Improved methods for the detection and quantification of SARS-CoV-2 RNA in wastewater

Abstract

Since the start of the COVID-19 pandemic, different methods have been used to detect the presence of genetic material of SARS-CoV-2 in wastewater. The use of wastewater for SARS-CoV-2 RNA detection and quantification showed different problems, associated to the complexity of the matrix and the lack of standard methods used to analyze the presence of an enveloped virus, such as coronavirus. Different strategies for the concentration process were selected to carry out the detection and quantification of SARS-CoV-2 RNA in wastewater: (a) aluminum hydroxide adsorption–precipitation, (b) pre-treatment with glycine buffer and precipitation with polyethylene-glycol (PEG) and (c) ultrafiltration (Centricon). Our results showed that the reduction of organic matter, using the pre-treatment with glycine buffer before the concentration with Centricon or aluminum hydroxide adsorption–precipitation, improved the recovery percentage of the control virus, Mengovirus (MgV) (8.37% ± 5.88 n = 43; 6.97% ± 6.51 n = 20, respectively), and the detection of SARS-CoV-2 in comparison with the same methodology without a pre-treatment. For the concentration with Centricon, the use of 100 mL of wastewater, instead of 200 mL, increased the MgV recovery, and allowed a positive detection of SARS-CoV-2 with N1 and N2 targets. The quantity of SARS-CoV-2 RNA detected in wastewater did not show a direct correlation with the number of confirmed cases, but the study of its upwards or downwards trend over time enabled the detection of an increase of epidemiological data produced in September 2020, January 2021 and April 2021.

Temporal Dynamics and Contribution of Phage Community to the Prevalence of Antibiotic Resistance Genes in a Full-Scale Sludge Anaerobic Digestion Plant

“…A modified PEG precipitation method was used to enrich virus-like particles (VLPs) from AD sludge samples . Briefly, 20 mL of sample was mixed with isochoric 0.5 M glycine solution and shaken on a rotatory shaker at 150 rpm for 2 h. The mixed solution was centrifuged at 20,000 g for 30 min, and the supernatant was filtrated with 0.45 and 0.22 μm poly­(ether sulfone) membrane successively.…”

“…A modified PEG precipitation method was used to enrich virus-like particles (VLPs) from AD sludge samples. 32 Briefly, 20 mL of sample was mixed with isochoric 0.5 M glycine solution and shaken on a rotatory shaker at 150 rpm for 2 h. The mixed solution was centrifuged at 20,000g for 30 min, and the supernatant was filtrated with 0.45 and 0.22 μm poly(ether sulfone) membrane successively. Then, 50% (w/v) PEG solution was added with a volume ratio of 3:7 to the supernatant, and NaCl was added with a final concentration of 1.5 M and reacted at 4 °C for 2 h. Finally, the solution was centrifuged at 20,000g for 30 min, and the precipitates were collected for downstream procedures.…”

Covid-19: Auswirkungen auf den Gastrointestinaltrakt

“…Die Viruslast im Abwasser prognostizierte die Dynamik der lokalen Inzidenz von SARS-CoV-2 etwa 3 Wochen vor den Daten die auf Atemwegsabstrichanalysen basieren [26]. Zudem ist diese Methode weniger anfällig für Störungen, wie beispielsweise durch Probleme bei der Abnahme der Proben oder bei Meldewegen [27]. In München konnte durch das Tropeninstitut die zunehmende Ausbreitung einer Virusvariante in der Münchner Bevölkerung detektiert werden, bereits Wochen bevor diese durch die Sequenzierung von Abstrich-Proben von Patient:innen festgestellt werden konnte.…”

Evaluation of variant calling algorithms for wastewater-based epidemiology using mixed populations of SARS-CoV-2 variants in synthetic and wastewater samples

“…Discrepancies between clinical and wastewater samples have been observed; in particular, the mixed strain nature of wastewater samples, the more degraded nature of viral genomes and, consequently, the inability to obtain a consensus genome sequence for each of the samples analysed. These differences can be accounted for by the nature and characteristics of the samples (wastewater vs clinical) and characteristics impacting the ability to extract and analyse the samples, such as virus titre, which is considerably lower in wastewater samples, and that, in turn, may affect variant calling, such as sample preparation, with additional steps such as centrifugation and filtration methods required to purify the samples from chemicals and other sources of contamination, and platform-dependent sequencing errors [11][12][13][14].…”