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. 2022 Oct 15:843:157101.
doi: 10.1016/j.scitotenv.2022.157101. Epub 2022 Aug 8.

The Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S): A rapid and cost-effective SARS-CoV-2 RNA detection method for routine wastewater surveillance

Hiroki Ando  1 , Ryo Iwamoto  2 , Hiroyuki Kobayashi  3 , Satoshi Okabe  1 , Masaaki Kitajima  4
Affiliations

The Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S): A rapid and cost-effective SARS-CoV-2 RNA detection method for routine wastewater surveillance

Hiroki Ando et al. Sci Total Environ. .

Erratum in

Abstract

Wastewater-based epidemiology has attracted attention as a COVID-19 surveillance tool. Here, we developed a practical method for detecting SARS-CoV-2 RNA in wastewater (the EPISENS-S method), which employs direct RNA extraction from wastewater pellets formed via low-speed centrifugation. The subsequent multiplex one-step RT-preamplification reaction with forward and reverse primers for SARS-CoV-2 and a reverse primer only for pepper mild mottle virus (PMMoV) allowed for qPCR quantification of the targets with different abundances in wastewater from the RT-preamplification product. The detection sensitivity of the method was evaluated using wastewater samples seeded with heat-inactivated SARS-CoV-2 in concentrations of 2.11 × 103 to 2.11 × 106 copies/L. The results demonstrated that the sensitivity of the EPISENS-S method was two orders of magnitude higher than that of the conventional method (PEG precipitation, followed by regular RT-qPCR; PEG-QVR-qPCR). A total of 37 untreated wastewater samples collected from two wastewater treatment plants in Sapporo, Japan when 1.6 to 18 new daily reported cases per 100,000 people were reported in the city (March 4 to July 8, 2021), were examined using the EPISENS-S method to confirm its applicability to municipal wastewater. SARS-CoV-2 RNA was quantified in 92 % (34/37) of the samples via the EPISENS-S method, whereas none of the samples (0/37) was quantifiable via the PEG-QVR-qPCR method. The PMMoV concentrations measured by the EPISENS-S method ranged from 2.60 × 106 to 1.90 × 108 copies/L, and the SARS-CoV-2 RNA concentrations normalized by PMMoV ranged from 5.71 × 10-6 to 9.51 × 10-4 . The long-term trend of normalized SARS-CoV-2 RNA concentration in wastewater was consistent with that of confirmed COVID-19 cases in the city. These results demonstrate that the EPISENS-S method is highly sensitive and suitable for routine COVID-19 wastewater surveillance.

Keywords: COVID-19; Pepper mild mottle virus; Quantification method; SARS-CoV-2; Wastewater-based epidemiology; qPCR.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masaaki Kitajima reports financial support was provided by Japan Science and Technology Agency. Masaaki Kitajima reports a relationship with Shionogi and Co Ltd. that includes: funding grants. Ryo Iwamoto reports a relationship with Shionogi and Co Ltd. that includes: employment. Hiroyuki Kobayashi reports a relationship with Shionogi and Co Ltd. that includes: employment. Satoshi Okabe reports a relationship with Shionogi and Co Ltd. that includes: funding grants. Masaaki Kitajima has patent pending to Shionogi & Co Ltd. Ryo Iwamoto has patent pending to Shionogi & Co Ltd.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
Detection flow of SARS-CoV-2 and PMMoV RNA in wastewater using the EPISENS-S and PEG-QVR-qPCR methods.
Fig. 2
Fig. 2
Standard curves of CDC N1 assay using plasmid DNA containing the target region. The blue plots denote the standard curve of regular qPCR (without preamplification), and the red plots denote that of qPCR following preamplification. Error bars denote the standard deviation of Ct values in duplicate. The standard deviation of preamp-qPCR was so small that they were almost invisible in the figure.
Fig. 3
Fig. 3
Concentrations of SARS-CoV-2 RNA normalized with PMMoV RNA in wastewater in Sapporo city, as determined using the EPISENS-S method, and newly reported COVID-19 cases in the city. The circle plots denote the normalized concentration of SARS-CoV-2 RNA in wastewater collected at WWTP A or B. The red line denotes the geometric mean normalized concentrations of SARS-CoV-2 RNA in wastewater collected from WWTPs A and B in the same week. The dotted line denotes the theoretical lower limit of detection of the EPISENS-S method.
Fig. 4
Fig. 4
Correlation of SARS-CoV-2 RNA concentration normalized by PMMoV RNA concentration, determined using the EPISENS-S method, and newly reported COVID-19 cases in Sapporo city (n = 15). The horizontal axis shows the 7-day (from 3 days before the day of sampling to 3 days after the sampling) moving average; the vertical axis represents the geometric normalized concentration of SARS-CoV-2 RNA in wastewater collected at two WWTPs in the same week. If SARS-CoV-2 RNA was not quantified from both samples collected at the WWTPs in the same week, the wastewater data were excluded from the statistical analysis.
Fig. S1
Fig. S1
Concentrations of SARS-CoV-2 RNA and PMMoV RNA in wastewater in Sapporo city determined with the EPISENS-S method and newly reported COVID-19 cases in the city. The circle plots denote concentration of SARS-CoV-2 RNA in wastewater collected at WWTP A or B. The red line denotes geometric mean concentrations of SARS-CoV-2 RNA in wastewater collected from WWTPs A and B in a week. The triangle plots and green line denote the PMMoV RNA concentrations at each WWTP and geometric mean concentrations of two WWTP in a week, respectively. The dotted lined denotes the theoretical lower limit of detection (93.1 copies/L) of the EPISENS-S method. The blue bars and black line denote the newly reported COVID-19 cases and 7-day moving average of the newly reported cases in Sapporo city, respectively
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