Development of polymerase chain reaction kit for detection of SARS-CoV-2 RNA in biological samples collected from animals

Today, global attention is drawn to the samecommon problem – spread of the novelCOVID-19 infection. From the end of December 2019 novelSARS-CoV-2 virus spreadover the majority of the countriesandon 11 March 2020 the World Health Organization announced pandemic. GlobalspreadofCOVID-19 was not limited to the human population and the rewasa need to test pet sand farm animals, whowere in contactwith the humans. The reare more and morere portson SARS-CoV-2 detected in minks, ferrets, dogs, cats, tigers, lions and other animals. Today the key methodofCOVID-19 diagnosis is polymerase chain reaction, butall currently availabletest-kits are intended for the virus detection in humans. The paper demonstrates data on the development of thereal-timePCR-basedmethodforSARS-CoV-2 RNA detection in the biological samples collected from animals. During the research, an optimal system of primers and aprobe were selected, reaction conditions were tested, basic validation specifications (sensitivity, specificity, reproducibility) wereset. The validation results demonstrated that the method met all the criteria of the high-quality measurement/test method sand it can be used for diagnostic tests. Thetest-kitwas based of the method in tended for SARS-CoV-2 RNA detection in animal biological samples and it was put into the veterinary practice. Animal populations in different regions of the Russian Federation were subjected to the screening tests in order to detect the novel coronavirus genome. NoSARS-CoV-2 was reported in her bivorous animals in the Russian Federation. TheFGBI “ARRIAH” experts detected only one positive pet animal. 

1. Zhou P., Yang X. L., Wang X. G., Hu B., Zhang L., Zhang W., et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020; 579 (7798): 270–273. DOI: 10.1038/s41586-020-2012-7.

2. Gorbalenya A. E., Baker S. C., Baric R. S., de Groot R. J., Drosten C., Gulyaeva A. A. The species Severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol. 2020; 5 (4): 536–544. DOI: 10.1038/s41564-020-0695-z.

3. Kiros M., Andualem H., Kiros T., Hailemichael W., Getu S., Geteneh A., et al. COVID-19 pandemic: current knowledge about the role of pets and other animals in disease transmission. Virol. J. 2020; 17 (1):143. DOI: 10.1186/s12985-020-01416-9.

4. Ciotti M., Ciccozzi M., Terrinoni A., Jiang W. C., Wang C. B., Bernardini S. The COVID-19 pandemic. Crit. Rev. Clin. Lab. Sci. 2020; 57 (6): 365–388. DOI: 10.1080/10408363.2020.1783198.

5. Hsih W. H., Cheng M. Y., Ho M. W., Chou C. H., Lin P. C., Chi C. Y., et al. Featuring COVID-19 cases via screening symptomatic patients with epidemiologic link during flu season in a medical center of central Taiwan. J. Microbiol. Immunol. Infect. 2020; 53 (3): 459–466. DOI: 10.1016/j. jmii.2020.03.008.

6. do Vale B., Lopes A. P., Fontes M. D. C., Silvestre M., Cardoso L., Coelho A. C. Bats, pangolins, minks and other animals – villains or victims of SARS-CoV-2? Vet. Res. Commun. 2021; 45 (1): 1–19. DOI: 10.1007/s11259- 021-09787-2.

7. CuiJ., Li F., Shi Z. L. Origin and evolution of pathogenic coronaviruses. Nat. Rev. Microbiol. 2019; 17 (3): 181–192. DOI: 10.1038/s41579-018-0118-9.

8. Ning S., Yu B., WangY., Wang F. SARS-CoV-2: origin, evolution, and targeting inhibition. Front. Cell. Infect. Microbiol. 2021; 11:676451. DOI: 10.3389/ fcimb.2021.676451.

9. Wang M. Y., Zhao R., Gao L. J., Gao X. F., Wang D. P., Cao J. M. SARS-CoV-2: structure, biology, and structure-based therapeutics development. Front. Cell. Infect. Microbiol. 2020; 10:587269. DOI: 10.3389/ fcimb.2020.587269.

10. Farag E. A., Islam M. M., Enan K., El-Hussein A. M., BansalD., Haroun M. SARS-CoV-2 at the human-animal interphase: A review. Heliyon. 2021; 7 (12):e08496. DOI: 10.1016/j.heliyon.2021.e08496.

11. Nikonova Z. B., Ovchinnikova E. V., Scherbakova L. O., Kozlov A. A., Zhestkov P. D., Zinyakov N. G., et al. Vyyavlenie vozbuditelya COVID-19 u zhivotnykh v 2020–2021 gg. = COVID-19 agent detection in animals in 2020–2021. Molekulyarnaya diagnostika: X Yubileinaya mezhdunarodnaya nauchno-prakticheskaya konferentsiya (9–11 noyabrya, 2021 g.). = Molecular Diagnosis: X Anniversary International Research-to-Practice Conference (9–11 November 2021). Moscow: Yulius; 2021; 2: 379–380. eLIBRARY ID: 47983412. (in Russ.)

12. Akimоva T. P., Semakina V. P., Mitrofanova M. N., Zhiltsova M. V., Vystavkina E. S., Isakova D. G., et al. SARS-CоV-2 spread in humans and animals. Veterinary Science Today. 2021; (2): 88–96. DOI: 10.29326/2304- 196X-2021-2-37-88-96.

