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Title
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An alternative approach for bioanalytical assay optimization for wastewater-based epidemiology of SARS-CoV-2
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Author
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Abstract
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| Wastewater-based epidemiology 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 optimization of a SARS-CoV-2 RNA concentration and detection procedure was performed with IWW samples with high viral SARS-CoV-2 RNA loads. 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, …). Centricon Plus-70 (100 kDa) centrifugal filter devices resulted in the lowest and most reproducible Ct-values for SARS-CoV-2 RNA. Lowering the molecular weight cut-off did not improve our limit of detection and quantification (approximately 100 copies/μL for all genes). Quantitative polymerase chain reaction (qPCR) was employed for the amplification of the N1, N2, N3 and E-gene fragments. This is one of the first studies to apply digital polymerase chain reaction (dPCR) for the detection of SARS-CoV-2 RNA in IWW. dPCR showed high variability at low concentration levels (100 copies/μL), indicating that variability in bioanalytical methods for wastewater-based epidemiology of SARS-CoV-2 might be substantial. dPCR results in IWW were in line with the results found with qPCR. On average, the N2-gene fragment showed high in-sample stability in IWW for 10 days of storage at 4 °C. Between-sample variability was substantial due to the low native concentrations in IWW. Additionally, the E-gene fragment proved to be less stable compared to the N2-gene fragment and showed higher variability. Freezing the IWW samples resulted in a 10-fold decay of loads of the N2- and E-gene fragment in IWW. |
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Language
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English
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Source (journal)
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The science of the total environment. - Amsterdam, 1972, currens
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Publication
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Amsterdam
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2021
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ISSN
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0048-9697
[print]
1879-1026
[online]
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DOI
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10.1016/J.SCITOTENV.2021.148043
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Volume/pages
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789
(2021)
, 11 p.
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Article Reference
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148043
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ISI
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000677550800005
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Pubmed ID
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34323818
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Medium
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E-only publicatie
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Full text (Publisher's DOI)
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Full text (open access)
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Full text (publisher's version - intranet only)
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