Real-time RT-PCR diagnostics of virus causing COVID-19
E. V. Goncharova,
A. E. Donnikov,
V. V. Kadochnikova,
S. A. Morozova, 
M. N. Boldyreva,
I. S. Galkina,
D. V. Blinov
https://doi.org/10.17749/2070-4909.2020.13.1.52-63
M. N. Boldyreva,
I. S. Galkina,
D. V. Blinov 
Abstract
Aim: the study was aimed to develop a reagent kit for the real-time RT-PCR diagnostics of virus causing COVID-19.
Materials and Methods. Three target sites were chosen in the genome SARS-CoV-2. The testing included 220 samples, 48 artificially created positive samples (made from patients’ biomaterial) and 172 clinical samples (scrapes from nasal and pharyngeal cavities, bronchoalveolar lavage, expectoration, endotracheal/nasopharyngeal aspirate, feces, post-mortem material), obtained from two medical centers. Preliminary, the obtained biomaterial was analyzed with a reagent kit of comparison. The evaluation was performed with a confidential interval CI 95%. The calculation of CI for the sensitivity and specificity was made based on the distribution of χ2.
Results. The authors developed a technology of novel coronavirus infection (COVID-19) real-time RT-PCR diagnostics for the application in practical healthcare and proposed the variants of testing at all the stages (preanalytical, analytical, and post-analytical, including automated results processing). The proposed reagent kit meets the requirements of the World Health Organization and the Ministry of Healthcare of the Russian Federation. The study results demonstrated high sensitivity and specificity. The sensitivity was 100% (95% CI) 95.6–100%; the specificity was 100% (95% CI) 96.7–100%.
Conclusion. The proposed reagent kit was registered in the RF as a medical product; the registration certificate No. RZN 2020/9948 dated 01.04.2020. The application of the reagent kit in network laboratories will provide patients with access to testing for the virus causing COVID-19 and contribute to quick differential diagnostics, improvement of pandemic control, and accurate statistics on the spread of the virus.
About the Authors
E. V. Goncharova
LLC “NPF DNA-Technology”.
Russian Federation
Elena V. Goncharova – PhD, Senior Researcher
Russian Federation
Elena V. Goncharova – PhD, Senior Researcher
A. E. Donnikov
FSBI “National Medical Research Center for Obstetrics, Gynecology and Perinatology n.а. Academician V.I. Kulakov” Ministry of Healthcare of the Russian Federation
Russian Federation
Andrei E. Donnikov – MD, PhD, Head of the Laboratory of Molecular Genetic Methods
Russian Federation
Andrei E. Donnikov – MD, PhD, Head of the Laboratory of Molecular Genetic Methods
V. V. Kadochnikova
LLC “NPF DNA-Technology”
Russian Federation
Vladislava V. Kadochnikova – PhD, Senior Researcher
Russian Federation
Vladislava V. Kadochnikova – PhD, Senior Researcher
S. A. Morozova
LLC “NPF DNA-Technology”
Russian Federation
Svetlana A. Morozova – Head of Registration
Russian Federation
Svetlana A. Morozova – Head of Registration
M. N. Boldyreva
LLC “NPF DNA-Technology”
Russian Federation
Margarita N. Boldyreva – MD, Dr Sci Med, Medical Director
Russian Federation
Margarita N. Boldyreva – MD, Dr Sci Med, Medical Director
I. S. Galkina
LLC “NPF DNA-Technology”
Russian Federation
Irina S. Galkina – PhD, Marketing Director
Russian Federation
Irina S. Galkina – PhD, Marketing Director
D. V. Blinov
Institute for Preventive and Social Medicine; Lapino Clinic Hospital, MD Medical Group.
Russian Federation
Russian Federation
Dmitry V. Blinov – MD, PhD, MBA, Head of Medical and Scientific Affairs, ; Neurologist
Researcher ID: E-8906-2017
References
1. The name of the disease caused by coronavirus (COVID-19), and the viral pathogen. [Electronic resource] URL: https://www.who. int/ru/emergencies/diseases/novel-coronavirus-2019/technicalguidance/naming-the-coronavirus-disease-(covid-2019)-and-thevirus-that-causes-it. Published 2020 g. Accessed: 10.03.2020.
2. WHO. Novel Coronavirus (2019-nCoV) Situation Report-7. WHO Bull. 2020; 27 JANUARY 2020: 1–7.
3. WHO. Coronavirus disease (COVID-19) pandemic. [Electronic resource] URL: https://www.who.int/emergencies/diseases/novelcoronavirus-2019. Published 2020 g. Accessed: 10.03.2020.
4. Tang X., Wu C., Li X. et al. On the origin and continuing evolution of SARS-CoV-2. Natl Sci Rev. 2020; DOI: https://doi.org/10.1093/nsr/nwaa036.
