Bulletin "Veterinary biotechnology"

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Veterynarna biotehnologija – Veterinary biotechnology, 2016, 29, 171-183 [in Ukrainian].

MUZYKINA L.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., HALKA I.V., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SYTIUK M.P., 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

ISHCHENKO L.M., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it., SPYRYDONOV V.G., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Ukrainian laboratory of quality and safety of agricultural products

VASILKIV O.B., e-mail: This email address is being protected from spambots. You need JavaScript enabled to view it.

Ternopil Regional State Laboratory of Veterinary Medicine

DEVELOPMENT AND VALIDATION OF METHOD OF VIRUS TESCHEN DISEASE RNA DETECTION BY REAL TIME PCR

Introduction. Enzootic encephalomyelitis (Teschen disease) in pigs is a contagious infectious disease of pigs with signs of CNS lesions, for laboratory diagnostics which are used as classical (RN, RIF) and modern (ELISA, PCR) methods.

The goal of the work. Develop and conduct a validation of methodology detection of RNA of Teschen disease virus in pathological material by Real-time PCR according to international requirements for use in laboratory diagnostics.

Materials and methods. The method was based on the identification of virus RNA of Teschen disease in the test sample, application of reverse transcription reaction to produce complementary DNA (cDNA), amplification of cDNA specific plot with using specific oligonucleotide primers along with Taq polymerase enzyme. We registered the result of amplification of cDNA of Teschen disease virus on FAM/Green fluorescence channel and the result of amplification of internal control sample (ICS) – on JOE/Yellow channel. We analyzed results of PCR by the presence or absence of fluorescence curve and threshold line intersection. Validation was conducted by the sensitivity, the convergence of the results, and specificity indices. We used for the sensitivity verification, virus-containing suspension of SNEV cell culture containing 1.4×109 GE/cm3 of Teschen disease virus strain «Bereznianskiy – 652». The number of virus genome equivalents per cm3. We used 8 concentrations, each one was tested in 10 fold rerun.

Results of research and discussion. Studies indicated that the confidence interval (CI) of the analytical sensitivity of the method by concentration 1.4×109–1.4×104 GE/cm3 was 100%. Only in 1.4×103 GE/cm3 concentration CI of method sensitivity was 40%, and in 1.4×102 GE/cm3 concentration – it was not sensitive at all. Limit of detection of tested test kit was 1.4×104 GE/cm3.

Conclusions and prospects for further research. Obtained results of determining of method sensitivity, specificity and affinity indicated the possibility of its use for the detection of Teschen disease RNA virus in biological material from domestic and wild pigs.

Keywords: virus, Teschen disease, validation, real time PCR.

