Department of Biology, Queensborough Community College, The City University of New York, New York City, New York, United States of America.
Biology Department, Queens College, The City University of New York, New York City, New York, United States of America.
PLoS One. 2021 Sep 23;16(9):e0257454. doi: 10.1371/journal.pone.0257454. eCollection 2021.
The following protocol describes our workflow for processing wastewater with the goal of detecting the genetic signal of SARS-CoV-2. The steps include pasteurization, virus concentration, RNA extraction, and quantification by RT-qPCR. We include auxiliary steps that provide new users with tools and strategies that will help troubleshoot key steps in the process. This protocol is one of the safest, cheapest, and most reproducible approaches for the detection of SARS-CoV-2 RNA in wastewater. Owing to a pasteurization step, it is safe for use in a BSL2 facility. In addition to making the protocol safe for the personnel involved, pasteurization had the added benefit of increasing the SARS-CoV-2 genetic signal. Furthermore, the RNA obtained using this protocol can be sequenced using both Sanger and Illumina sequencing technologies. The protocol was adopted by the New York City Department of Environmental Protection in August 2020 to monitor SARS-CoV-2 prevalence in wastewater in all five boroughs of the city. In the future, this protocol could be used to detect a variety of other clinically relevant viruses in wastewater and serve as a foundation of a wastewater surveillance strategy for monitoring community spread of known and emerging viral pathogens.
本方案描述了我们处理废水以检测 SARS-CoV-2 遗传信号的工作流程。该流程包括巴氏消毒、病毒浓缩、RNA 提取以及 RT-qPCR 定量。我们还提供了辅助步骤，为新用户提供有助于解决流程关键步骤的工具和策略。本方案是检测废水中 SARS-CoV-2 RNA 的最安全、最便宜且重现性最高的方法之一。由于采用了巴氏消毒步骤，因此可在生物安全 2 级设施中安全使用。此外，巴氏消毒不仅提高了 SARS-CoV-2 的遗传信号，还有助于确保操作人员的安全。此外，使用本方案获得的 RNA 可以使用 Sanger 和 Illumina 测序技术进行测序。该方案于 2020 年 8 月被纽约市环境保护局采用，以监测该市五个行政区废水中 SARS-CoV-2 的流行情况。在未来，本方案可用于检测废水中的多种其他具有临床相关性的病毒，并为监测已知和新兴病毒病原体在社区传播的废水监测策略奠定基础。