5. Maclean O.A., Orton R., Singer J.B., Robertson D.L. Response to “On the origin and continuing evolution of SARS-CoV-2”. [Electronic resource] URL: http://virological.org/t/response-to-on-the-origin-and-continuingevolution-of-sars-cov-2/418. Published 2020 g. Accessed: 10.03.2020.
6. WHO. COVID-19 coding in ICD-10. [Electronic resource] URL: http://www9.who.int/classifications/icd/COVID-19-coding-icd10.pdf. Published 2020 g. Accessed: 26.03.2020.
7. John Hopkins University. Coronavirus Resourse Center. [Electronic resource] URL: https://coronavirus.jhu.edu/map.html. Published 2020 g. Accessed: 26.03.2020.
8. WHO. WHO Director-General’s opening remarks at the media briefing on COVID-19 - 11 March 2020. [Electronic resource] URL: https://www.who.int/dg/speeches/detail/who-director-general-sopening-remarks-at-the-media-briefing-on-covid-19---11march-2020. Accessed: 23.03.2020.
9. Decree of the Government of the Russian Federation of January 31, 2020 No. 66 “On Amending the List of Diseases of Danger to Others”. 2020. (In Russ).
10. Dong Y., Mo X., Hu Y. et al. Epidemiological Characteristics of 2143 Pediatric Patients With 2019 Coronavirus Disease in China. Pediatrics. 2020; DOI: https://doi.org/10.1542/peds.2020-0702.
11. Aylward B., Liang W. Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19). WHO-China Jt Mission Coronavirus Dis 2019. 2020; February 2019; 16–24. [Electronic resource] URL: https:// www.who.int/docs/default-source/coronaviruse/who-china-joint-missionon-covid-19-final-report.pdf. Accessed: 23.03.2020.
12. Burke R.M., Midgley C.M., Dratch A. et al. Active Monitoring of Persons Exposed to Patients with Confirmed COVID-19 – United States, January-February 2020. MMWR Morb Mortal Wkly Rep. 2020; 69 (9): 245–246. DOI: https://doi.org/10.15585/mmwr.mm6909e1.
13. Bai Y., Yao L., Wei T. et al. Presumed Asymptomatic Carrier Transmission of COVID-19. JAMA. 2020; DOI: https://doi.org/10.1001/ jama.2020.2565.
14. Liu Y., Yan L.-M., Wan L. i dr. Viral dynamics in mild and severe cases of COVID-19. Lancet Infect Dis. 2020; DOI: https://doi. org/10.1016/S1473-3099(20)30232-2.
15. Zhou F., Yu T., Du R. et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020; 395 (10229): 1054–1062. DOI: https://doi.org/10.1016/S0140-6736(20)30566-3.
16. Lauer S.A., Grantz K.H., Bi Q. et al. The Incubation Period of Coronavirus Disease 2019 (COVID-19) From Publicly Reported Confirmed Cases: Estimation and Application. Ann Intern Med. 2020; DOI: https://doi.org/10.7326/M20-0504.
17. Adhikari S.P., Meng S., Wu Y.-J. et al. Epidemiology, causes, clinical manifestation and diagnosis, prevention and control of coronavirus disease (COVID-19) during the early outbreak period: a scoping review. Infect Dis Poverty. 2020; 9 (1): 29. DOI: https://doi. org/10.1186/s40249-020-00646-x.
18. Dhama K., Sharun K., Tiwari R., Sircar S., Bhat S. Coronavirus Disease 2019 – COVID-19. 2020; [Electronic resource] URL: https:// www.preprints.org/manuscript/202003.0001. Accessed: 23.03.2020.
19. Guan W., Ni Z., Hu Y. et al. Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med. 2020; DOI: https://doi. org/10.1056/NEJMoa2002032.
20. Lai C.C., Shih T.P., Ko W.C., Tang H.J., Hsueh P.R. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus disease-2019 (COVID-19): The epidemic and the challenges. Int J Antimicrob Agents. 2020; 55 (3): 105924. DOI: https://doi.org/10.1016/j. ijantimicag.2020.105924.
21. Sun J., He W.T., Wang L. et al. COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives. Trends Mol Med. 2020: 1–13. DOI: https://doi.org/10.1016/j.molmed.2020.02.008.
22. Wang L., Wang Y., Ye D., Liu Q. A review of the 2019 Novel Coronavirus (COVID-19) based on current evidence. Int J Antimicrob Agents. 2020:105948. DOI: https://doi.org/10.1016/j.ijantimicag.2020.105948.
