power microbiome kit | Qiagen | Qiagen Sciences, Inc., Germantown, MD, USA | |
| QuantStudio 5 Real-Time PCR System | Thermo Fisher Scientific | A34322 | Life Technologies, Carlsbad, CA, USA |
| Qubit 1X dsDNA High Sensitivity (HS) assay kit | Invitrogen | Q33231 | Thermo Fisher Scientific |
| Qubit 4 Fluorometer, with WiFi | Invitrogen | Q33238 | Thermo Fisher Scientific |
| Qubit RNA High Sensitivity (HS) assay kit | Invitrogen | Q32855 | Thermo Fisher Scientific |
| RNAse A | Invitrogen | EN0531 | Thermo Fisher Scientific |
| RNeasy PowerMicrobiome Kit | Qiagen | 26000-50 | Qiagen Sciences, Inc., Germantown, MD, USA |
| Skim milk powder | Difco (BD Life Sciences) | DF0032173 | Fisher Scientific, Fair Lawn, NJ, USA |
| Sodium phosphate buffer | Alfa Aesar | Alfa Aesar, Ottawa, ON, Canada | |
| Synthetic seawater | VWR | RC8363-1 | RICCA chemical company |
| Synthetic single-stranded DNA gBlock | IDT | Integrated DNA Technologies, Inc., Coralville, IA, USA | |
| VacuCap 90 Vacuum Filtration Devices – 0.1 µm, 90 mm, gamma-irradiated | Pall | 4621 | Pall Corporation, Ann Arbor, MI |
| VacuCap 90 Vacuum Filtration Devices – 0.2 µm, 90 mm, gamma-irradiated | Pall | 4622 | Pall Corporation, Ann Arbor, MI |
| β-mercaptoethanol | Gibco | 21985023 | Fisher Scientific, Fair Lawn, NJ, USA |
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Citazione di questo articolo
Yanaç, K., Francis, J., Zambrano-Alvarado, J., Yuan, Q., Uyaguari-Díaz, M. Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration. J. Vis. Exp. (193), e65058, doi:10.3791/65058 (2023).