20.500.12556/DiRROS-19431 Evaluation of methods and processes for robust monitoring of SARS-CoV-2 in wastewater The SARS-CoV-2 pandemic has accelerated the development of virus concentration and molecular-based virus detection methods, monitoring systems and overall approach to epidemiology. Early into the pandemic, wastewater-based epidemiology started to be employed as a tool for tracking the virus transmission dynamics in a given area. The complexity of wastewater coupled with a lack of standardized methods led us to evaluate each step of the analysis individually and see which approach gave the most robust results for SARS-CoV-2 monitoring in wastewater. In this article, we present a step-by-step, retrospective view on the method development and implementation for the case of a pilot monitoring performed in Slovenia. We specifically address points regarding the thermal stability of the samples during storage, screening for the appropriate sample concentration and RNA extraction procedures and real-time PCR assay selection. Here, we show that the temperature and duration of the storage of the wastewater sample can have a varying impact on the detection depending on the structural form in which the SARS-CoV-2 target is present. We found that concentration and RNA extraction using Centricon filtration units coupled with Qiagen RNA extraction kit or direct RNA capture and extraction using semi-automated kit from Promega give the most optimal results out of the seven methods tested. Lastly, we confirm the use of N1 and N2 assays developed by the CDC (USA) as the best performing assays among four tested in combination with Fast Virus 1-mastermix. Data show a realistic overall process for method implementation as well as provide valuable information in regards to how different approaches in the analysis compare to one another under the specific conditions present in Slovenia during a pilot monitoring running from the beginning of the pandemic. waste water method evolution virus detection SARS-CoV-2 odpadna voda SARS-Cov-2 razvoj metode zaznavanje virusa true false true Angleški jezik Slovenski jezik Neznano 2024-07-17 03:59:02 2024-07-17 10:11:11 2024-07-18 03:54:02 0000-00-00 00:00:00 2022 0 0 str. 384-400 Vol. 14 23 Aug 2022 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2022-08-23 579 1867-0334 10.1007/s12560-022-09533-0 119616515 https://link.springer.com/article/10.1007/s12560-022-09533-0 1 6090723c-43cf-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26797 RAZ_Maksimovic_Olivera_2022.pdf RAZ_Maksimovic_Olivera_2022.pdf 1 DE49AA11C9A1134C49C9787FF19F93D4 ffda9dc809c2dc85fdae70abad30abc7e1ca707987fb6a78055b2ae1e8b044f4 698364ef-4403-11ef-b487-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=26820 Nacionalni inštitut za biologijo 0 0 0