SARS-CoV-2 RNA Isolation Method from Sewage Sludge, Application in Field Samples and Comparison with Bacteriophage Loads

Yann Quesnelle; Suzanne Trancart; Hélène Bouras; Maryline Houssin

doi:10.47277/JETT/10(4)241

Yann Quesnelle SARS-CoV-2 RNA Isolation Method from Sewage Sludge, Application in Field Samples and Comparison with Bacteriophage Loads
Suzanne Trancart LABÉO, Research department, 1 Route de Rosel, 14053 Caen Cedex 4, France
Hélène Bouras LABÉO, Research department, 1 Route de Rosel, 14053 Caen Cedex 4, France
Maryline Houssin LABÉO, Research department, 1 Route de Rosel, 14053 Caen Cedex 4, France

DOI: https://doi.org/10.47277/JETT/10(4)241

Keywords: SARS-CoV-2, Sewage sludge, Viral concentration, RNA isolation

Abstract

Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is mainly transmitted through the respiratory tract. It can also be found in faeces leading to its detection in wastewater and potentially in sewage sludge. This one can be used in agriculture as a soil amendment. In France, the spreading of sludge is controlled in order to limit the dissemination of pathogenic microorganisms including SARS-CoV-2 since the pandemic. However, the control only concerns the analysis of bacteriophages. The present study was carried out to assess the presence of the virus in sewage sludge and compare with bacteriophages results. It describes the validation of a method for the isolation of SARS-CoV-2 RNA for detection by RT-PCR, using a surrogate virus. Two virus concentration methods and three nucleic acid extraction methods were compared. After validation, the most efficient method was applied to field samples (n=34) from Normand sewage treatment plants during the pandemic. Then, the results were compared with bacteriophage loads. According to our results, PEG precipitation followed by a nucleic acid extraction based on cleared lysate with phenol:chloroform:isoamyl alcohol, then concentrated and purified on anion-exchange column was selected. This process resulted in a yield of 39.6±37.3%. The field study confirmed the presence of SARS-CoV-2 in both primary and hygienized sludges. The comparative analysis suggested that the study of the effectiveness of sanitation on bacteriophages does not appear representative of that on SARS-CoV-2. In addition to the bacteriophages test, a direct search for the SARS-CoV-2 is recommended to evaluate the sanitation of sludge.

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