Direct RT-qPCR assay for SARS-CoV-2 variants of concern (Alpha, B.1.1.7 and Beta, B.1.351) detection and quantification in wastewater

Abstract

Less than a year following the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak, variants of concern have emerged in the form of variant Alpha (B.1.1.7, the British variant) and Beta (B.1.351, the South Africa variant). Due to their high infectivity and morbidity, it has become clear that it is crucial to quickly and effectively detect these and other variants. Here, we report improved primers-probe sets for reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) for SARS-CoV-2 detection including a rapid, cost-effective, and direct RT-qPCR method for detection of the two variants of concern (Alpha, B.1.1.7 and Beta, B.1.351). All the developed primers-probe sets were fully characterized, demonstrating sensitive and specific detection. These primer-probe sets were also successfully employed on wastewater samples aimed at detecting and even quantifying new variants in a geographical area, even prior to the reports by the medical testing. The novel primers-probe sets presented here will enable proper responses for pandemic containment particularly, considering the emergence of variants of concern.

RT-qPCR assays for SARS-CoV-2 variants of concern in wastewater reveals compromised vaccination-induced immunity

“…Considering that the different SARS-CoV-2 variants share very high similarity in their N gene, we employed N gene detection in prospect of general detection of all the SARS-CoV-2 RNA traces in the wastewater. Using a CDC's standard detection set (CDC's N2) and our previously published designed primer set ( Yaniv et al, 2021 ) (Improved N3), we documented the SARS-CoV-2 RNA levels in wastewater and monitored these values together with the corresponding reported active cases and percent vaccination in the city populations ( Fig. 1 ).…”

“…This reappearance of increased morbidity continues throughout September 2021, despite vaccination percentages (second dose) of nearly 70% of the city's population. Previously, we reported the emergence of the Alpha variant of concern (B.1.1.7) in Israel by the end of January 2021 using RT-qPCR assay we developed ( Yaniv et al, 2021 ). Soon after appearance, the alpha variant dominated the original variant to such an extent that within one month all SARS-CoV-2 RNA traces in wastewater corresponded solely to the Alpha variant.…”

“…Previously we developed direct RT-qPCR detection assays for the Alpha variant (B.1.17, British) and Beta variant (B.1.315, South Africa) ( Yaniv et al, 2021 ). The possibility that the new outbreak is a result of the invasion of a novel variant of concern that differs from the Alpha or Beta was suggested.…”

“…RT-qPCR was executed as previously described ( Yaniv et al, 2021 ). Reaction final volume is 20 µL with primers concentration of 0.5 µM and probe concentration of 0.2 µM.…”

“…Such an assay can help assess a variant's frequency within a population and contribute to policy guidelines. Indeed a number of publications targeting variants of concern detection using RT-qPCR ( Barreto et al, 2020 ; Bivins et al, 2021 ; Gand et al, 2021 ; Vega-Magaña et al, 2021 ; Vogels et al, 2021 ; H. Wang et al, 2021 ; Yaniv et al, 2021 ) as well as available commercial kits (e.g., TaqPath ThermoScientific, GT molecular, PerkinElmer) have emerged for individual assessments.…”