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The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines. Version 1 included the following assays: N1, PMMoV, BCoV, and SOC. Version 2 was amended to include the following assays: crAssphage, Bacteroides HF183 rRNA, and human 18S rRNA.

Clinical and surveillance testing for the SARS-CoV-2 virus relies overwhelmingly on RT-qPCR-based diagnostics, yet several popular assays require 2–3 separate reactions or rely on detection of a single viral target, which adds significant time, cost, and risk of false-negative results. Furthermore, multiplexed RT-qPCR tests that detect at least two SARS-CoV-2 genes in a single reaction are typically not affordable for large scale clinical surveillance or adaptable to multiple PCR machines and plate layouts. We developed a RT-qPCR assay using the Luna Probe Universal One-Step RT-qPCR master mix with publicly available primers and probes to detect SARS-CoV-2 N gene, E gene, and human RNase P (LuNER) to address these shortcomings and meet the testing demands of a university campus and the local community. This cost-effective test is compatible with BioRad or Applied Biosystems qPCR machines, in 96 and 384-well formats, with or without sample pooling, and has a detection sensitivity suitable for both clinical reporting and wastewater surveillance efforts.

The following protocol describes the reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods that we have used at the University of California, Berkeley in the Nelson Lab and COVID-WEB (covid-web.org) for downstream analysis of extracted wastewater samples for the purpose of SARS-CoV-2 surveillance. We have been successfully using this protocol paired with the Sewage, Salt, Silica, and SARS-CoV-2 (4S) method. We are including multiple assays that we have used throughout this project, although not all of them may be in active use. This method may need to be adapted based on your lab's application, available equipment, and safety requirements. It is intended for use in a BSL2+ lab with extra precautions for processing environmental specimens for SARS-CoV-2 as described in the CDC Interim Laboratory Biosafety Guidelines.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

The following protocol describes the "4S" (Sewage, Salt, Silica and SARS-CoV-2) workflow applied to using dry silica powder as an RNA-binding matrix instead of silica spin columns. This offers an even more economical alternative, requiring only centrifugation to extract RNA from wastewater. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.

This protocol describes the procedure of the "4S" (Sewage, Salt, Silica and SARS-CoV-2) method for SARS-CoV-2 RNA extraction from wastewater. Offering a highly efficient, modular and economical alternative to existing wastewater RNA purification methods, this procedure lowers the barrier to entry for SARS-CoV-2 wastewater-based epidemiology. This procedure is intended to be carried out in a BSL2+ laboratory space, with precautions when handling raw wastewater samples.