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

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.

SARS-CoV-2 RNA in Wastewater and Bivalve Mollusk Samples of Campania, Southern Italy

“…In addition, wastewater analysis provides insights into infectious trends in the population regardless of the availability of clinical testing data [ 21 ]. Environmental monitoring of SARS-CoV-2 has been extensively described also in various Italian regions [ 22 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ], confirming it as a powerful tool for tracking the spread of SARS-CoV-2 variants/subvariants in the population [ 32 , 33 , 34 ].…”

Long-term monitoring of COVID 19 prevalence in raw and treated wastewater in Salvador, a largest capital of Brazilian Northeast

“…The recovery of the genetic material of SARS-CoV-2 by concentration using PEG 8000 (which is based on the precipitation analyzes) has shown a better yield when compared to other techniques, such as occulation or ultracentrifugation 30,43,44 . Therefore, a lower concentration of organic matter (or reaction inhibitors) in the treated samples, as well as the application of a concentration technique capable of increasing the e ciencies of viral recovery may explain the superior performance of the positivity rate of the treated samples that were concentrated by PEG 8000 compared to the others analyzed in this study.…”

Wastewater-Based Epidemiology of SARS-CoV-2 and Other Respiratory Viruses: Bibliometric Tracking of the Last Decade and Emerging Research Directions

“…Our keyword analysis revealed that scientists are actively investigating the issues of the basic complexities of wastewater and viral dilutions by using several sample pre-treatment and virus concentration steps [51][52][53]. Inter-laboratory comparisons for identifying the best concentration principles were also published [54]. Upon an in-depth manual analysis, we noted that most of the studies utilized a smaller wastewater volume (<250 mL); however, quite a few studies reported that an increased wastewater sample volume can enhance the overall sensitivity of WBE and that the use of ultracentrifugation techniques for virus concentration is apparently the most common one [55,56].…”