Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR

Dimitrakopoulos, Lampros; Kontou, Aikaterini; Strati, Αreti; Galani, Aikaterini; Kostakis, Marios; Kapes, Vasileios; Lianidou, Evi; Τhomaidis, Νikolaos S.; Markou, Athina

doi:10.1016/j.cscee.2022.100224

Cited by 14 publications

(6 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In contrast to previous work that reported detectable viral RNA in both the influent and treated sewage (Westhaus et al, 2021), here we found no quantifiable viral RNA after the secondary clarification process. Although viral RNA detection is influenced by the sensitivity of our extraction and quantification approach (Dimitrakopoulos et al, 2022), our results support the effectiveness of several different wastewater treatment sizes and designs in removing SARS-CoV-2 RNA before discharge to receiving water bodies at the reported detection limits. Nevertheless, molecular methods for concentrating viral RNA in wastewater continue to evolve with research knowledge and improved biotechnology tools.…”

Section: Discussionsupporting

confidence: 67%

The fate of SARS-CoV-2 viral RNA in coastal New England wastewater treatment plants

et al. 2023

View full text Add to dashboard Cite

Municipal sewage carries SARS-CoV-2 viruses shed in the human stool by infected individuals to wastewater treatment plants (WWTPs). It is well-established that increasing prevalence of COVID-19 in a community increases the viral load in its WWTPs. Despite the fact that wastewater treatment facilities serve a critical role in protecting downstream human and environmental health through removal or inactivation of the virus, little is known about the fate of the virus along the treatment train. To assess the efficacy of differing WWTP size and treatment processes in viral RNA removal we quantified two SARS-CoV-2 nucleocapsid (N) biomarkers (N1 and N2) in both liquid and solids phases for multiple treatment train locations from seven coastal New England WWTPs. SARS-CoV-2 biomarkers were commonly detected in the influent, primary treated, and sludge samples (returned activated sludge, waste activated sludge, and digested sludge), and not detected after secondary clarification processes or disinfection. Solid fractions had 470 to 3,700-fold higher concentrations of viral biomarkers than liquid fractions, suggesting considerably higher affinity of the virus for the solid phase. Our findings indicate that a variety of wastewater treatment designs are efficient at achieving high removal of SARS CoV-2 from effluent; however, quantifiable viral RNA was commonly detected in wastewater solids at various points in the facility. This study supports the important role municipal wastewater treatment facilities serve in reducing the discharge of SARS-CoV-2 viral fragments to the environment and highlights the need to better understand the fate of this virus in wastewater solids.

show abstract

Section: Discussionsupporting

confidence: 67%

The fate of SARS-CoV-2 viral RNA in coastal New England wastewater treatment plants

et al. 2023

View full text Add to dashboard Cite

show abstract

“…However, it is important to note that the removal of solids on the efficacy of the virus concentration method is still unclear, as suggested by Pecson et al ( 2021 ). Still, very good results in concentration measurements have also been obtained in other studies with methods using PEG precipitation which are shown to work better than filtration and adsorption methods (Dimitrakopoulos et al 2022 ).…”

Section: Resultsmentioning

confidence: 82%

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater

Cutrupi,

Rossi,

Cadonna

et al. 2023

Environ Sci Pollut Res

View full text Add to dashboard Cite

Crucial information on the pandemic’s spread has been gathered by monitoring the trend of SARS-CoV-2 in wastewater. This surveillance has highlighted that the initial concentration is a critical step of the analytical procedure due to the low viral titer that may be present in this matrix. This paper presents the results of the evaluation of two different wastewater concentration protocols to determine the most efficient and cost-effective. The two methods tested were the following: (a) a biphasic separation system with PEG-dextran and (b) a PEG/NaCl precipitation protocol. Other aspects of the detection method were also investigated including the influence of storage temperature on virus recovery and the heat treatment of pasteurization, which aims to make samples safer for operators and the environment. The PEG/NaCl precipitation method was found to perform better than the biphasic separation system, allowing for more sensitive identification of the presence of the virus and the detection of a higher viral titer than that identified with the biphasic separation in all results. Storage of the samples at 4.3±0.2°C for up to 3 weeks did not adversely affect the virus titer and the pasteurization pre-treatment increases operator safety and maintains the identification of the viral concentration. Graphical abstract

show abstract

“…Analytical recoveries (%) for each virus were estimated based on the following Eq. ( 3 ) described elsewhere [ 22 ]: …”

Section: Resultsmentioning

confidence: 99%

“…24-h composite flow proportional raw wastewater samples were collected at the Wastewater Treatment Plant (WWTP) of Attica, the region of Greece that includes the greater Athens area and its suburbs. 37 raw wastewater samples were collected and total nucleic acids were extracted as previously described [ 22 , 23 ]. Wastewater samples were collected in pre-cleaned 1L high-density polyethylene bottles and transported to the laboratory at 4 °C, where they were processed immediately upon arrival.…”

Section: Methodsmentioning

confidence: 99%

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay

Zafeiriadou,

Kaltsis,

Thomaidis

et al. 2024

Hum Genomics

Self Cite

View full text Add to dashboard Cite

Background After the occurrence of the COVID-19 pandemic, detection of other disseminated respiratory viruses using highly sensitive molecular methods was declared essential for monitoring the spread of health-threatening viruses in communities. The development of multiplex molecular assays are essential for the simultaneous detection of such viruses even at low concentrations. In the present study, a highly sensitive and specific multiplex one-step droplet digital PCR (RT-ddPCR) assay was developed for the simultaneous detection and absolute quantification of influenza A (IAV), influenza B (IBV), respiratory syncytial virus (RSV), and beta-2-microglobulin transcript as an endogenous internal control (IC B2M). Results The assay was first evaluated for analytical sensitivity and specificity, linearity, reproducibility, and recovery rates with excellent performance characteristics and then applied to 37 wastewater samples previously evaluated with commercially available and in-house quantitative real-time reverse transcription PCR (RT-qPCR) assays. IAV was detected in 16/37 (43%), IBV in 19/37 (51%), and RSV in 10/37 (27%) of the wastewater samples. Direct comparison of the developed assay with real-time RT-qPCR assays showed statistically significant high agreement in the detection of IAV (kappa Cohen’s correlation coefficient: 0.834, p = 0.001) and RSV (kappa: 0.773, p = 0.001) viruses between the two assays, while the results for the detection of IBV (kappa: 0.355, p = 0.27) showed good agreement without statistical significance. Conclusions Overall, the developed one-step multiplex ddPCR assay is cost-effective, highly sensitive and specific, and can simultaneously detect three common respiratory viruses in the complex matrix of wastewater samples even at low concentrations. Due to its high sensitivity and resistance to PCR inhibitors, the developed assay could be further used as an early warning system for wastewater monitoring.

show abstract

Evaluation of viral concentration and extraction methods for SARS-CoV-2 recovery from wastewater using droplet digital and quantitative RT-PCR

Cited by 14 publications

References 42 publications

The fate of SARS-CoV-2 viral RNA in coastal New England wastewater treatment plants

The fate of SARS-CoV-2 viral RNA in coastal New England wastewater treatment plants

Evaluation of concentration procedures, sample pre-treatment, and storage condition for the detection of SARS-CoV-2 in wastewater

Simultaneous detection of influenza A, B and respiratory syncytial virus in wastewater samples by one-step multiplex RT-ddPCR assay

Contact Info

Product

Resources

About