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

Fuzzen, Meghan; Harper, Nathanael B.J.; Dhiyebi, Hadi A.; Srikanthan, Nivetha; Hayat, Samina; Bragg, Leslie M.; Peterson, Shelley; Yang, Ivy; Sun, Jianxian; Edwards, Elizabeth; Giesy, John P.; Mangat, Chand S.; Graber, Tyson E.; Delatolla, Robert; Servos, Mark R.

doi:10.1016/j.scitotenv.2023.163292

Cited by 10 publications

(12 citation statements)

References 44 publications

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“…The effect of primer/probe-template mismatch on quantification in qPCR has been investigated in other targets for SARS-CoV-2 ( Bozidis et al, 2022 ; Hasan et al, 2021 ). Especially, mutation in the probe binding region was found to largely impact qPCR amplification by delaying amplification or causing failure, which were also used to design mutation/variant detection methods such as the N200 assays to quantify Delta/Omicron/Universal SARS2 ( Fuzzen et al, 2023 ), the D3Ll assay to detect Alpha variant ( Graber et al, 2021 ), and the various assays targeting the S gene to measure Alpha/Beta/Gamma variants ( Liu et al, 2023 ; Peterson et al, 2022 ; Vega-Magaña et al, 2021 ). N gene dropout or failure was also reported using some commercial SASR-CoV-2 RT-qPCR kits when mutations were found in the N200 region (28881–28883, 28896–28898, in B.1.1.318 variant) by sequencing ( Bozidis et al, 2022 ).…”

Section: Resultsmentioning

confidence: 99%

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region

Sun,

Yang,

Peng

et al. 2024

Water Research X

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Section: Resultsmentioning

confidence: 99%

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region

Sun,

Yang,

Peng

et al. 2024

Water Research X

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“…Successful detection and discrimination of all six Omicron subvariants in a single assay was a challenge that limited previous studies. − Our Omicron triplex assay is advantageous because, by utilizing three target mutations, all six Omicron subvariants (BA.1/BA.3, BA.2/XBB, and BA.4/BA.5) can be discriminated. Additionally, our triplex assay only requires a commonly available PCR instrument, uses fewer reagents, and is more versatile and cost-effective.…”

Section: Resultsmentioning

confidence: 99%

“…Using combinations of the HV 69–70 deletion, N501Y, and T478K targets to detect Omicron can broaden the range of Omicron subvariants detected, but this assay is not capable of differentiating BA.1 from BA.4/5 . Additionally, targeting two mutations in the N gene and the two mutations E484A and S477N in the S gene can detect all Omicron subvariants, but these assays cannot differentiate among all the subvariants, because these mutations are shared by all the Omicron subvariants. , …”

Section: Resultsmentioning

confidence: 99%

Quantification and Differentiation of SARS-CoV-2 Variants in Wastewater for Surveillance

et al. 2023

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Wastewater surveillance plays an important role in the monitoring of infections of SARS-CoV-2 at the community level. We report here the determination of SARS-CoV-2 and differentiation of its variants of concern in 294 wastewater samples collected from two major Canadian cities from May 2021 to March 2023. The overall method of analysis involved extraction of the virus and viral components using electronegative membranes, in situ stabilization and concentration of the viral RNA onto magnetic beads, and direct analysis of the viral RNA on the magnetic beads. Multiplex reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays, targeting specific and naturally selected mutations in SARS-CoV-2, enabled detection and differentiation of the Alpha, Beta, Gamma, Delta, and Omicron variants. An Omicron triplex RT-qPCR assay targeting three mutations, HV 69−70 deletion, K417N, and L452R, was able to detect and differentiate the Omicron BA.1/BA.3, BA.2/XBB, and BA.4/5. This assay had efficiencies of 90−104% for all three mutation targets and a limit of detection of 28 RNA copies per reaction. Analyses of 294 wastewater samples collected over a two-year span showed the concentrations and trends of Alpha, Beta, Gamma, Delta, and Omicron variants as they emerge in two major Canadian cities participating in the wastewater surveillance program. The trends of specific variants were consistent with clinical reports for the same period. At the beginning of each wave, the corresponding variants were detectable in wastewater. For example, RNA concentrations of the BA.2 variant were as high as 10 4 copies per 100 mL of wastewater collected in January 2022, when approximately only 50−60 clinical cases of BA.2 infection were reported in Canada. These results show that the strategy and highly sensitive assays for the variants of concern in wastewater are potentially useful for the detection of newly emerging SARS-CoV-2 variants and other viruses for future community biomonitoring.

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“…The final volume was ∼140 μL and the RNA concentrations were determined by qPCR using methods described elsewhere. 39…”

Section: Methodsmentioning

confidence: 99%

Digital microfluidics with distance-based detection – a new approach for nucleic acid diagnostics

Ho,

Sathishkumar,

Sklavounos

et al. 2024

Lab Chip

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

Cited by 10 publications

References 44 publications

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region

Underestimation of SARS-CoV-2 in wastewater due to single or double mutations in the N1 qPCR probe binding region

Quantification and Differentiation of SARS-CoV-2 Variants in Wastewater for Surveillance

Digital microfluidics with distance-based detection – a new approach for nucleic acid diagnostics

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