Protocol for safe, affordable, and reproducible isolation and quantitation of SARS-CoV-2 RNA from wastewater

Abstract

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.

Fig 1. Repeatability of protocol: Pearson correlation of replicate measurements (n = 2) of copy number yield for the N1 target from fourteen 24-hr composite wastewater samples and a negative control, demonstrating the reproducibility of our protocol.

Sample collection and initial processing was performed on the same day.

Fig 2. Effect of pasteurization: Copy number yield for the N1 target obtained from three 24-hr composite wastewater samples processed either with or without pasteurization (n = 5 for WWTP1 and 2 for WWTP 2 & 3).

Each point is the mean of three technical replicate measurements. Horizontal black lines show means and asterisks show significance level in pairwise comparisons where ns = P > 0.05; * = P ≤ 0.05; ** = P ≤ 0.01; *** = P ≤ 0.001; **** = P ≤ 0.0001.

Fig 3. Effect of storage at 4°C in PEG/NaCl: Following initial processing (pasteurization, preliminary centrifugation, and filtering), 24-hr composite samples from 3 different wastewater treatment plants were stored at 4°C in a PEG/NaCl solution for precipitation and concentration of virions.

Each point is the mean of two technical replicate measurements from a 24-hour composite sample. Horizontal black lines show means. See Fig 2 legend for explanation of pairwise comparison.

Fig 4. Effect of storage at 4°C on unpasteurized samples: Following initial processing (pasteurization, preliminary centrifugation, and filtering), 24-hr composite samples (n = 8) were stored at 4°C for 72 hrs.

Each point is the mean of two technical replicate measurements from a 24-hour composite sample. Horizontal black lines show means. See Fig 2 legend for explanation of pairwise comparisons.

Fig 5. Effect of safranin staining: Copy number yield for the N1 target for 24-hr composite wastewater samples obtained from 3 wastewater treatment plants.

Samples processed with safranin are labeled by dark blue circles; controls are labeled with light blue circles. Points are technical replicate measurements from a 24-hour composite sample. Horizontal black lines show means. See Fig 2 legend for explanation of pairwise comparisons.

Fig 6. Effect of TRIzol extraction: Copy number yield for the N1 target for 24-hr composite wastewater samples obtained from 5 wastewater treatment plants.

Each point in the mean of 2 technical replicate measurements from a 24-hour composite sample. Horizontal black lines show means. See Fig 2 legend for explanation of pairwise comparisons.

Fig 7. Effect of different RT-qPCR enzymes: Copy number yield for the N1 target for 24-hr composite wastewater samples obtained from 6 wastewater treatment plants (WWTP).

RT-qPCR assays performed with the TaqPath 1-Step RT-qPCR enzyme recommended by the CDC [20] are compared to RT-qPCR assays performed with One Step PrimeScript III enzyme. Each point in the mean of 2 technical replicate measurements from a 24-hour composite sample. Horizontal black lines show means. See Fig 2 legend for explanation of pairwise comparisons.