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环境科学

废水和空气样本中SARS-CoV-2 RNA的定量和全基因组表征

Published: June 30, 2023
doi:
10.3791/65053
, , , , , , , , ,
1Institute of Sustainable Processes,University of Valladolid, 2Department of Chemical Engineering and Environmental Technology,University of Valladolid, 3Department of Public Health Microbiology,National Laboratory of Health, Environment and Food, 4ICBAS – School of Medicine and Biomedical Sciences,Porto University, 5Epidemiology Research Unit (EPIUnit),Instituto de Saúde Pública da Universidade do Porto, 6Laboratório para a Investigação Integrativa e Translacional em Saúde Populacional (ITR)

Summary

该协议旨在量化废水和空气样本中的SARS-CoV-2 RNA，用于基于废水的流行病学研究，并评估室内和室外气溶胶中SARS-CoV-2的暴露风险。该协议还描述了用于 SARS-CoV-2 全基因组表征的平铺扩增子长模板测序方法。

Abstract

在许多国家，基于废水的流行病学已成为一种有前途且有效的SARS-CoV-2和其他传染病监测系统。该过程通常涉及废水浓缩、核酸提取、选定基因组片段的扩增以及扩增基因组片段的检测和定量。这种方法同样可用于检测和量化空气样本中的传染性病原体，例如SARS-CoV-2。最初，据推测，SARS-CoV-2 主要通过与感染者在说话、打喷嚏、咳嗽、唱歌或呼吸时产生的飞沫密切接触传播。然而，越来越多的研究报告说，医疗机构的空气中存在SARS-CoV-2 RNA，将空气传播确定为病毒的可行途径。本研究提出了一系列既定的协议，以促进废水和空气样本中病毒的环境检测、定量和测序。

Introduction

2019 年 12 月，出现了一种名为 COVID-19 的新型疾病，由以前未知的冠状病毒 SARS-CoV-2引起 1.由此产生的全球大流行给全球临床和公共卫生实验室带来了重大挑战，因为大量个人需要检测以准确评估病毒在社区中的传播和流行情况。然而，在许多地区，及时和空间全面地达到必要的测试水平在经济上是不可行的 2,3.目前基于个体临床诊断的监测系统在很大程度上依赖于症状严重程度和个体报告，以及这些症状与人群中流行的现有疾病重叠的程度4,5,6,7,8,9,10。因此，大量无症状病例导致疾病负担被严重低估7,11。

由于这些挑战，COVID-19监测的基于废水的流行病学（WBE）被提议作为补充监测策略。WBE于2001年首次被描述12，最初用于追踪可卡因和其他非法药物13。这种方法依赖于这样的假设，即可以计算出废水中稳定并由人类排泄的任何物质的初始浓度8,12。WBE已在许多国家成功实施，作为SARS-CoV-2 3,8,14,15,16的补充和有效监测系统。在水生环境中检测人类病毒的大多数方法都遵循以下步骤：浓度、核酸提取、所选基因组片段（或多个片段）的扩增以及扩增基因组片段的检测/定量3.

检测和定量SARS-CoV-2的另一个重要环境是在空气样本中。最初，SARS-CoV-2 被认为主要通过密切接触感染者在说话、打喷嚏、咳嗽、唱歌或呼吸时产生的气溶胶产生的呼吸道飞沫传播17.然而，一些研究开始报告空气中存在 SARS-CoV-2 RNA，尤其是在医疗机构和其他封闭空间中 18,19,20,21。当病毒浓度足够高时，在医院和其他封闭空间的室内采集的空气样本中发现了 SARS-CoV-2 活力的证据22,23,2 4。户外研究通常没有发现SARS-CoV-2的证据，除了在拥挤的户外空间21,25,26,27,28,29。截至目前，SARS-CoV-2的空气传播已被公认为一种传播方式30,31。最近的一项回顾性研究表明，室外和室内之间存在差异，在室外，在拥挤区域之外，空气传播的风险最小，而室内则在通风不良的环境中可能存在更大的风险，其中可能存在强源（即感染人数）。最近的一项综合评价研究强调了室外环境与室内环境中空气传播风险之间的巨大差异，特别是在通风不良的拥挤地区。该研究表明，在室外环境中，空气传播的风险很小，因为室外环境中有较大的空气可用于稀释和分散病毒颗粒32。这些发现对与COVID-19相关的公共卫生政策和指南具有重要意义。通过认识到室内和室外环境之间传播风险的显著差异，政策制定者可以制定更有效的策略来减轻病毒的传播并保护公众健康。

