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. 2023 May 1;49(5):213-220.
doi: 10.14745/ccdr.v49i05a07.

Real-time quantitative reverse transcription polymerase chain reaction detection of SARS-CoV-2 Delta variant in Canadian wastewater

Shelley Peterson  1 , Jade Daigle  1 , Codey Dueck  1 , Audra Nagasawa  2 , Michael Mulvey  1   3 , Chand S Mangat  1
Affiliations

Real-time quantitative reverse transcription polymerase chain reaction detection of SARS-CoV-2 Delta variant in Canadian wastewater

Shelley Peterson et al. Can Commun Dis Rep. .

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are associated with increased infectivity, severity, and mortality of coronavirus disease 2019 (COVID-19) and have been increasingly detected in clinical and wastewater surveillance in Canada and internationally. In this study, we present a real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay for detection of the N gene D377Y mutation associated with the SARS-CoV-2 Delta variant in wastewater.

Methods: Wastewater samples (n=980) were collected from six cities and 17 rural communities across Canada from July to November 2021 and screened for the D377Y mutation.

Results: The Delta variant was detected in all major Canadian cities and northern remote regions, and half of the southern rural communities. The sensitivity and specificity of this assay were sufficient for detection and quantitation of the Delta variant in wastewater to aid in rapid population-level screening and surveillance.

Conclusion: This study demonstrates a novel cost-effective RT-qPCR assay for tracking the spread of the SARS-CoV-2 Delta variant. This rapid assay can be easily integrated into current wastewater surveillance programs to aid in population-level variant tracking.

Keywords: B.1.617.2; Delta; SARS-CoV-2; qPCR; variant; wastewater.

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Conflict of interest statement

Competing interests None.

Figures

Figure 1
Figure 1
Standard curves for D377Y assays in the presence of the alternate genotype for each allelea Abbreviations: Ct, cycle threshold; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; V, variant; WT, wild-type a Standard curves for real-time quantitative reverse transcription polymerase chain reaction SARS-CoV-2 wild-type and D377Y variant B.1.617 assays against tenfold dilutions of DNA oligonucleotide controls in the presence of 100, 500 and 1,000 copies/µL of the alternate genotype for each allele
Figure 2
Figure 2
Standard curvesa,b,c for the D377Y real-time polymerase chain reaction assays performed with serial dilutions of synthetic DNA oligonucleotides for the wild-type and D377Y variant alleles Abbreviations: Ct, cycle threshold; V, variant; WT, wild-type a For all standard curves, equations for the lines and R2 values are indicated b (A) Standard curve for the WT assay using WT template and cross-reactivity with the V template c (B) Standard curve for the V assay using V template (solid line) and cross-reactivity with the WT template (dashed line)
Figure 3
Figure 3
Detection of SARS-CoV-2 Delta variant in wastewater from Canadian cities and rural areas using real-time quantitative reverse transcription polymerase chain reactiona,b Abbreviations: AB, Alberta; BC, British Columbia; NL, Newfoundland and Labrador; NS, Nova Scotia; NT, Northwest Territories; NU, Nunavut; ON, Ontario; QC, Québec; RNA, ribonucleic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2 a Red line depicts copies/ml (cp/mL) of D377Y mutation indicative of Delta variant presence b Black line is the SARS-CoV-2 concentration using the average of the Centers for Disease Control and Prevention N1 and N2 cp/mL. Sites tested over a period of less than one month or with fewer than three samples in which SARS-CoV-2 was detected by N1 or N2 are not shown. Dashed line represents the limit of quantification of the assay. Sites are described in Table A2
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