A Recurrent Mutation at Position 26340 of SARS-CoV-2 Is Associated with Failure of the E Gene Quantitative Reverse Transcription-PCR Utilized in a Commercial Dual-Target Diagnostic Assay

Abstract

Control of the ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic requires accurate laboratory testing to identify infected individuals while also clearing essential staff to continue to work. At the current time, a number of quantitative real-time PCR (qRT-PCR) assays have been developed to identify SARS-CoV-2, targeting multiple positions in the viral genome. While the mutation rate of SARS-CoV-2 is moderate, given the large number of transmission chains, it is prudent to monitor circulating viruses for variants that might compromise these assays. Here, we report the identification of a C-to-U transition at position 26340 of the SARS-CoV-2 genome that is associated with failure of the cobas SARS-CoV-2 E gene qRT-PCR in eight patients. As the cobas SARS-CoV-2 assay targets two positions in the genome, the individuals carrying this variant were still called SARS-CoV-2 positive. Whole-genome sequencing of SARS-CoV-2 showed all to carry closely related viruses. Examination of viral genomes deposited on GISAID showed this mutation has arisen independently at least four times. This work highlights the necessity of monitoring SARS-CoV-2 for the emergence of single-nucleotide polymorphisms that might adversely affect RT-PCRs used in diagnostics. Additionally, it argues that two regions in SARS-CoV-2 should be targeted to avoid false negatives.

Keywords: E gene; SARS-CoV-2; qRT-PCR.

FIG 1

(A) Screen shot from IGV (Integrative Genomics Viewer; http://software.broadinstitute.org/software/igv/home) shows the VCFs (lines represent SNPs) for the eight viruses as well as a BAM file showing the reads and coverage for one virus. The sampling date for each virus is indicated. Six of the viruses have identical patterns of SNPs and are underlined. The zoomed-in section shows the SNP at 26340, and the blue rectangle labeled E_Sarbeco_P1 corresponds to the region covered by the E gene probe in Corman et al. (9). (B) Sanger sequencing of four samples carrying the C-to-U transition at position 26340. The top chromatogram shows a virus carrying the wild-type sequence. (For convenience, U is displayed as T.)

FIG 2

(A) Phylogenetic tree generated by Nextstrain (12) with the viruses carrying the U allele at 26340 highlighted in yellow. The six viruses with identical patterns of SNPs are underlined. (B) Expanded view of the eight Belgian samples. (C) Expanded view of the seven English samples.