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Article

Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy

by
Laura Pellegrinelli
1,*,
Sara Castiglioni
2,
Clementina E. Cocuzza
3,
Barbara Bertasi
4,
Valeria Primache
1,
Silvia Schiarea
2,
Giulia Salmoiraghi
2,
Andrea Franzetti
5,
Rosario Musumeci
3,
Michela Tilola
4,
Elisa Galuppini
4,
Giorgio Bertanza
6,
Marialuisa Callegari
7,
Fabrizio Stefani
8,
Andrea Turolla
9,
Emanuela Ammoni
10,
Danilo Cereda
10,
Elena Pariani
1,
Sandro Binda
1 and
the WBE Study Group
†
1
Department of Biomedical Sciences of Health, University of Milan, 20133 Milan, Italy
2
Department of Environmental Sciences, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, 20156 Milan, Italy
3
Department of Medicine and Surgery, University of Milano-Bicocca, 20900 Monza, Italy
4
Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna “B. Ubertini”, 25124 Brescia, Italy
5
Department of Earth and Environmental Sciences, University of Milano-Bicocca, 20126 Milano, Italy
6
Dipartimento di Ingegneria Civile, Architettura, Territorio, Ambiente e di Matematica, Università di Brescia, 25123 Brescia, Italy
7
Dipartimento di Scienze e Tecnologie Alimentari per una Filiera Agro-Alimentare Sostenibile (DiSTAS), Università Cattolica Sacro Cuore, 29122 Piacenza, Italy
8
Water Research Institute-National Research Council (IRSA-CNR), 20861 Brugherio, Italy
9
Department of Civil and Environmental Engineering (DICA)—Environmental Section, Politecnico di Milano, 20133 Milan, Italy
10
DG Welfare, Regione Lombardia, 20124 Milan, Italy
*
Author to whom correspondence should be addressed.
†
Members of the ‘WBE Study Group’ are listed in acknowledgments.
Water 2022, 14(5), 833; https://doi.org/10.3390/w14050833
Submission received: 9 February 2022 / Revised: 2 March 2022 / Accepted: 4 March 2022 / Published: 7 March 2022
(This article belongs to the Special Issue Pathogen Detection and Identification in Wastewater)
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Abstract

(1) Background: The surveillance of SARS-CoV-2 RNA in urban wastewaters allows one to monitor the presence of the virus in a population, including asymptomatic and symptomatic individuals, capturing the real circulation of this pathogen. The aim of this study was to evaluate the performance of different pre-analytical and analytical methods for identifying the presence of SARS-CoV-2 in untreated municipal wastewaters samples by conducting an inter-laboratory proficiency test. (2) Methods: three methods of concentration, namely, (A) Dextran and PEG-6000 two-phase separation, (B) PEG-8000 precipitation without a chloroform purification step and (C) PEG-8000 precipitation with a chloroform purification step were combined with three different protocols of RNA extraction by using commercial kits and were tested by using two primers/probe sets in three different master mixes. (3) Results: PEG-8000 precipitation without chloroform treatment showed the best performance in the SARS-CoV-2 recovery; no major differences were observed among the protocol of RNA extraction and the one-step real-time RT-PCR master mix kits. The highest analytic sensitivity was observed by using primers/probe sets targeting the N1/N3 fragments of SARS-CoV-2. (4) Conclusions: PEG-8000 precipitation in combination with real-time RT-PCR targeting the N gene (two fragments) was the best performing workflow for the detection of SARS-CoV-2 RNA in municipal wastewaters.
Keywords: environmental surveillance; urban wastewater; PEG-8000 precipitation; SARS-CoV-2 environmental surveillance; urban wastewater; PEG-8000 precipitation; SARS-CoV-2

Share and Cite

MDPI and ACS Style

Pellegrinelli, L.; Castiglioni, S.; Cocuzza, C.E.; Bertasi, B.; Primache, V.; Schiarea, S.; Salmoiraghi, G.; Franzetti, A.; Musumeci, R.; Tilola, M.; et al. Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy. Water 2022, 14, 833. https://doi.org/10.3390/w14050833

AMA Style

Pellegrinelli L, Castiglioni S, Cocuzza CE, Bertasi B, Primache V, Schiarea S, Salmoiraghi G, Franzetti A, Musumeci R, Tilola M, et al. Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy. Water. 2022; 14(5):833. https://doi.org/10.3390/w14050833

Chicago/Turabian Style

Pellegrinelli, Laura, Sara Castiglioni, Clementina E. Cocuzza, Barbara Bertasi, Valeria Primache, Silvia Schiarea, Giulia Salmoiraghi, Andrea Franzetti, Rosario Musumeci, Michela Tilola, and et al. 2022. "Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy" Water 14, no. 5: 833. https://doi.org/10.3390/w14050833

APA Style

Pellegrinelli, L., Castiglioni, S., Cocuzza, C. E., Bertasi, B., Primache, V., Schiarea, S., Salmoiraghi, G., Franzetti, A., Musumeci, R., Tilola, M., Galuppini, E., Bertanza, G., Callegari, M., Stefani, F., Turolla, A., Ammoni, E., Cereda, D., Pariani, E., Binda, S., & the WBE Study Group. (2022). Evaluation of Pre-Analytical and Analytical Methods for Detecting SARS-CoV-2 in Municipal Wastewater Samples in Northern Italy. Water, 14(5), 833. https://doi.org/10.3390/w14050833

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