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. 2021 Feb 10;755(Pt 1):142939.
doi: 10.1016/j.scitotenv.2020.142939. Epub 2020 Oct 14.

Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater

Mohammed Hakim Jafferali  1 , Kasra Khatami  2 , Merve Atasoy  2 , Madeleine Birgersson  3 , Cecilia Williams  1 , Zeynep Cetecioglu  4
Affiliations

Benchmarking virus concentration methods for quantification of SARS-CoV-2 in raw wastewater

Mohammed Hakim Jafferali et al. Sci Total Environ. .

Abstract

Wastewater-based epidemiology offers a cost-effective alternative to testing large populations for SARS-CoV-2 virus, and may potentially be used as an early warning system for SARS-CoV-2 pandemic spread. However, viruses are highly diluted in wastewater, and a validated method for their concentration and further processing, and suitable reference viruses, are the main needs to be established for reliable SARS-CoV-2 municipal wastewater detection. For this purpose, we collected wastewater from two European cities during the Covid-19 pandemic and evaluated the sensitivity of RT-qPCR detection of viral RNA after four concentration methods (two variants of ultrafiltration-based method and two adsorption and extraction-based methods). Further, we evaluated one external (bovine corona virus) and one internal (pepper mild mottle virus) reference virus. We found a consistently higher recovery of spiked virus using the modified ultrafiltration-based method. This method also had a significantly higher efficiency (p-value <0.01) for wastewater SARS-CoV-2 detection. The ultracentrifugation method was the only method that detected SARS-CoV-2 in the wastewater of both cities. The pepper mild mottle virus was found to function as a potentially suitable internal reference standard.

Keywords: Bovine corona virus; Municipal wastewater; Pepper mild mottle virus; RT-qPCR; SARS-CoV-2; Virus concentration method.

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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
RT-qPCR detection of SARS-CoV2, PMMoV, and BCoV in wastewater sample concentrated by different methods. Method 1 or 1.A performed significantly better than method 2 and 2.A for all samples. Fold change values were generated for RT-qPCR wastewater samples concentrated by four different methods from Stockholm and North of Italy using the 2−ΔCt method, and normalized to input (RNA extracted from corresponding 8 ml wastewater) with Stockholm 1 sample and Method 1.A set at value 1. A. SARS-CoV-2 N gene B. nPMMoV C. Recovery rate of spiked BCoV. Error bars: SD. Student's t-test were used to calculate statistical significance, and is indicated for comparison between Method 1 and 2, and 1A and 2A. (*p < 0.05, **p > 0.01, ***p < 0.001).
Fig. 2
Fig. 2
RT-qPCR efficiency in wastewater sample concentrated by different methods. A. RT-qPCR standard curves for cultured SARS-CoV-2 and BCoV RNA. B. Standard curves for RT-qPCR efficiency determination of PMMoV in Stockholm 3 and North of Italy 1 wastewater samples, concentrated by three different methods (as indicated). E refers to the qPCR efficiency. Samples generated by Method 1 and Method 1.A had the most efficient amplification, indicative of little qPCR inhibition. Error bar: SD.
Fig. 3
Fig. 3
Internal and external references for comparing virus levels between methods and samples. A. PMMoV detection normalized to input and recovery rate of spiked BCoV. B. SARS-CoV-2 N gene detection normalized to input and internal PMMoV reference. C. SARS-CoV-2 N gene detection normalized to input and recovery rate of spiked BCoV. Student's t-test were used to calculate statistical significance (*p < 0.05, **p > 0.01, ***p < 0.001).
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