23. Rajgor D.D., Lee M.H., Archuleta S., Bagdasarian N., Quek S.C. The many estimates of the COVID-19 case fatality rate. Lancet Infect Dis. 2020; DOI: https://doi.org/10.1016/S1473-3099(20)30244-9.
24. Wang Y., Liu Y., Liu L., Wang X., Luo N., Ling L. Clinical outcome of 55 asymptomatic cases at the time of hospital admission infected with SARS-Coronavirus-2 in Shenzhen, China. J Infect Dis. 2020; DOI: https://doi.org/10.1093/infdis/jiaa119.
25. Hu Z., Song C., Xu C. et al. Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing, China. Sci China Life Sci. 2020; DOI: https://doi. org/10.1007/s11427-020-1661-4.
26. Temporary guidelines. Prevention, diagnosis and treatment of new coronavirus infection (COVID-19). Version 4. Ministry of Health of the Russian Federation. Moscow; 2020 (In Russ).
27. WHO. Coronavirus disease (COVID-19) technical guidance: Surveillance and case definitions. [Electronic resource] URL: https:// www.who.int/emergencies/diseases/novel-coronavirus-2019/technicalguidance/surveillance-and-case-definitions. Accessed: 12.03.2020.
28. Wang C.J., Ng C.Y., Brook R.H. Response to COVID-19 in Taiwan. JAMA. 2020; DOI: https://doi.org/10.1001/jama.2020.3151.
29. Decree of the President of the Russian Federation of March 25, 2020 No. 206 “On the announcement of non-working days in the Russian Federation” Russian Newspaper – Federal Issue. 2020; 66 (8120) (In Russ).
30. Tellis G.J., Sood A., Sood N. How Long Should Social Distancing Last? Predicting Time to Moderation, Control, and Containment of COVID-19. SSRN Electron J. 2020; DOI: https://doi.org/10.2139/ ssrn.3562996.
31. Guriev S. GDP or people’s lives. Vedomosti (In Russ). [Electronic resource] URL: https://www.vedomosti.ru/opinion/ articles/2020/03/27/826385-vvp-lyudei?fbclid=IwAR1ftEZN050VJd 3cU9mnPtx8RC3lwDNlADJVFv5D9AxanZEIqme2a0ApU4Y. Accessed: 26.03.2020.
32. Coronavirus and FMCG trends in Russia: new reality and new opportunities. Nielsen 2020. (In Russ).
33. WHO. Laboratory testing for coronavirus disease 2019 (COVID-19) in suspected human cases: interim guidance, 2 March 2020. [Electronic resource]. URL: https://apps.who.int/iris/ handle/10665/331329. Accessed: 23.03.2020
34. WHO. Coronavirus disease (COVID-19) technical guidance: Laboratory testing for 2019-nCoV in humans. [Electronic resource] URL: https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ technical-guidance/laboratory-guidance. Accessed: 12.02.2020.
35. Reagent kit for detecting 2019-nCoV coronavirus RNA by PCR with hybridization-fluorescence detection “Vektor-PTsRrv-2019-nCoVRG” po TU 21.20.23-088-05664012-2020. [Electronic resource] URL: https://nevacert.ru/reestry/med-reestr/rzn-2020-9677-41240. Accessed: 11.03.2020. (In Russ).
36. Konrad R., Eberle U., Dangel A. et al. Rapid establishment of laboratory diagnostics for the novel coronavirus SARS-CoV-2 in Bavaria, Germany, February 2020. Eurosurveillance. 2020; 25 (9). DOI: https://doi.org/10.2807/1560-7917.ES.2020.25.9.2000173.
37. Pfefferle S., Reucher S., Nörz D., Lütgehetmann M. Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Eurosurveillance. 2020; 25 (9). DOI: https://doi.org/10.2807/1560-7917.ES.2020.25.9.2000152.
38. Won J., Lee S., Park M. et al. Development of a Laboratory-safe and Low-cost Detection Protocol for SARS-CoV-2 of the Coronavirus Disease 2019 (COVID-19). Exp Neurobiol. March 2020. DOI: https:// doi.org/10.5607/en20009.
39. Registration certificate No. RZN 2020/9948 for a medical device “Reagent kit for detecting RNA of SARS-CoV coronaviruses and similar SARS-CoV by real-time reverse transcription and polymerase chain reaction (SARS-CoV-2/SARS-CoV). 2020. (In Russ).
Review
For citations:
Goncharova E.V., Donnikov A.E., Kadochnikova V.V., Morozova S.A., Boldyreva M.N., Galkina I.S., Blinov D.V. Real-time RT-PCR diagnostics of virus causing COVID-19. FARMAKOEKONOMIKA. Modern Pharmacoeconomics and Pharmacoepidemiology. 2020;13(1):52-63. (In Russ.) https://doi.org/10.17749/2070-4909.2020.13.1.52-63
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