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. 2023 Jul 10:881:163292.
doi: 10.1016/j.scitotenv.2023.163292. Epub 2023 Apr 7.

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays

Meghan Fuzzen  1 , Nathanael B J Harper  2 , Hadi A Dhiyebi  2 , Nivetha Srikanthan  2 , Samina Hayat  2 , Leslie M Bragg  2 , Shelley W Peterson  3 , Ivy Yang  4 , J X Sun  4 , Elizabeth A Edwards  4 , John P Giesy  5 , Chand S Mangat  3 , Tyson E Graber  6 , Robert Delatolla  7 , Mark R Servos  2
Affiliations

An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays

Meghan Fuzzen et al. Sci Total Environ. .

Abstract

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

Keywords: Omicron; RT-PCR; SARS-CoV-2; VOC; Wastewater based epidemiology.

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Conflict of interest statement

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mark Servos reports financial support was provided by Ontario Ministry of the Environment Conservation and Parks. Mark Servos reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Mark Servos reports financial support was provided by Global Water Futures.

Figures

Unlabelled Image
Graphical abstract
Fig. 1
Fig. 1
N200 assay amplicon with the location of the N200 forward and reverse primers and all probes are shown on a portion of the N gene of the SARS-CoV-2 genome. The sequence, nucleotide numbers, and amino acid displayed are based on the SARS-CoV-2 reference genome (Wuhan; NC_045512.2; figure generated using Benchling.com).
Fig. 2
Fig. 2
SARS-CoV-2 signal measured in municipal wastewater using the N200 assay with the Universal probe in a multiplexed rection (Log scale, copies/mL). Some samples were included that were above the limit of detection (LOD) but below the limit of quantification (LOQ) as indicated in the figure. Samples from the Peel (GE Booth and Clarkson), York (Humber AMF and Warden) and Waterloo (Waterloo and Kitchener) Regions were collected during the period encompassing both the introduction of Omicron into these communities and the concomitant reduction of the endemic Delta VOC.
Fig. 3
Fig. 3
Application of the N200 assay to monitor the relative abundance (%) of the mutations R203M (black circles- presumed Delta) or R203K + G204R (grey squares – presumed Omicron) in municipal wastewater influent. Influent was collected from the Region of Peel (A, B), York Region (C, D) and Waterloo Region (E, F) using composite (A–B, D–F) and grab (C) sampling. Lines drawn spline smoothed curve calculated using GraphPad PRISM (v 9.5.0).
Fig. 4
Fig. 4
Comparison of whole genome sequencing (WGS) data from clinical samples (triangles) and qPCR wastewater data (squares) during the transition from Delta (blue) to Omicron (red) in each of three regions; Peel Public Health (A), York Region Public Health (B), and Region of Waterloo Public Health (C). Clinical whole genome sequencing data points represent the frequency of each variant over a four-week period within a single public health unit. Wastewater qPCR data points represent the mean frequency of each variant at all sites assessed within a region within the same four-week period. The date on the x-axis represents the end of the four-week period.
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