(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.

The current global pandemic due to the SARS-CoV-2 has pushed the limits of global health systems across all aspects of clinical care, including laboratory diagnostics. Supply chain disruptions and rapidly-shifting markets have resulted in flash-scarcity of commercial laboratory reagents; this has motivated health care providers to search for alternative workflows to cope with the international increase in demand for SARS-CoV-2 testing. The aim of this study is to present a reproducible workflow for real time RT-PCR SARS-CoV-2 testing using OT-2 open-source liquid-handling robots (Opentrons, NY). We have developed a framework that includes a code template which is helpful for building different stand-alone robotic stations, capable of performing specific protocols. Such stations can be combined together to create a complex multi-stage workflow, from sample setup to real time RT-PCR. Using our open-source code, it is easy to create new stations or workflows from scratch, adapt existing templates to update the experimental protocols, or to fine-tune the code to fit specific needs. Using this framework, we developed the code for two different workflows and evaluated them using external quality assessment (EQA) samples from the European Molecular Genetics Quality Network (EMQN). The affordability of this platform makes automated SARS-CoV-2 PCR testing accessible for most laboratories and hospitals with qualified bioinformatics personnel. This platform also allows for flexibility, as it is not dependent on any specific commercial kit, and thus it can be quickly adapted to protocol changes, reagent, consumable shortages, or any other temporary material constraints.