13. Parolin C., Virtuoso S., Giovanetti M., Angeletti S., Ciccozzi M., Borsetti A. Animal hosts and experimental models of SARS-CoV-2 infection. Chemotherapy. 2021; 66 (1–2): 8–16. DOI: 10.1159/000515341.

14. Meekins D. A., Gaudreault N. N., Richt J. A. Natural and experimental SARS-CoV-2 infection in domestic and wild animals. Viruses. 2021; 13 (10):1993. DOI: 10.3390/v13101993.

15. Michelitsch A., Wernike K., Ulrich L., Mettenleiter T. C., Beer M. SARS-CoV-2 in animals: From potential hosts to animal models. Adv. Virus. Res. 2021; 110: 59–102. DOI: 10.1016/bs.aivir.2021.03.004.

16. Sharun K., Dhama K., Pawde A. M., Gortázar C., Tiwari R., Bonilla-Aldana D. K., et al. SARS-CoV-2 in animals: potential for unknown reservoir hosts and public health implications. Vet. Q. 2021; 41 (1): 181–201. DOI: 10. 1080/01652176.2021.1921311.

17. Prince T., Smith S. L., Radford A. D., Solomon T., Hughes G. L., Patterson E. I. SARS-CoV-2 infections in animals: reservoirs for reverse zoonosis and models for study. Viruses. 2021; 13 (3):494. DOI: 10.3390/ v13030494.

18. Maurin M., Fenollar F., Mediannikov O., Davoust B., Devaux C., Raoult D. Current status of putative animal sources of SARS-CoV-2 infection in humans: wildlife, domestic animals and pets. Microorganisms. 2021; 9 (4):868. DOI: 10.3390/microorganisms9040868.

19. Hossain M. G., Javed A., Akter S., Saha S. SARS-CoV-2 host diversity: An update of natural infections and experimental evidence. J. Microbiol. Immunol. Infect. 2021; 54 (2): 175–181. DOI: 10.1016/j.jmii.2020.06.006.

20. Dhar B. C. Diagnostic assay and technology advancement for detecting SARS-CoV-2 infections causing the COVID-19 pandemic. Anal. Bioanal. Chem. 2022; 414 (9): 2903–2934. DOI: 10.1007/s00216-022-03918-7.

21. Kabir M. A., Ahmed R., Iqbal S. M. A., Chowdhury R., Paulmurugan R., Demirci U., Asghar W. Diagnosis for COVID-19: current status and future prospects. Expert Rev. Mol. Diagn. 2021; 21 (3): 269–288. DOI: 10.1080/ 14737159.2021.1894930.

22. Chen Q., He Z., Mao F., Pei H., Cao H., Liu X. Diagnostic technologies for COVID-19: a review. RSC Adv. 2020; 10 (58): 35257–35264. DOI: 10.1039/ d0ra06445a.

23. Filchakova O., Dossym D., Ilyas A., Kuanysheva T., Abdizhamil A., Bukasov R. Reviewof COVID-19 testing and diagnostic methods. Talanta. 2022; 244:123409. DOI: 10.1016/j.talanta.2022.123409.

24. CormanV. M., LandtO., KaiserM., MolenkampR., MeijerA., ChuD. K., etal. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020; 25 (3):2000045. DOI: 10.2807/1560-7917.ES.2020.25.3.2000045.

25. Long C., Xu H., Shen Q., Zhang X., Fan B., Wang C., et al. Diagnosis of the coronavirus disease (COVID-19): rRT-PCR or CT? Eur. J. Radiol. 2020; 126:108961. DOI: 10.1016/j.ejrad.2020.108961.

26. Kevadiya B. D., Machhi J., Herskovitz J., Oleynikov M. D., Blomberg W. R., Bajwa N., et al. Diagnostics for SARS-CoV-2 infections. Nat. Mater. 2021; 20 (5): 593–605. DOI: 10.1038/s41563-020-00906-z.

27. Smyrlaki I., Ekman M., Lentini A., Rufino de Sousa N., Papanicolaou N., Vondracek M., et al. Massive and rapid COVID-19 testing isfeasible by extraction-free SARS-CoV-2 RT-PCR. Nat. Commun. 2020; 11 (1):4812. DOI: 10.1038/s41467-020-18611-5.

28. Gribanov O. G., Shcherbakov A. V., Perevozchikova N. A., Gusev A. A. The use of Aerosil A-300 and GF/F (GF/C) filters for purification of DNA fragments, plasmid DNA and RNA. Biochemistry (Moscow). 1996; 61 (6): 1064–1070. Available at: https://biochemistrymoscow.com/ru/ archive/1996/61-06-1064/#_pdf. (in Russ.)

29. Nosyrev P., Nosyreva M., Rasskazova T., Korneeva N. Praktikum po GMP. Validatsiya analiticheskikh metodik: teoriya i praktika = GMP Workshop. Validation of analytical methods: theory and practice. Remedium. 2003; 10: 69–71; 11: 62–64. (in Russ.)

30. Polyakov I. V., Sokolova N. S. Practical guide on medical statistics. Leningrad: Meditsina; 1975. 151 р. (in Russ.)