REFERENCES

  1. Romanenko, V.P. (2001). Hvoroba Teshena (enzootychnyj encefalomijelit svynej) [Teschen disease (porcine enzootic encephalomyelitis)]. Kyiv [in Ukrainian].
  2. Romanenko, V.P., Biloshtan, V.B., Muzykina, L.M. et al. (2007). Kerovanist infekcijnym procesom pry enterovirusnyh hvorobah svynej [Controllability of infectious process of enteroviral disease in pigs]. Veterynarna Biotehnologija – Veterinary biotechnology, 10, 188–195 [in Ukrainian].
  3. Romanenko, V.P. (2013). Suchasni metody i zasoby diagnostyky ta profilaktyky hvoroby Teshena svynej [Modern methods and means of diagnosis and prevention of Teschen disease in pigs]. Veterynarna medycyna Ukrainy – Veterinary medicine of Ukraine, 1, 10–13 [in Ukrainian].
  4. Romanenko, V.P. (2010). Enterovirusy svynej i ih rol u pryrodnomu seredovyshhi [Enteroviruses of pigs and their role in the environment]. Visnyk agrarnoi nauky – Journal of Agricultural Science, 12, 27-30.
  5. Romanenko, V.P., & Opanasenko, V.N. (1972). Poshyrennja enterovirusiv svynej v umovah zony Polissja URSR [The spread of swine enteroviruses in terms of area of Polissja of Ukrainian SSR]. Mikrobiologichnyj zhurnal – Microbiological journal, 1, 121–122 [in Ukrainian].
  6. Romanenko, V.P. (1974). Hvoroba Teshena [Teschen disease]. Kyiv: Urozhaj [in Ukrainian].
  7. Romanenko, V.F., Katasonov, N.S., Petrishhe, Ju.I., & Shveb, I.V. (1974). Jenzooticheskij jencefalomielit svinej (bolezn Teshena) [Enzootic encephalomyelitis of pigs (Teschen disease)]. Veterinarija – Veterinary science, 2, 61–62 [in Russian].
  8. Romanenko, V.F., Pruss, O.G., Bokun, A.A. et al. (1993). Klassifikacija jenterovirusov svinej [Classification of porcine enterovirus]. Vіsnik agrarnoi nauki - Journal of Agricultural Science, 1, 94–101 [in Russian].
  9. Yamada, M., Kozakura, R., Ikegami, R. et al. (2004). Enterovirus encephalomyelitis in pigs in Japan caused by porcine teschovirus. Veterinary Record, 155 (10), 304–306.
  10. Sereika, V., Lelesius, R., Zienius, D. (2007). Seroprevalence of antibodies against porcine teschovirus 1 in Lithuania. Archives of Virology, 152 (5), 929–940.
  11. Prodelalova, J., Malenovska, H., Valicek, L. (2009). Genotyping of porcine teschoviruses isolated from 1960 to 1980 in the former Czechoslovakia and new Porcine teschovirus isolates obtained from piglets with diarrhoea. Veterinarni Medicina, 54 (10), 451–466.
  12. Bangari, D.S., Pogranichniy, R.M., Gillespie, T., & Stevenson, G.W. (2010). Genotyping of Porcine teschovirus from nervous tissue of pigs with and without polioencephalomyelitis in Indiana. Journal of Veterinary Diagnostic Investigation, 22, 4, 594–597.
  13. Zhang, C.-F., Cui, S.-J., Hu, S. et al. (2010). Isolation and characterization of the first Chinese strain of porcine Teschovirus-8. Journal of Virological Methods, 167 (2), 208–213.
  14. Sozzi, E., Barbieri, I., Lavazza, A. et al. (2010). Molecular characterization and phylogenetic analysis of VP1 of porcine enteric picornaviruses isolates in Italy. Transboundary and Emerging Diseases, 57 (6), 434–442.
  15. Teschovirus encephalomyelitis (previously enterovirus encephalomyelitis or Teschen / Talfan disease) (2008). OIE Terrestrial Manual, Chapter 2.8.10., 1146–1152. www.oie.int. Retrieved from http://www.oie.int/fileadmin/Home/eng/Health_standards/tahm/2.08.10TESCHOVIRUS_ENCEPH.pdf.
  16. Zell, R., Krumbholz, A., Henke, A. et al. (2000). Detection of porcine enteroviruses by nRT-PCR: differetion of CPE groups I-III specific primer sets. J. Virol. Methods, 88, 2, 205–218.
  17. Stegnіj, B.T., Gerіlovich, A.P., Limans’ka, O.Ju. et al. (2010). Polіmerazna lancjugova reakcіja u prakticі veterinarnoi medicini ta bіologіchnih doslіdzhennjah [Polymerase chain reaction in the practice of veterinary medicine and biological research]. Harkіv [in Ukrainian].
  18. Sytiuk, M.P., Spyrydonov, V.G., Postoienko, V.O. et al. (2014). Molecular and genetic characteristics isolates virus of Porcine enzooticencephalomyelitis isolated from wild boar and domestic swine on the territory of Ukraine. Bіologіja tvarin – Animal biology, 16, 4, 133–142.
  19. Muzykіna, L.M., Romanenko, V.P., Sytiuk, M.P. et al. (2015). Molekuljarno-genetichna ocіnka polіantigennih іzoljatіv enterovіrusіv svinej [Molecular and genetic evaluation of poliantyhennyh isolates of porcine enteroviruses]. Veterynarna Biotehnologija – Veterinary biotechnology, 27, 210–219 [in Ukraine].
  20. «Development and optimisation of Nucleadacid detection assays» (2014). OIE Validation Guidelines – 3.6.3.

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