Surveillance of SARS-CoV-2 in wastewater by quantitative PCR and digital PCR: a case study in Shijiazhuang city, Hebei province, China

Abstract

In this study, we reported the first long-term monitoring of SARS-CoV-2 in wastewater in Mainland China from November 2021 to October 2023. The city of Shijiazhuang was employed for this case study. We developed a triple reverse transcription droplet digital PCR (RT-ddPCR) method using triple primer-probes for simultaneous detection of the N1 gene, E gene, and Pepper mild mottle virus (PMMoV) to achieve accurate quantification of SARS-CoV-2 RNA in wastewater. Both the RT-ddPCR method and the commercial multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) method were implemented for the detection of SARS-CoV-2 in wastewater in Shijiazhuang City over a 24-month period. Results showed that SARS-CoV-2 was detected for the first time in the wastewater of Shijiazhuang City on 10 November 2022. The peak of COVID-19 cases occurred in the middle of December 2022, when the concentration of SARS-CoV-2 in the wastewater was highest. The trend of virus concentration increases and decreases forming a "long-tailed" shape in the COVID-19 outbreak and recession cycle. The results indicated that both multiplex RT-ddPCR and RT-qPCR are effective in detecting SARS-CoV-2 in wastewater, but RT-ddPCR is capable of detecting low concentrations of SARS-CoV-2 in wastewater which is more efficient. The SARS-CoV-2 abundance in wastewater is correlated to clinical data, outlining the public health utility of this work.HighlightsFirst long-term monitoring of SARS-CoV-2 in wastewater in Mainland ChinaCOVID-19 outbreak was tracked in Shijiazhuang City from outbreak to containmentWastewater was monitored simultaneously using RT-ddPCR and RT-qPCR methodsTriple primer-probe RT-ddPCR detects N1 and E genes of SARS-CoV-2 and PMMoV.

Keywords: COVID-19; RT-ddPCR; SARS-CoV-2; Wastewater; monitoring.

Graphical abstract

Figure 1.

(a) The temporal distribution of SARS-CoV-2 in wastewater using RT-ddPCR during the COVID-19 epidemic from November 2021 to October 2023. (b) The temporal distribution of SARS-CoV-2 in wastewater Since China's Zero-COVID strategy ends from November 2022 to October 2023. And positive rate of COVID-19 in sentinel hospitals in China (%) published by China CDC.

Figure 2.

(a) Linear regression to model the relationship between positive rate of COVID-19 in sentinel hospitals in China and concentration of SARS-CoV-2 in wastewater. R² Linear (N1) = 0.401, R² Linear (E) = 0.462. (b) Linear regression to model the relationship between positive rate of COVID-19 in sentinel hospitals in China and SARS-CoV-2 N/ORF1ab cycle threshold in wastewater. R² Linear (N) = 0.395, R² Linear (ORF1ab) = 0.399.