Development of a reagent kit for the detection of SARS-CoV-2 virus RNA using real-time reverse transcription polymerase chain reaction
AbstractWhen lack of therapeutics for special prophylactic and current of new and existing viral infection the means development for early reveal and identification of agent has the priority significance.
The aim of the work is to develop and test a set of reagents for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) based on reverse transcription-polymerase chain reaction in real-time.
Material and methods. The study used 2 strains of SARS-CoV-2 virus, 1 strain of middle east respiratory syndrome coronavirus (MERS-CoV), 4 strains of influenza A virus, 1 strain of respiratory syncytial virus, Haemophilus influenza type B, Human respirovirus 1, Rhinovirus and 3 strains of human coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63), from which isolated preparations of nucleic acids. The complementary deoxyribonucleic acid (cDNA) nucleotide sequences of the SARS-CoV-2 virus genomic RNA were aligned using the Muscle program (v.3.6). Identification of species-specific differences was carried out by viewing aligned nucleotide sequences. Computer analysis of chromatograms after sequencing was carried out using the Sequencher program (v.4.0.5) “Gene Codes”, USA. Sequence analysis was performed using the Genome Analysis Toolkit (v.3.6) Statistical processing of the results was carried out, the average value and standard deviation were calculated.
Results. When designing a set of reagents, most important element is selecting specific components, to which for the real-time RT-PCR the oligonucleotids primers and probes. As the gene, where the hybridization site is located, the open reading frames 1ab (OFR 1ab) gene is selected. Oligonucleotids primers and probe, used in PCR have the specific hybridization area – positions of nucleotides residues 5327–5518 in the genome of SARS-CoV-2 virus in the region of the specified gene.
Conclusion. Advantages of the tactical and technical characteristics of the developed set compared to analogues in Russia and abroad consist of the sensitivity of real time RT-PCR method by using of developed kit is 1×103 genome-equivalents per 1 sm3, that permits minimizes the risks of false negative results in the study of clinical samples from individuals with suspected COVID-19.
Keywords:reverse transcription; real-time polymerase chain reaction; set of reagents; primers; and probes; sensitivity of method; specify of method; reproduction of method; coronaviruses; SARS-CoV-2 virus; COVID-19
Funding. The study had no sponsor support.
Conflict of interest. The authors declare no conflict of interest.
Contribution. All authors took placed in collection and analyze of experimental data; elaborate the conception of conducted research – Kazantsev A.V., Borisevich S.V.; writing of text of article and carring out of statistical calculation of experimental data – Karulina N.V.; editing of text of article – Borisevich S.V.
For citation: Kazantsev A.V., Alekseev Ya.I., Chukhralya O.V., Varlamov D.A., Karulina N.V., Sizikova T.E., Paveliev D.I., Petrov A.A., Manoshkin A.V., Kuzubov A.V., Khmurenko S.N., Lebedev V.N., Kutaev D.A., Melnikov D.G., Borisevich S.V. Development of a reagent kit for the detection of SARS-CoV-2 virus RNA using real-time reverse transcription polymerase chain reaction. Infektsionnye bolezni: novosti, mneniya, obuchenie [Infectious Diseases: News, Opinions, Training]. 2023; 12 (4): 25–32. DOI: https://doi.org/10.33029/2305-3496-2023-12-4-25-32 (in Russian)
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