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.

Keywords: Molecular probes; Reverse transcriptase polymerase chain reaction; SARS-CoV-2; Variants of concern; Wastewater-based epidemiological monitoring.

Fig. 1

N gene detection designs and characterization. (a) Improved and new primers-probe sets design along SARS-CoV-2 N gene. Numbers in red indicate bp region amplified in RT-qPCR with the relevant primers-probe set. (b) List of Improved and new primers-probe sets sequences. (c) Calibration curves for all examined primers-probe sets for N gene detection. Resulted Ct value from RT-qPCR plotted against Log copy number tested. Error bars present standard deviation for six replicates. (d) List of Limit of detection, Linear regression R², and Y intercept values extracted from the resulted calibration curves. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Lower detection limit of N gene examined primers–probe sets in wastewater matrix. RNA extracted from negative detection wastewater sample (No virus) served as wastewater matrix and spiked with known concentrations of SARS-CoV-2. Matrix was spiked with different concentration of N gene template (10⁰–10² copies per μL) or Non-Template Control (NTC, water). ND - not detected. Solid lines indicate the median and dashed lines indicate the detection limit as decided by clinical guidelines (Ct > 40).

Fig. 3

SARS-CoV-2 detection in wastewater using primers-probe sets Improved N3 and CDC's N2. For each sample out of 148 different samples in September–December 2020, ΔCt was calculated. ΔCt is comprised from reduction of the resulted Ct for Improved N3 from the resulted Ct for CDC's N2. For samples where CDC's N2 resulted in a signal while Improved N3 did not, meaning only CDC's N2 detected, an arbitrary value of −5 was assigned and colored in blue. For samples where Improved N3 resulted in a signal while CDC's N2 did not, meaning only Improved N3 detected, an arbitrary value of +5 was assigned and colored in red. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

S gene detection designs and characterization for variants of concern. (a) Designed detection sets for differentiation and identification of SARS-CoV-2 Alpha variant, B.1.1.7 and Beta variant, B.1.351. Design was based on sequence differences in the S gene between the variants. (b) List of primers and probes sequences for variants detection. (c) Calibration curves for primers-probe sets for variants of concern detection. Resulted Ct value from RT-qPCR plotted against Log copy number tested. Error bars present standard deviation for six replicates. (d) List of Limit of detection, Linear regression R², and Y intercept values extracted from the resulted calibration curves. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Lower detection limit of SΔ69, SΔ241 and S1 primers–probe sets in wastewater matrix. RNA extracted from negative detection wastewater sample (No virus) served as wastewater matrix and spiked with known concentrations of SARS-CoV-2. Matrix was spiked with different concentration of S gene template (10⁰–10² copies per μL) or Non-Template Control (NTC, water). For SΔ69 set and SΔ241 set, the S gene deletion template corresponded to Δ69–70 deletion site in the Alpha strain and Δ241–243 deletion site in the Beta strain. For S1 set, the S gene template corresponded to NC_045512.2 original sequence. ND - not detected. Solid lines indicate the median and dashed lines indicate the detection limit as decided by clinical guidelines (Ct > 40).

Fig. 6

SARS-CoV-2 variants detection in Beer Sheva wastewater over time. Samples collected between November 2020 and March 2021 from WWTP were tested for N gene (black line), S1 (blue) and SΔ69 (orange) detection. Consist of levels for SARS-CoV-2 overall detection (N gene), whereas Alpha strain detection manifested by the end of January 2021 and became dominant over the original variant within a month. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)