Abstract
(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.
Highlights
Amid the pandemic of SARS-CoV-2 [1], communities have faced the rapid spread of the virus and its related disease—called COVID-19—affecting the testing capacity of public health systems and microbiological laboratories [2,3]
The wastewaterbased epidemiology (WBE) approach has been used for decades to detect poliovirus and development a surveillance system through the implem toThe track other viruses—able to of persist long enough in untreated wastewater to allow reliable detection—in that the sewage system can blend viral in shedding variation approach mayconsideration serve to monitor viral transmission the community a among single individuals and over the course of their infection, into an average amount that warning system, allowingunder timely interventions to several face studies new have pathogen represents the entire community investigation
The WBE approach has been used for decades to d and have shown a good correlation between the number of active COVID-19 cases and to track other viruses—able towastewater persistsamples long enough incities untreated the SARS-CoV-2
Summary
Amid the pandemic of SARS-CoV-2 [1], communities have faced the rapid spread of the virus and its related disease—called COVID-19—affecting the testing capacity of public health systems and microbiological laboratories [2,3]. Since SARS-CoV-2 is shed by feces in the early stage of infection and can cause asymptomatic infection in a large proportion of individuals, it is an ideal target for WBE. This strategy may allow to: (i) estimate the real prevalence of SARS-CoV-2 infection at a population level, (ii) monitor SARS-CoV-2 spread after the implementation of containment measures and restrictions and (iii) provide an early warning of virus re-introduction [4–6]. Surveillance of SARS-CoV-2 in sewage may provide timely indications on SARS-CoV-2 infection dynamics, overcoming the lag in monitoring exclusively COVID-19 symptoms and tests, since the onset of symptoms might be 2 weeks apart from viral infection [7–9]. This approach of surveillance can overcome test availability and indications that can result under pressure during the surge of new outbreaks
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