方法文章

Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

DOI：

10.3791/65053

⸱

2023年6月30日

 ,  ,  ,  ,  ,  ,  ,  ,  , 
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)

本文内容

摘要

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This protocol aims to quantify SARS-CoV-2 RNA in wastewater and air samples to be used for wastewater-based epidemiology studies and to assess the exposure risk to SARS-CoV-2 in indoor and outdoor aerosols. This protocol also describes a tiled amplicon long-template sequencing approach for SARS-CoV-2 whole genome characterization.

摘要

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Wastewater-based epidemiology has emerged as a promising and efficacious surveillance system for SARS-CoV-2 and other infectious diseases in many nations. The process typically involves wastewater concentration, nucleic acid extraction, amplification of selected genomic segments, and detection and quantification of the amplified genomic segment. This methodology can similarly be leveraged to detect and quantify infectious agents, such as SARS-CoV-2, in air samples. Initially, SARS-CoV-2 was presumed to spread primarily through close personal contact with droplets generated by an infected individual while speaking, sneezing, coughing, singing, or breathing. However, a growing number of studies have reported the presence of SARS-CoV-2 RNA in the air of healthcare facilities, establishing airborne transmission as a viable route for the virus. This study presents a composite of established protocols to facilitate environmental detection, quantification, and sequencing of viruses from both wastewater and air samples.

引言

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In December 2019, a novel disease called COVID-19 emerged, caused by a previously unknown coronavirus, SARS-CoV-21. The resulting global pandemic has presented a significant challenge to clinical and public health laboratories worldwide, as a large number of individuals require testing to accurately assess virus transmission and prevalence in the community. However, in many regions, achieving the necessary level of testing in a timely and spatially comprehensive manner is economically unfeasible2,3. Current surveillance systems based on individual clinical diagnostics rely heavily on sy....

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方案

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All methods described here have been published elsewhere and contain small modifications from the original methods.

1. Wastewater collection and sample pre-processing

NOTE: Due to the low concentrations of SARS-CoV-2 RNA in environmental samples, the implementation of a concentration step is crucial for a successful detection33,34,35. Described here is the first reported method for the detection of SARS-CoV-2 in wastewater36.

  1. Collection and concentration of wastewater ....

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结果

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The results summarized in Table 3 show examples of the detection and quantification of SARS-CoV-2 RNA in wastewater and air samples using the method described in this article. Wastewater samples were collected from wastewater treatment plants in Spain and Slovenia and were considered positive if the Ct was less than 40 in at least two of the three replicates, with quantification considered valid if the Ct had a variation of less than 5%. In Spain and Portugal, indoor and outdoor air samples were collecte.......

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讨论

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Microbial and viral detection and quantification using (RT-)qPCR methods have garnered widespread acceptance due to their remarkable sensitivity. However, these techniques face numerous challenges when analyzing environmental samples. Wastewater samples contain an abundance of inhibitory substances that can skew measurements and generate misleading results. To tackle these limitations and enhance precision, a complex protocol was conceived, designed, and implemented. This protocol was tailored by combining protocols from.......

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披露

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The authors have no competing economic interests or other conflicts of interest.

致谢

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This work was performed with financial support from the Regional Government of Castilla y Leon and the FEDER program (projects CLU 2017-09, UIC315 and VA266P20).

....

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材料

本文使用的材料清单
姓名公司目录编号评论
接头+A25+A2：D19+A2：D20+A2+A2：D19牛津纳米孔EXP-AMII001测序
AllPrep PowerViral DNA/RNA 试剂盒Qiagen28000-50RNA 提取试剂盒
AMPure XPBeckman CoulterA63880PCR 纯化、NGS 纯化、PCR 纯化
ARTIC SARS-CoV-2 扩增子检测组合IDT10011442SARS-CoV-2 基因组扩增
Blunt/TA 连接酶预混液NEBM0367S文库制备
CENTRICON PLUS­70 10KDA。Fisher Scientific10296062浓度过滤器
CORIOLIS紧凑型空气采样器Bertin Technologies083-DU001采样器
Duran 实验室瓶MerckZ305200-10EA采样瓶
流通池 （R9.4.1）Oxford NanoporeFLO-MIN106D测序
通用实验室消耗品（试管、qPCR 板等）
连接测序试剂盒Oxford NanoporeSQK-LSK109测序
LunaScript RT SuperMix 套件NEBE3010 cDNA 合成
Mengovirus 提取对照试剂盒BiomérieuxKMG浓度控制
Nalgene 通用长期储存 低温管Thermofisher5011-0012样品储存
天然条形码扩展 1-12（无 PCR）牛津纳米孔EXP-NBD104条形码
NEBNext Ultra II 末端修复/dA-尾模块NEBE7595DNA 修复
NEBNext VarSkip Short SARS-CoV-2 引物混合物NEBE7658SARS-CoV-2 基因组扩增
NEBNext 快速连接反应缓冲液NEBB6058S测序 
磷酸盐缓冲盐水MerckP4474收集缓冲液
磷酸盐缓冲盐水 （PBS， 1X），无菌过滤ThermofisherJ61196。空气样品的 AP 洗脱
Q5 热启动高保真 2X 预混液NEBM0494S热启动 DNA 聚合酶
Qubit RNA HS 检测试剂盒ThermofisherQ32852RNA 定量
SARS-CoV-2 RUO qPCR 引物 &探针试剂盒IDT10006713引物-探针混合物和 qPCR 阳性对照
TaqPath 一步法 RT-qPCR 预混液ThermofisherA15299RT-qPCR 试剂盒
空气： 的

参考文献

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  1. Naming the Coronavirus Disease (COVID-19) and the Virus that Causes it. World Health Organization. , Available from: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/technical-guidance/naming-the-coronavirus-disease-(cover-2019)-and-the-virus-that-causes-it (2020).
  2. Lab Workplace Safety. Centers for Disease Control and Prevention. , Available from: http....

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标签

RNA RT qPCR

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