Bulletin "Veterinary biotechnology"

  • Print

Cite:  Molozhanova A.V., & Nychyk S.A. (2024). Provedennia validatsii z vyznachennia analitychnoi spetsyfichnosti izotermichnoi petlevoi amplifikatsii (RT-LAMP) dlia diahnostyky virusu SARS-CoV-2 u tvaryn [Validation to determine the analytical specificity of loop-mediated isothermal amplification (RT-LAMP) for the diagnosis of the SARS-CoV-2 virus in animals]. Veterynarna biotekhnolohiia – Veterinary biotechnology, 44, 96-102. https://doi.org/10.31073/vet_biotech44-08 [in Ukrainian].

 

MOLOZHANOVA A.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., NYCHYK S.A., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Institute of Veterinary Medicine of the NAAS

 

VALIDATION TO DETERMINE THE ANALYTICAL SPECIFICITY OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (RT-LAMP) FOR THE DIAGNOSIS OF THE SARS-COV-2 VIRUS IN ANIMALS

   Introduction. COVID-19 is the first global pandemic caused by the SARS-CoV-2 coronavirus, the third virus from the Coronaviridae family to cause fatal infections in humans after SARS-CoV and MERS-CoV.

   The COVID-19 pandemic has also affected animals. Coronaviruses can infect both humans and various species of animals, and cases of SARS-CoV-2 infection in pets, minks, zoo animals, and wildlife have been reported. Interspecies transmission of coronaviruses between different hosts forms a complex ecosystem that requires careful study.

   The emergence of the SARS-CoV-2 pandemic has highlighted the need for rapid, simple and cost-effective tests, such as RT-LAMP, to diagnose emerging pathogens.

   The goal of the work was to conduct a validation phase to determine the analytical specificity of RT-LAMP for the detection of SARS-CoV-2 virus.

   Materials and methods. Primer sets for RT-LAMP were used for the study, designed for gene regions: nucleocapsid gene N-gene and envelope gene S-gene of SARS-CoV-2.

   To study the analytical specificity of RT-LAMP, the following biological material was used: samples of isolated SARS-CoV-2 virus RNA provided by the Center for Public Health of the Ministry of Health of Ukraine, as well as RNA/DNA from three additional pathogens.

   The reaction mixture was prepared immediately before the test by mixing 10X LAMP buffer – 2.5 μL; 100 μM MgSO4 (NEB) – 1.13 μL; 10 μM dNTPs mixture – 3.5 μL; 10X primer mixture – 2.5 μL; Bst 1.0 polymerase – 1 μL; RTx - 0.5 μL; Nuclease-free water – 2.87 μL; SYBR Green solution (intercalator) – 1 μL (per sample). The isolated DNA/RNA of the experimental and control samples was added in a volume of 5.0 μL. The total volume was 25 μL.

   Research results and discussion. Analytical specificity is defined as the ability of the assay to distinguish the target DNA from the DNA of other infectious agents. According to the results of the studies, there were no cross-reactions with strains of other viruses, which indicates the specificity of the designed test system. At the same time, specific cDNA was detected in samples of biological material containing SARS-CoV-2 virus. This indicates the ability of our selected primers to accurately recognize the target site of SARS-CoV-2 virus and bind to it on the principle of complementarity.

   Conclusions and prospects for further research. The results of our studies showed the absence of nonspecific reactions with strains of additional viruses, which indicates the specificity of RT-LAMP. The obtained results indicate the possibility of its use in laboratory diagnostics.

Keywords: COVID-19, SARS-CoV-2, RT-LAMP, LAMP.

