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. 2022 Jul 28;11(8):845.
doi: 10.3390/pathogens11080845.

A Methodological Approach to Water Concentration to Investigate the Presence of SARS-CoV-2 RNA in Surface Freshwaters

Marina Tesauro  1   2 , Mara Terraneo  3 , Michela Consonni  1 , Clara Fappani  3   4 , Daniela Colzani  3 , Caterina Stevanin  3 , Antonella Amendola  2   3 , Daniele Masseroni  5 , Elisabetta Tanzi  2   3
Affiliations

A Methodological Approach to Water Concentration to Investigate the Presence of SARS-CoV-2 RNA in Surface Freshwaters

Marina Tesauro et al. Pathogens. .

Abstract

During the COVID-19 public health emergency, an increasing number of studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide, but little is known about the presence of the virus in surface freshwaters. The aim of the current study was to develop and validate an appropriate and scalable methodological approach for the concentration and detection of SARS-CoV-2 from surface freshwater samples, collected within the Milan rural network subjected to flood spillways activity. Overall, both surface water and distilled water samples spiked with inactivated SARS-CoV-2 were used to validate the concentration method for pathogens determination. Two pre-filtration systems, filter paper and Sartolab® P20 (Sartorius, Germany) and two concentration methods, two-phase (PEG-dextran method) separation and tangential flow ultrafiltration (UF), were compared. The effects of pre-filtration and concentration on viral nucleic acid recovery were assessed through real time RT-PCR targeting SARS-CoV-2 and the internal viral control PMMoV (Pepper Mild Mottle Virus). Our results showed that UF is more sensitive than the PEG-dextran method in viral acid nucleic recovery from surface water samples. Better results were obtained pre-filtering samples with Sartolab® P20 and extracting the nucleic acids with undiluted silica, rather than diluted as required by the standard protocol. The proposed method will be used for the monitoring of surface waters in the Milan area.

Keywords: SARS-CoV-2; flood spillways; surface freshwater.

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Conflict of interest statement

The authors declare no conflict of interest. The funder had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Flowchart of tests carried out on concentration methods, PEG-Dextran (P-D) method vs. tangential flow ultrafiltration (UF), starting from different types of water (distilled and freshwater) spiked with Inactivated SARS-CoV-2 Whole Virus (pellet).
Figure 2
Figure 2
Study domain: the rural canal, Roggia Gamberina, located in the south-west of the city of Milan (Italy). The numbers 1–9 indicate the location of the spillways (with the courtesy of D. Masseroni).
See this image and copyright information in PMC

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