Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives

Alygizakis, Nikiforos; Markou, Athina; Rousis, Nikolaos I.; Galani, Aikaterini; Avgeris, Margaritis; Adamopoulos, Panagiotis G.; Scorilas, Andreas; Lianidou, Evi; Paraskevis, Dimitrios; Tsiodras, Sotirios; Tsakris, Athanassios; Dimopoulos, Meletios-Athanasios; Thomaidis, Nikolaos S.

doi:10.1016/j.trac.2020.116125

Cited by 98 publications

(90 citation statements)

References 49 publications

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“…While PEG precipitation resulted in detection levels of SARS-CoV-2 above the LLOQ for the E-gene, yields for the N1 and N2 gene were generally low or these genes remained undetected. This was in line with previous studies that reported poor recoveries of SARS-CoV-2 with PEG precipitation, possibly due to the co-concentration of PCR-inhibitors because of the small MWCO with this concentration method [21]. Ct-values measured with the other centrifugal methods were also low, as presented in Figure S4.…”

Section: Resultssupporting

confidence: 91%

“…The number of washing steps to remove PCR inhibitors may vary between the kits and different elution solvents are used for the extraction of SARS-CoV-2 RNA from the concentrate. These commercialized kits have been proposed in other WBE applications for the extraction of SARS-CoV-2 in IWW [21].…”

Section: Methodsmentioning

confidence: 99%

“…Pre-existing concentration methods for SARS-CoV-2 RNA primarily include ultracentrifugation, ultrafiltration, charged filter membranes or PEG precipitation [20]. Nevertheless, most of these methods assess efficiency of recovery (RE) by spiking non-enveloped human enteric viruses to IWW [21–23]. Only a limited number of papers on method development and optimization investigate RE through spiking of enveloped viruses (e.g.…”

Section: Introductionmentioning

confidence: 99%

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An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Jacobs

et al. 2021

Preprint

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Wastewater-based epidemiology could be applied to track down SARS-CoV-2 outbreaks at high spatio-temporal resolution and could potentially be used as an early-warning for emergence of SARS-CoV-2 circulation in the general population. Epidemiological surveillance of SARS-CoV-2 could play a role in monitoring the spread of the virus in the population and controlling possible outbreaks. However, sensitive sample preparation and detection methods are necessary to detect trace levels of SARS-CoV-2 RNA in influent wastewater (IWW). Unlike predecessors, method development of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 loads (in combination with seeding IWW with a surrogate coronavirus). This is of importance since the SARS-CoV-2 genome in IWW might have already been subject to in-sewer degradation into smaller genome fragments or might be present in a different form (e.g. cell debris,etc). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering pore sizes did not improve our limit of detection and quantification. Real-time polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E_Sarbeco-gene. This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. Interestingly, qPCR results were comparable with dPCR results suggesting that qPCR is a valid method. In this study, dPCR was also used as a proxy to assess the precision of qPCR. In this light, dPCR showed high variability at low concentration levels (100 copies/microliter), indicating that variability in bioanalytical assays for SARS-CoV-2 RNA might be substantial. On average, the N2-gene showed high in-sample stability in IWW for 10 days of storage at 4 degrees. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene proved to be less stable compared to the N2-gene and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene in IWW. Although WBE can already aid in filling some knowledge gaps in the epidemiological surveillance of SARS-CoV-2, future WBE studies should aim to further validate and standardize bioanalytical assays, especially with regards to methodological limitations.

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Section: Resultssupporting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Jacobs

et al. 2021

Preprint

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“…Some studies stored the composite sample immediately at -20°C or -80 °C until further analysis ( Table 4 ). A recent review stated that freezing and de-freezing the sample could potentially lead to degradation of the genetic material of SARS-CoV-2 by the RNases in wastewater [92] . The potential decay of SARS-CoV-2 in wastewater during storage at -20°C or -80°C and the impact of freezing and de-freezing require further investigation and may potentially lead to under-estimation of COVID-19 prevalence.…”

Section: Key Uncertainties For Estimating Sars-cov-2 Prevalence‘mentioning

confidence: 99%

Uncertainties in estimating SARS-CoV-2 prevalence by wastewater-based epidemiology

Zhang

Shi

et al. 2021

Chemical Engineering Journal

162

139

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“…Sampling and detecting the SARS-CoV-2 and its RNA in wastewater are undoubtedly the hottest of research in 2020. Although the SARS-CoV-2 RNAs in wastewater have been widely reported, the method of sampling, storage, concentration, isolation, and detection are not the same in each study [43] . Obviously, as a key step in WEB system, the analytical protocols need more evaluation and investigation, which will be discussed in detail in later section of the review.…”

Section: Bibliometric Tracking Of the Investigation Trends Since Outbmentioning

confidence: 97%

Water science under the global epidemic of COVID-19: Bibliometric tracking on COVID-19 publication and further research needs

Zhao

Wei

et al. 2021

Journal of Environmental Chemical Engineering

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Analytical methodologies for the detection of SARS-CoV-2 in wastewater: Protocols and future perspectives

Cited by 98 publications

References 49 publications

An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

An alternative approach for bioanalytical assay development for wastewater-based epidemiology of SARS-CoV-2

Uncertainties in estimating SARS-CoV-2 prevalence by wastewater-based epidemiology

Water science under the global epidemic of COVID-19: Bibliometric tracking on COVID-19 publication and further research needs

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