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. 2021 Apr 1:763:144587.
doi: 10.1016/j.scitotenv.2020.144587. Epub 2020 Dec 18.

Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater

Kouichi Kitamura  1 , Kenji Sadamasu  2 , Masamichi Muramatsu  3 , Hiromu Yoshida  3
Affiliations

Efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater

Kouichi Kitamura et al. Sci Total Environ. .

Abstract

In the context of the coronavirus disease 2019 (COVID-19) pandemic, environmental surveillance for the detection of SARS-CoV-2 has become increasingly important. Studies have demonstrated that the SARS-CoV-2 RNA is present in the feces of infected individuals; further, its presence in wastewater has been reported. However, an optimized method for its detection in sewage has not yet been adequately investigated. Therefore, in this study, the efficient detection of SARS-CoV-2 RNA in the solid fraction of wastewater was investigated using two quantitative PCR assays. In particular, wastewater samples were collected from a manhole located in the commercial district of a metropolitan region in Japan, where COVID-19 is highly prevalent, and two wastewater treatment plants (WWTPs). The samples were concentrated using four separate methods, namely, electronegative membrane adsorption, polyethylene glycol precipitation, ultrafiltration, and solid precipitation. Each method revealed a significant concentration of pepper mild mottle virus (PMMoV) RNA, which is an indicator virus for wastewater. As expected, non-enveloped PMMoV RNA was enriched in the supernatant fraction such that relatively low concentrations were detected in the solid fraction of the wastewater samples. In contrast, higher SARS-CoV-2 RNA concentrations were consistently detected in the solid fractions compared with the supernatant fractions based on the other methods that were investigated in this study. Spearman's correlation tests showed that the SARS-CoV-2 RNA concentrations in wastewater samples from the WWTP were significantly correlated with the number of COVID-19 cases recorded during the data collection period. These results demonstrate that viral recovery from the solid fraction is an effective method for SARS-CoV-2 RNA surveillance in an aqueous environment.

Keywords: COVID-19; Environmental surveillance; SARS-CoV-2; Sewage; Wastewater.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Quantification of SARS-CoV-2 RNA in the solid fraction of the wastewater samples. Samples were collected weekly from Manhole A (red), WWTP B (blue), and WWTP C (green). Two different RT-qPCR assays, (A) NIID_N2 and (B) CDC_N1N2, were performed. Values are presented as the number of SARS-CoV-2 RNA gene copies per litter of wastewater (gc/L). Dots represent weekly collected samples and dotted lines represent the limit of quantification. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2
Fig. 2
Quantification of PMMoV RNA in wastewater. Samples were collected weekly from Manhole A (red), WWTP B (blue), and WWTP C (green). RNAs were recovered using four methods: electronegative membrane adsorption, PEG precipitation, ultrafiltration, and solid precipitation. Values are presented as the number of PMMoV RNA gene copies per liter of wastewater (gc/L). Dots represent weekly collected samples. Average and standard error of mean (SEM) are indicated using bars, and details are shown in Table S2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 3
Fig. 3
Comparison of RT-qPCR assays (NIID_N2, & CDC_N1N2) based on copy number. Magenta circles represent the solid fractions of weekly collected samples from the three sample collection sites. Gray squares represent dilution series of RNA from SARS-CoV-2 isolate. Values are presented as the number of SARS-CoV-2 RNA gene copies per reaction of the RT-qPCR assays. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4
Fig. 4
Summary of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater. (A) SARS-CoV-2 RNA concentrations in Manhole A and WWTP B. Daily new cases of COVID-19 based on (B) date reported and (C) date of onset in a metropolitan region in Japan. The red circles and blue squares indicate SARS-CoV-2 RNA concentrations in samples obtained from Manhole A and WWTP B, respectively. The gray bars and black lines represent new cases on each day and the seven-day average, respectively. (D) Correlation between COVID-19 cases (seven-day average) and SARS-CoV-2 RNA concentrations analyzed using Spearman's rank correlation test. *p < 0.05, **p < 0.005. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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