SARS-CoV-۲ RNA Isolation Method from Sewage Sludge, Application in Field Samples and Comparison with Bacteriophage Loads Service Unavailable

Severe Acute Respiratory Syndrome CoronaVirus ۲ (SARS-CoV-۲) 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-۲ 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-۲ 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=۳۴) 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 ۳۹.۶±۳۷.۳%. The field study confirmed the presence of SARS-CoV-۲ 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-۲. In addition to the bacteriophages test, a direct search for the SARS-CoV-۲ is recommended to evaluate the sanitation of sludge.Severe Acute Respiratory Syndrome CoronaVirus ۲ (SARS-CoV-۲) 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-۲ 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-۲ 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=۳۴) 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 ۳۹.۶±۳۷.۳%. The field study confirmed the presence of SARS-CoV-۲ 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-۲. In addition to the bacteriophages test, a direct search for the SARS-CoV-۲ is recommended to evaluate the sanitation of sludge.

کلیدواژه ها:

SARS-CoV-۲ ، Sewage sludge ، Viral concentration ، RNA isolation