A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2

Abstract

The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/μL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.

Keywords: COVID-19; N501Y variants; One-step RT-qPCR; SARS-CoV-2; Variant of concern.

Fig. 1

Determination of limit of detection (LoD) of the N501Y RT-qPCR and detection of low-frequency N501Y variants. (1A) The concentration (copies/μL) of serial dilutions of B.1.1.7 sample. (1B) Amplification curves of N501 (wildtype allele) for ten-fold serial dilutions of B.1.1.7. (1C) Amplification curves of Y501 (N501Y allele) for ten-fold serial dilutions of B.1.1.7. (1D) The concentration (copies/μL) of serial dilutions of B.1.36.27 (non-N501Y variant) sample. (1E) Amplification curves of N501 (wildtype allele) for ten-fold serial dilutions of B.1.36.27. (1 F) Amplification curves of Y501 (N501Y allele) for ten-fold serial dilutions of B.1.36.27. The LoD of this assay was determined as 1.5 copies/μL for both N501 and Y501 alleles. (1 G) Wildtype and N501Y containing SARS-CoV-2 RNA were mixed at ratios from 9:1 (allele frequency = 90 %) to 1:9 (allele frequency = 10 %). (1H, 1I) Amplification curves of mutant: wildtype RNA mixtures. N501Y mutation was successfully detected at an allele frequency as low as 10 % in the RNA mixtures.