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

(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.