有多种方法和方案可用于检测、定量和测序来自不同环境样本的 SARS-CoV-2。本文旨在介绍一套成熟的协议，允许具有不同能力水平的实验室对废水和空气样品中的病毒进行环境检测、定量和测序。

Protocol

这里描述的所有方法都已在其他地方发表，并包含对原始方法的小修改。 1、废水收集及样品预处理 注：由于环境样品中SARS-CoV-2 RNA的浓度较低，因此实施浓度步骤对于成功检测至关重要33,34,35。这里描述的是第一种报道的在废水中检测SARS-CoV-2的方法36。</p…

Representative Results

表 3 中总结的结果显示了使用本文所述方法检测和定量废水和空气样品中 SARS-CoV-2 RNA 的示例。从西班牙和斯洛文尼亚的废水处理厂收集废水样品，如果三次重复中至少有两次的Ct低于40，则认为阳性，如果Ct的变异小于5%，则定量被认为是有效的。在西班牙和葡萄牙，收集了室内和室外空气样本，并应用了相同的规则。空气样本使用重复样本，而不是废水样本使用一式三份，因为该研?…

Discussion

使用（RT-）qPCR方法进行微生物和病毒检测和定量分析因其卓越的灵敏度而获得了广泛的认可。然而，这些技术在分析环境样品时面临许多挑战。废水样品中含有大量的抑制性物质，这些物质可能会扭曲测量结果并产生误导性结果。为了解决这些限制并提高精度，构思、设计和实施了一个复杂的协议。该协议是通过结合科学文献中的协议而量身定制的，并通过在过程的每个阶段采用一系列严格的控…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是在卡斯蒂利亚-莱昂地区政府和FEDER计划（CLU 2017-09，UIC315和VA266P20项目）的财政支持下进行的。

Materials

Adapter+A25+A2:D19+A2:D20+A2+A2:D19 Oxford Nanopore EXP-AMII001 Sequencing
AllPrep PowerViral DNA/RNA Kit Qiagen 28000-50 RNA extraction kit
AMPure XP Beckman Coulter A63880 PCR Purification, NGS Clean-up, PCR clean-up
ARTIC SARS-CoV-2 Amplicon Panel IDT 10011442 SARS-CoV-2 genome amplification
Blunt/TA Ligase Master Mix NEB M0367S Library preparation
CENTRICON PLUS­70 10KDA. Fisher Scientific 10296062 Concentration filters
CORIOLIS COMPACT AIR SAMPLER Bertin Technologies 083-DU001 Air sampler
Duran laboratory bottles Merck Z305200-10EA Sampling Bottles
Flow Cell (R9.4.1) Oxford Nanopore FLO-MIN106D Sequencing
General labarotory consumables (tubes, qPCR plates, etc)
Ligation Sequencing Kit Oxford Nanopore SQK-LSK109 Sequencing
LunaScript RT SuperMix Kit NEB E3010  cDNA synthesis
Mengovirus extraction control Kit Biomérieux KMG Concentration control
Nalgene General Long-Term Storage Cryogenic Tubes Thermofisher 5011-0012 Sample storage
Native Barcoding Expansion 1-12 (PCR-free Oxford Nanopore EXP-NBD104 Barcoding
NEBNext Ultra II End Repair/dA-Tailing Module NEB E7595 DNA repair
NEBNext VarSkip Short SARS-CoV-2 Primer Mixes NEB E7658 SARS-CoV-2 genome amplification
NEBNext Quick Ligation Reaction Buffer NEB B6058S Sequencing 
Phosphate buffered saline Merck P4474 Collection buffer
Phosphate-buffered saline (PBS, 1X), sterile-filtered Thermofisher J61196.AP Elution of air samples
Q5 Hot Start High-Fidelity 2X Master Mix NEB M0494S hot start DNA polymerase
Qubit RNA HS Assay Kit Thermofisher Q32852 RNA quantitation
SARS-CoV-2 RUO qPCR Primer & Probe Kit IDT 10006713 Primer-Probe mix and qPCR positive control
TaqPath 1-Step RT-qPCR Master Mix Thermofisher A15299 RT-qPCR kit
DOWNLOAD MATERIALS LIST

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Tags

Wastewater-based EpidemiologySARS-CoV-2COVID-19Air SamplingViral RNART-qPCRMengovirusN1 GeneN2 GeneRNA ExtractionCentrifugal UltrafiltrationComposite Wastewater SampleMolecular Biology

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Gonçalves, J., Gomes da Silva, P., Koritnik, T., Bosilj, M., Torres-Franco, A., Diaz, I., Rodriguéz, E., Marcos, E., Mesquita, J. R., García-Encina, P. Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples. J. Vis. Exp. (196), e65053, doi:10.3791/65053 (2023).
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