REFERENCES

  1. da Costa, V.G., Moreli, M.L., & Saivish, M.V. (2020). The emergence of SARS, MERS and novel SARS-2 coronaviruses in the 21st century. Archives of Virology, 165(7), 1517-1526. doi: 10.1007/s00705-020-04628-0.
  2. Song, Z., Xu, Y., Bao, L., Zhang, L., Yu, P., Qu, Y., Zhu, H., Zhao, W., Han, Y., Qin, C. (2019). From SARS to MERS, Thrusting Coronaviruses into the Spotlight. Viruses., 11(1), 59. https://doi.org/10.3390/v11010059.
  3. Zhu, Z., Lian, X., Su, X., Wu, W., Marraro, G.A., Zeng, Y. (2020). From SARS and MERS to COVID-19: a brief summary and comparison of severe acute respiratory infections caused by three highly pathogenic human coronaviruses. Respir Res, 21(1), 224. doi: 10.1186/s12931-020-01479-w.
  4. Zeidler, A., & Karpinski, T.M. (2020). SARS-CoV, MERS-CoV, SARS-CoV-2 Comparison of Three Emerging Coronaviruses. Jundishapur J Microbiol., 13(6), e103744. https://doi.org/10.5812/jjm.103744.
  5. Chandler, J.C., Bevins, S.N., Ellis, J.W., Linder, T.J., Tell, R.M., Jenkins-Moore, M., Root, J.J., Lenoch, J.B., Robbe-Austerman, S., DeLiberto, T.J., et al. (2021). SARS-CoV-2 exposure in wild white-tailed deer (Odocoileus virginianus). Proc Natl Acad Sci U S A., 118(47),  e2114828118. doi: 10.1073/pnas.2114828118.
  6. Cui, S.J., Liu, Y.M., Zhao, J.C., Peng, X.M., Lu, G.L., Shi, W.X., Pan, Y., Zhang, D.T., Yang, P., Wang, Q.Y. (2022). An updated review on SARS-CoV-2 infection in animals. Viruses-Basel., 14(7), 1527. doi: 10.3390/v14071527.
  7. Ferasin, L., Fritz, M., Ferasin, H., Becquart, P., Corbet, S., Gouilh, M.A., Legros, V., Leroy, E.M.. (2021). Infection with SARS-CoV-2 variant B.1.1.7 detected in a group of dogs and cats with suspected myocarditis. Vet Rec., 189(9), e944. doi: 10.1002/vetr.944.
  8. Fernandez-Bellon, H., Rodon, J., Fernandez-Bastit, L., Almagro, V., Padilla-Sole, P., Lorca-Oro, C., Valle, R., Roca, N., Grazioli, S., Trogu, T., et al. (2021). Monitoring natural SARS-CoV-2 infection in lions (Panthera leo) at the Barcelona Zoo: viral dynamics and host responses. Viruses-Basel., 13(9), 1683. doi: 10.3390/v13091683.
  9. Fritz, M., de Riols, de Fonclare D., Garcia, D., Beurlet, S., Becquart, P., Rosolen, S.G., Briend-Marchal, A., & Leroy, E.M. (2022). First evidence of natural SARS-CoV-2 infection in domestic rabbits. Vet Sci., 9(2), 49. doi: 10.3390/vetsci9020049.
  10. Gaudreault, N.N., Trujillo, J.D., Carossino, M., Meekins, D.A., Morozov, I., Madden, D.W., Indran, S.V., Bold, D., Balaraman, V., Kwon, T., et al. (2020). SARS-CoV-2 infection, disease and transmission in domestic cats. Emerg Microbes Infect., 9(1), 2322-2332. doi: 10.1080/22221751.2020.1833687.
  11. Hamer, S.A., Pauvolid-Corrêa, A., Zecca, I.B., Davila, E., Auckland, L.D., Roundy, C.M., Tang, W., Torchetti, M.K., Killian, M.L., Jenkins-Moore, M., et al. (2021). SARS-CoV-2 infections and viral isolations among serially tested cats and dogs in households with infected owners in Texas, USA. Viruses, 13(5), 938. doi: 10.3390/v13050938.
  12. Hayashi, T., Abiko, K., Mandai, M., Yaegashi, N., Konishi, I. (2020). Highly conserved binding region of ACE2 as a receptor for SARS-CoV-2 between humans and mammals. Vet Q., 40(1), 243-249. doi: 10.1080/01652176.2020.1823522.
  13. Hoppe, J.M., Füeßl, L.U., Hartmann, K., Hofmann-Lehmann, R., Graf, A., Krebs, S., Blum, H., Badell, I., Keppler, O.T., Muenchhoff, M. (2023). Secondary zoonotic dog-to-human transmission of SARS-CoV-2 suggested by timeline but refuted by viral genome sequencing. Infection., 51(1), 253-259. doi: 10.1007/s15010-022-01902-y.
  14. Gozalo, A.S., Clark, T.S., & Kurtz, D.M. (2023). Coronaviruses: Troubling Crown of the Animal Kingdom. Comp Med., 73(1), 6-44. doi: 10.30802/AALAS-CM-21-000092.
  15. Ruiz-Aravena, M., McKee, C., Gamble, A., Lunn, T., Morris, A., Snedden, C.E., Yinda, C.K., Port, J.R., Buchholz, D.W., Yeo, Y.Y., Faust, C., Jax, E., Dee, L., Jones, D.N., Kessler, M.K., Falvo, C., Crowley, D., Bharti, N., Brook, C.E., Aguilar, H.C., Peel, A.J., Restif, O., Schountz, T., Parrish, C.R., Gurley, E.S., Lloyd-Smith, J.O., Hudson, P.J., Munster, V.J., Plowright, R.K. (2022). Ecology, evolution and spillover of coronaviruses from bats. Nat Rev Microbiol., 20(5), 299-314. doi: 10.1038/s41579-021-00652-2.
  16. Hartenian, E., Nandakumar, D., Lari, A., Ly, M., Tucker, J.M., Glaunsinger, B.A. (2020). The molecular virology of coronaviruses. J Biol Chem., 295(37), 12910-12934. doi: 10.1074/jbc.REV120.013930.
  17. Liu, Q., & Wang, HY. (2021). Porcine enteric coronaviruses: an updated overview of the pathogenesis, prevalence, and diagnosis. Vet Res Commun., 45, 75-86. https://doi.org/10.1007/s11259-021-09808-0.
  18. Turlewicz-Podbielska, H., & Pomorska-Mól, M. (2021). Porcine Coronaviruses: Overview of the State of the Art. Virol. Sin., 36, 833-851 https://doi.org/10.1007/s12250-021-00364-0.
  19. Oguzoglu, T.C., Koc, B.T., & Akkutay-Yoldar, A.Z. (2021). Triple viral infections in the same cats: Feline coronavirus, feline parvovirus, feline foamy virus. Rev MVZ Cordoba, 26 (3) e2182. doi: 10.21897/rmvz.2182.
  20. Vlasova, A.N., Saif, L.J. (2021). Bovine coronavirus and the associated diseases. Front. Veterinary Sci., 8, 643220. doi: 10.3389/fvets.2021.643220.
  21. Becherer, L., Borst, N., Bakheit, M., Frischmann, S., Zengerleab, R. & von Stetten, F. (2020). Loop-mediated isothermal amplifcation (LAMP) – Review and classifcation of methods for sequence-specifc detection. Analytical Method., 12, 717-746. https://doi.org/10.1039/C9AY02246E.
  22. Notomi, T., Okayama, H., Masubuchi, H., Yonekawa, T., Watanabe, K., Amino, N. & Hase, T. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res., 28(12), e63. doi: 10.1093/nar/28.12.e63.
  23. Kubo, S., Niimi, H. & Kitajima, I. (2023). Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA. Analytical Biochemistry, 664, 115029. https://doi.org/10.1016/j.ab.2022.115029.

 Download full text in PDF