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SARS-CoV-2 variant with mutations in N gene affecting detection by widely used PCR primers.

ABSTRACT: While most of the spontaneous mutations in the viral genome have no functional, diagnostic, or clinical consequences, some have. In February 2021, we noticed in Southern Finland coronavirus disease 2019 cases where two commercial polymerase chain reaction (PCR) analyses failed to recognize the used N gene target but recognized the other target gene of severe acute respiratory syndrome coronavirus 2. Complete viral genome sequence analysis of the strains revealed several mutations that were not found at that time in public databases. A short 3 bp deletion and three subsequent single nucleotide polymorphisms in the N gene were found exactly at the site where an early published and widely used N gene-based PCR primer is located, explaining the negative results in the N gene PCR. Later the variant strain was identified as a member of the B.1.1.318 Pango lineage that had first been found from Nigerian samples collected in January 2021. This strain shares with the Beta variant the S gene E484K mutation linked to impaired vaccine protection, but differs from this variant in several other ways, for example by deletions in the N gene region. Mutations in the N gene causing diagnostic resistance and on the other hand E484K mutation in the causing altered infectivity warrants careful inspection on virus variants that might get underdiagnosed.

SUBMITTER: Laine P

PROVIDER: S-EPMC8661661 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The emergence of the B.1.617.2 (Delta) variant of the severe acute syndrome coronavirus (SARS-CoV-2) that emerged in 2019 (COVID-19), resulted in a surge of cases in India and has expanded and been detected across the world, including in the United States. The B.1.617.2 (Delta) variant has been seen to be twice more transmissible coupled with potential increases in disease severity and immune escape. As a result, case numbers and hospitalisations are once again on the rise in the USA. On 16 July 2021, the Centers for Disease Control and Prevention (CDC) reported a 7-day average 69.3% increase in new cases and a 35% increase in hospitalisations. Although the gold standard for SARS-CoV-2 variants identification remains genomic sequencing, this approach is not accessible to many clinical laboratories. The main goal of this study was to validate and implement the detection of the B.1.617.2 (Delta) variant utilising an open reverse transcription polymerase chain reaction (RT-PCR) platform by explicitly detecting the S-gene target failure (SGTF) corresponding to the deletion of two amino acids (ΔE156/ΔF157) characteristic of B.1.617.2 (Delta) variant. This approach was conceived as a rapid screening of B.1.617.2 (Delta) variant in conjunction with CDC's recommended N1 (nucleocapsid gene), N2, and RP (human RNase P) genes, as a pre-screening tool prior to viral genomic sequencing. We assessed 4,937 samples from 5 July to 5 September 2021. We identified the B.1.617.2 (Delta) variant in 435 of 495 positive samples (87.8%); the additional positive samples (7 samples, 1.4%) were found to belong to the B.1.1.7 (Alpha, UK) lineage and the remaining 53 samples (10.7%) were reported as 'other' lineages. Whole genome sequencing of 46 randomly selected samples validated the strains identified as positive and negative for the B.1.617.2 (Delta) variant and confirmed the S gene deletion in addition to B.1.617.2 characteristic mutations including L452R, T478K, P681R and D950N located in the spike protein. This modality has been used as routine testing at the Riverside University System Health (RUHS) Medical Center as a method for detection of B.1.617.2 (Delta) to pre-screen samples before genome sequencing. The assay can be easily implemented in clinical laboratories, most notably those with limited economic resources and access to genomic platforms.

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SARS-CoV-2 variant with mutations in N gene affecting detection by widely used PCR primers.

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