Development of pyrosequencing-based assay for genotyping of different coronavirus variants

BACKGROUND: The rapid spread of SARS-CoV-2 virus, which caused the COVID-19 pandemic, and the emergence of new co-circulating antigenic variants require the development and update of subtyping kits and protocols. Pyrosequencing-based protocols are promising approach for detection of single nucleotide polymorphisms. AIM: In this study we designed the assays for genotyping Variants of Concern of the SARS-CoV-2 coronavirus using polymerase chain reaction, followed by determination of the virus variant in the sample by pyrosequencing. MATERIALS AND METHODS: Pyrosequencing assays were designed based on alignment of SARS-CoV-2 sequences. Testing was performed using RNA of SARS-CoV-2 viruses of different lineages (alpha, beta, gamma, delta, and omicron). Pyrosequencing was performed using the PyroMark Q24 system. RESULTS: The protocols of sample preparation and pyrosequencing were developed and tested for sequencing of regions encoding substitutions in amino acid positions: L18F, T19R, T20N; A67V, 69-70; G142D, 143-145; 156-157, R158G; 242-244; K417N/T; L452R; S477N, T478K, E484A/K/Q; H655Y; N679K, P681H/R. The specificity of the system was also evaluated in reactions with a negative control sample (RNA isolated from human nasal swab). CONCLUSIONS: In this study, we developed and initially tested protocol for detecting coronavirus variants (alpha, beta, gamma, delta, and omicron) from samples collected from cell culture, based on the PCR technique, followed by genotyping of the variants by pyrosequencing with PyroMark Q24. The developed protocols may be adjusted to the current epidemiological situation by increasing the number of detectable sites.