Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity.

Detection of SARS-CoV-2 Variants Mu, Beta, Gamma, Lambda, Delta, Alpha, and Omicron in Wastewater Settled Solids Using Mutation-Specific Assays Is Associated with Regional Detection of Variants in Clinical Samples

“…Lee et al ( 14 ) reported a 3-fold-higher increase in a characteristic mutation in Alpha in wastewater from January to March 2021, comparable to an increased fraction in Alpha sequences from clinical samples deposited in GISAID during the same time period. Graber et al ( 15 ) reported agreement between wastewater trends of a characteristic mutation from Alpha and data aggregated at the city level on Alpha circulation based on clinical specimens. Yaniv et al ( 18 ) reported lack of detection of a characteristic mutation in Alpha in wastewater when clinical data suggested that it was not circulating.…”

“…Heijnen et al ( 13 ) applied a commercial digital RT-PCR assay to wastewater influent samples to detect a single nucleotide polymorphism (SNP) (mutation N501Y) present in Beta and Alpha. Lee et al ( 14 ) and Graber et al ( 15 ) applied RT-qPCR assays that detect mutations present in Alpha to wastewater samples. Yaniv et al developed RT-qPCR assays for Gamma and Delta ( 16 ) and for Alpha and Beta ( 17 ) and applied them to 4 and 10 wastewater samples, respectively, as proof of concept.…”

RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern

“…Concentrations of SARS-CoV-2, PMMoV, F-specific RNA bacteriophages group II (FRNAPH-II), and AQHRP were quantified by use of TaqMan RT-qPCR assays. Quantitative VOC assays, including N.D3L.GAT.CTA, 32 S.P681R.CCT.CGT, and S.T20N.ACC.AAC (TaqMan SARS-CoV-2 Mutation Panel, ThermoFisher, CA, USA), were adopted for detecting B.1.1.7 (α), P.1 (γ), and B.1.617 (including δ) lineages. Synthetic quantitative RNA standards were aliquoted for single use, and their nominal concentrations were confirmed by ddPCR in eight replicates.…”

Quantitative detection of SARS-CoV-2 Omicron BA.1 and BA.2 variants in wastewater through allele-specific RT-qPCR

“…However, poor sensitivity towards low-frequency variants commonly found during the "introduction" of a VOC in a population and the lack of quantitative modelling of the data generated limits widespread application (Van Poelvoorde et al, 2021). RT-qPCR-based methods capable of targeting variant-specific mutants in the SARS-CoV-2 genome have been successfully demonstrated for variant identification in clinical samples (Wang et al, 2021) and we and others have recently adapted and validated use of such assays for quantification of multiple variants in wastewater samples (Graber et al, 2021;Lee et al, 2021aLee et al, , 2021bLee et al, , 2021cYaniv et al, 2021). RT-qPCR methods enable differentiation of specific variant-linked mutations and are more sensitive than sequence-based approaches, allowing for their quantitation, and providing readily interpretable results within hours and using widely available methods and workflows.…”