Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based RT-qPCR

Abstract

The coronavirus disease 2019 (COVID-19) pandemic is unceasingly spreading across the globe, and recently a highly transmissible Omicron SARS-CoV-2 variant (B.1.1.529) has been discovered in South Africa and Botswana. Rapid identification of this variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive and time-consuming genomic sequencing. In this study, we propose an alternative RT-qPCR approach for the detection of the Omicron BA.1 variant using a low-cost and rapid SYBR Green method. We have designed specific primers to confirm the deletion mutations in the spike (S Δ143-145) and the nucleocapsid (N Δ31-33) which are characteristics of this variant. For the evaluation, we used 120 clinical samples from patients with PCR-confirmed SARS-CoV-2 infections, and displaying an S-gene target failure (SGTF) when using TaqPath COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. Our results showed that all the 120 samples harbored S Δ143-145 and N Δ31-33, which was further confirmed by whole-genome sequencing of 10 samples, thereby validating our SYBR Green-based protocol. This protocol can be easily implemented to rapidly confirm the diagnosis of the Omicron BA.1 variant in COVID-19 patients and prevent its spread among populations, especially in countries with high prevalence of SGTF profile.

Keywords: B.1.1.529; SARS-CoV2 RT-PCR; SGTF; variants of concern.

Figure 1.

The Localization and design of the primers used. The nine nucleotides deleted in S gene and in N gene of Omicron variant are marked in bold.

Figure 2.

Calibration curves of SYBR Green-based qPCR primers Spike del 143-145, N del 31-33, Spike control and TaqPath kit. Serially 10-fold diluted RNA containing the mutations S Δ143-145 and N Δ31-33 were amplified and analyzed. (a) The threshold cycle (Ct) mean values were plotted against the Log starting quantity (ng/μl). slope, R2 and Efficiency (E) were determined. Each dilution was assayed in triplicate.

Figure 3.

The melting curves for the products amplified with the SYBR Green-based qPCR protocol in S-gene target failure samples using the primers: (a) Spike del 143-145; (b) N del 31-33. The melting curves in Delta samples using the primers: (c) Spike control and Spike del 143-145 or N del 31-33 reveal specific melt peak for each primer set.

Figure 4.

Analyses of the correlation between the Ct values of ORF1ab gene tested by TaqPath™ assay and Ct values of N del 31-33, Spike del 142-145 and Spike control genes tested by our assay. Pearson’s correlation coefficient (R) was used to test the strength of correlation. Black dots denote individual samples (n = 120). P-values <0.05 are considered as significant.