Development of a test system and a method for detecting ribonucleic acid of severe acute respiratory syndrome coronavirus 2 using real-time polymerase chain reaction

Abstract

Given the rapid spread of coronavirus disease 2019 (COVID-19) globally, test systems are needed for its diagnosis, timely treatment, and introduction of quarantine measures. In the shortest possible time, a diagnostic system based on real-time reverse-transcription polymerase chain reaction to detect the ribonucleic acid of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal and oropharyngeal smears was developed and registered. The method determines the nucleocapsid and small-membrane protein genes and the human PGK1 gene, acting as internal control reactions. The nucleotide sequences of SARS-CoV-2 were analyzed, and primers were selected. The conditions for carrying out real-time reverse-transcription polymerase chain reaction and the composition of a set of reagents were set. The diagnostic sensitivity and specificity of the kit were tested on biological samples, with the addition of inactivated SARS-CoV-2. The high analytical characteristics of the developed set of reagents were demonstrated, with a sensitivity of at least 103 GE/mL and a specificity of 100%, and no false-positive or false-negative results were recorded. The high specificity of the test system was shown on a representative sample of genetic materials of respiratory viral pathogens. Clinical and laboratory tests of the diagnostic SARS-CoV-2 test were conducted in the N.F. Gamalei National Research Center for Epidemiology and Microbiology. A set of reagents for the detection of ribonucleic acid of SARS-CoV-2 through on real-time reverse-transcription polymerase chain reaction for in vitro diagnostics SARS-CoV-2 test was registered in the Russian Federation as a medical device (Registration certificate no. RZN 2020/10632, dated 06/03/2020).