Assessing sensitivity and reproducibility of RT-ddPCR and RT-qPCR for the quantification of SARS-CoV-2 in wastewater

[Display omitted] •Wastewater surveillance methods captured the emergence of SARS-CoV-2 presence in a community.•In a direct comparison of workflows, RT-ddPCR demonstrated superior analytical sensitivity.•RT-ddPCR was resistant to inhibition in a wastewater matrix and was reproducible across laborat...

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Published in:Journal of virological methods Vol. 297; p. 114230
Main Authors: Ciesielski, Mark, Blackwood, Denene, Clerkin, Thomas, Gonzalez, Raul, Thompson, Hannah, Larson, Allison, Noble, Rachel
Format: Journal Article
Language:English
Published: Netherlands Elsevier B.V 01.11.2021
Subjects:
COVID-19
detection limit
Humans
Molecular methods
monitoring
North Carolina
nucleic acids
nucleocapsid proteins
pandemic
pathogens
PCR
Real-Time Polymerase Chain Reaction
Reproducibility of Results
RNA, Viral - genetics
sanitation
SARS-CoV-2
Severe acute respiratory syndrome coronavirus 2
Virginia
Wastewater
North Carolina
Virginia
SARS-CoV-2
Wastewater
Molecular methods
PCR
ISSN:0166-0934, 1879-0984, 1879-0984
Online Access:Get full text
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Summary:[Display omitted] •Wastewater surveillance methods captured the emergence of SARS-CoV-2 presence in a community.•In a direct comparison of workflows, RT-ddPCR demonstrated superior analytical sensitivity.•RT-ddPCR was resistant to inhibition in a wastewater matrix and was reproducible across laboratories. Throughout the COVID-19 global pandemic there has been significant interest and investment in using Wastewater-Based Epidemiology (WBE) for surveillance of viral pathogen presence and infections at the community level. There has been a push for widescale implementation of standardized protocols to quantify viral loads in a range of wastewater systems. To address concerns regarding sensitivity, limits of quantification, and large-scale reproducibility, a comparison of two similar workflows using RT-qPCR and RT-ddPCR was conducted. Sixty raw wastewater influent samples were acquired from nine distinct wastewater treatment plants (WWTP’s) served by the Hampton Roads Sanitation District (HRSD, Virginia Beach, Virginia) over a 6-month period beginning March 9th, 2020. Common reagents, controls, master mixes and nucleic acid extracts were shared between two individual processing groups based out of HRSD and the UNC Chapel Hill Institute of Marine Sciences (IMS, Morehead City, North Carolina). Samples were analyzed in parallel using One-Step RT-qPCR and One-Step RT-ddPCR with Nucleocapsid Protein 2 (N2) specific primers and probe. Influent SARS-CoV-2 N2 concentrations steadily increased over time spanning a range from non-detectable to 2.13E + 05 copies/L. Systematic dilution of the extracts indicated that inhibitory components in the wastewater matrices did not significantly impede the detection of a positive N2 signal for either workflow. The RT-ddPCR workflow had a greater analytical sensitivity with a lower Limit of Detection (LOD) at 0.066 copies/μl of template compared to RT-qPCR with a calculated LOD of 12.0 copies/μL of template. Interlaboratory comparisons using non-parametric correlation analysis demonstrated that there was a strong, significant, positive correlation between split extracts when employing RT-ddPCR for analysis with a ρ value of 0.86.
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ISSN:0166-0934
1879-0984
1879-0984
DOI:10.1016/j.jviromet.2021.114230