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Evaluation of Methods and Processing for Robust Monitoring of SARS-CoV-2 in Wastewater

Maksimović, O., Lengar, Ž., Kogej, Z., Bačnik K., Bajde, I., Milavec, M., Županič, A., Mehle, N., Kutnjak, D., Ravnikar, M., Gutierrez-Aguirre, I., Food Environ. Virol. 2022 Dec.; 384-400. doi: 10.1007/s12560-022-09544-0 Water based epidemiology was employed as a tool for tracking the transmission dynamics of SARS-CoV-2 in a given area. In this paper, we describe the evaluation of the methods that are currently used in the Slovenian national wastewater monitoring scheme for SARS-CoV-2. Specifically we compared several methods for virus concentration, different RT-qPCR assays and tested the stability of the virus over time in different temperature regiments.

The complexity of wastewater and lack of standardised methods led us to evaluate critical steps of the analysis prior to large-scale sampling efforts. We showed that temperature and duration of storage have a varying impact depending on the structural form the virus is present in the water sample (whole particle vs. free RNA). The two best performing methods for concentration (out of seven tested in total), in our laboratory set up, were Centricon filtration units coupled with Qiagen RNA extraction kit and direct RNA capture and extraction with semi-automated Promega kit. Lastly, we confirmed the use of N1 and N2 assays (CDC, USA) as best performing, among the tested assays in combination with Fast Virus 1 mastermix. Data presented in the paper shows a realistic process for method implementation in fast developing situations such as the ongoing pandemic. 

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