SARS-CoV-2 E Gene Variant Alters Analytical Sensitivity Characteristics of Viral Detection Using a Commercial Reverse Transcription-PCR Assay

Abstract

Diagnostic assays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are essential for patient management, infection prevention, and the public health response for coronavirus disease 2019 (COVID-19). The efficacy and reliability of these assays are of paramount importance in both tracking and controlling the spread of the virus. Real-time reverse transcription-PCR (RT-PCR) assays rely on a fixed genetic sequence for primer and probe binding. Mutations can potentially alter the accuracy of these assays and lead to unpredictable analytical performance characteristics and false-negative results. Here, we identify a G-to-U transversion (nucleotide 26372) in the SARS-CoV-2 E gene in three specimens with reduced viral detection efficiency using a widely available commercial assay. Further analysis of the public GISAID repository led to the identification of 18 additional genomes with this mutation, which reflect five independent mutational events. This work supports the use of dual-target assays to reduce the number of false-negative PCR results.

Keywords: COVID-19; RT-PCR; SARS-CoV-2; mutation.

FIG 1

Detection of samples with discordant PCR values between E gene and ORF1a/b assays. Five samples (arrows) with E gene variants were identified by a significant deviation in the ΔC_T values between the ORF1ab and E gene targets in the Roche cobas assay compared to other dual-target-positive samples tested from May through August 2020 (n = 3,150). Three of the samples (filled circles) contained a sufficient concentration of virus for further genomic characterization.

FIG 2

Sixteen mutations shared across samples USA/MO-WUSTL_C/2020, USA/MO-WUSTL_D/2020, and USA/MO-WUSTL_E/2020. Samples USA/MO-WUSTL_C/2020, USA/MO-WUSTL_D/2020, and USA/MO-WUSTL_E/2020 shared 16 mutations present across the entire SARS-CoV-2 genome. The mutation of interest in the E gene is highlighted in orange.

FIG 3

Multiple-sequence alignment of 21 SARS-CoV-2 genomes with the same mutation in the E gene. Twenty-one SARS-CoV-2 genomes downloaded from GISAID, labeled by their GISAID “virus name,” were aligned to the reference SARS-CoV-2 genome, highlighted in yellow. The indicated position (POS) is the position of the mutation of interest in the E gene.

FIG 4

Nextstrain phylogenetic tree. Sixty-seven genomes are shown, with genomes containing 26372G>U (21 in total) highlighted in red. Genomes with the U genotype arose independently five different times.