Late in December 2019, an outbreak of an unknown coronavirus, later identified as SARS-CoV- 2, emerged in the city of Wuhan, China. It causes a dangerous respiratory coronavirus disease in humans - COVID-19. Objective. To detect cases of the disease and prevent its spread across the Russian Federation it is necessary to create an effective diagnostic test system. Material and methods. Based on the analysis of the alignment of the SARS-CoV-2 nucleotide sequences, primers and a probe for RT-PCR were selected, and the analysis conditions were optimized. Results. The diagnostic system was developed and registered in the shortest possible time in real-time RT-PCR format for detecting SARS-CoV-2 coronavirus RNA in smears from the nasopharynx and oropharynx, sputum and feces. The high specificity of the system was verified on a representative set of viruses and microorganisms, the analytical sensitivity was 1x103 copies / ml in smears from the mucous membrane of the nasopharynx and oropharynx and sputum, 5x104 copies / ml in fecal samples. Diagnostic sensitivity and specificity established during clinical trials on samples from patients with confirmed COVID-19 infection, from patients with a different etiology of a disease and clinically healthy people were to 100% (range 94.2-100% with a confidence level of 95%).

The emergence of novel SARS-CoV-2 genetic variants with increased transmissivity and reduced antibody neutralization efficiency is a threat to global public health. Reverse transcription polymerase chain reaction (RT-PCR) with the use of fluorescent probes, which make it possible to detect the single nucleotide substitutions, is a technique suitable for screening the SARS-CoV-2 RNA-containing samples for the already known functionally significant mutations in the S-gene, identification of which allows to define and differentiate the most epidemiologically significant genetic variants. The study was aimed to develop an assay for the large-scale monitoring of the spread of the SARS-CoV-2 top-priority variants. Based on the whole-genome alignment of the SARS-CoV-2 sequences, deposited in the GISAID database, primers and LNA-modified probes were selected to detect mutations in the S gene, typical for the Alpha, Beta/Gamma and Delta variants of concern (VOC). The developed reagent kit for detection of the key mutations in the SARS-CoV-2 S gene by the real time RT-PCR has good analytical and diagnostic characteristics and was authorized as a medical device (reagent) for in vitro use. The results of detecting the VOC and the key mutations with the use of the developed reagent kit were consistent with the data of the whole genome sequencing of 1,500 SARS-CoV-2 RNA samples. The developed reagent kit and the subsequent SARS-CoV-2 RNA sequencing assay used to perform the epidemiological monitoring of SARS-CoV-2 variants made it possible to promptly report the emergence of the Delta genetic variant in Russia, and to trace the dynamic changes in the prevalence of Delta in Moscow Region in April–September 2021.

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