Development and application of an RT‒PCR assay for the identification of the delta and omicron variants of SARS-COV-2.

The emergence of mutations in the coronavirus genome provides opportunities for occurrence new strains with higher transmissibility, severity and duration of the disease poses. In 2020, a new variant of the coronavirus SARS-COV-2 - Delta was identified in India. This genetic variant has spread rapidly and became dominant in many countries, including Russia. In November 2021, a new outbreak of COVID-19 occurred in Africa driven by a variant SARS-COV-2 named later Omicron. Both variants had increased transmissibility compared to previously encountered variants and quickly, replacing its around the world. To promptly monitor the epidemiological situation in the country, to assess the spread of dominant genetic variants of the virus and to take appropriate measures, we have developed an RT‒PCR reagent kit for the identification of Delta and Omicron by detecting a corresponding combination of major mutations. The minimum set of mutations was chosen which allows to differentiate Delta and Omicron variants, in order to increase the analysis productivity and reduce costs. Primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Delta and Omicron. Similar approach can be implemented for the rapid development of assays for differentiating important SARS-COV-2 variants or for other viruses genotyping for epidemiological surveillance or for diagnostic use in order to assist in making clinical decisions. It was demonstrated that the results of VOC Delta and Omicron detection and their typical mutations were concordant with genotyping based on WGS results for all 847 samples of SARS-CoV-2 RNA. The kit has high analytical sensitivity (1х103 copies/mL of SARS-CoV-2 RNA) for each of the detected genetic variants and possesses 100% analytic specificity for microorganism panel testing. The diagnostic sensitivity (95% confidence interval) obtained during pivotal trials was 91.1-100% for Omicron and 91.3-100% for Delta, while the diagnostic specificity with a 95% confidence interval was 92.2-100%. The use of a set of reagents in combination with sequencing of SARS-CoV-2 RNA as part of epidemiological monitoring made it possible to quickly track the dynamics of changes in Delta and Omicron prevalence in the Moscow region in the period from December 2021 to July 2022.

[Replication protein RepN encoded by the RC plasmid of thermophilic bacterium Thermoanaerobacterium saccharolyticum: mutational analysis and deletion mapping of domains responsible for its lethal effect]. / Replikatsionnyi belok RepN, kodiruemyi RC-plazmidoi termofil'nykh bakterii Thermoanaerobacterium saccharolyticum: mutatsionnyi analiz i deletsionnoe kartirovanie domenov, otvetstvennykh za ego letal'noe deistvie.

Amino acid sequence analysis of the product encoded by repN of Thermoanaerobacterium saccharolyticum (Clostridium thermosaccharolyticum) pNB2, which is capable of rolling-circle (RC) replication, revealed all known motifs conserved among replication (Rep) proteins that initiate RC replication of plasmids related to pC194/pUB110. Using the T7 expression system in Escherichia coli, RepN was identified as a 35K protein. Its lethal effect on bacterial cells was unusually high for a protein of the kind. Mutation analysis of the potential active centers (Y85F and Y211F) showed that the lethal effect of RepN is not associated with its putative topoisomerase (relaxase) activity. On evidence of deletion mapping, the lethal effect was attributed to the N- and C-terminal domains, each accounting for about 30% of the total protein. The RepN fragments essential for the lethal effect were found to share a motif, which showed no appreciable homology to known conserved motifs. The high lethal effect of RepN was assumed to result from duplication of the motif and to play an adaptive role, providing for the stable maintenance of the AT-rich plasmid in thermophilic bacterial cells.