DETERMINATION OF PRIMER AND PROBE SPECIFICITY IN THE DEVELOPMENT OF A MULTIPLEX REAL-TIME PCR TEST SYSTEM FOR THE DIAGNOSIS OF AVIAN INFLUENZA AND NEWCASTLE DISEASE

This study presents the results of evaluating the specificity of primers and probes developed for a multiplex real-time PCR test system designed for the detection of avian influenza and Newcastle disease. To assess specificity, RNA samples from Newcastle disease virus, avian influenza virus type A, and infectious bronchitis virus of chickens were used. As positive controls, plasmid DNAs containing fragments of the M gene (Newcastle disease virus) and NP gene (avian influenza virus) were employed, while deionized water served as a negative control. The optimized amplification program included a reverse transcription step, denaturation, and 45 cycles of real-time PCR with registration of fluorescent signals.

The results demonstrated that the developed primers and probes provided selective detection of the target genomic fragments of viruses without cross-reactions or false-positive signals. Additionally, a comparison with commercial kits PCR-GRIPP-A-FACTOR and PCR-NEWCASTLE-FACTOR confirmed the reliability and reproducibility of the obtained data.

Therefore, the developed test system is characterized by high specificity and can be recommended for practical use in veterinary diagnostics.

1. Kazakhstanskaya sel'skokhozyaystvennaya gazeta Agrariy Kazakhstana. Ptitsevodstvo Kazakhstana: sostoyanie i problemy razvitiya [Elektronnyy resurs]. – Rezhim dostupa: http://abkaz.kz/pticevodstvo-kazaxstana-sostoyanie-i-problemy-razvitiya/ (data obrashcheniya: 25.11.2020).

2. Bogoyavlenskiy A. P., Berezin V. E. Virusnye infektsii v promyshlennom ptitsevodstve i problemy ikh diagnostiki // Biotekhnologiya. – Sektsiya 2.

3. Usilenie podgotovlennosti i ukreplenie sposobnosti k otvetnym deystviyam v sluchaye vysokopatogennogo ptichego grippa (i drugikh zoonoznykh zabolevaniy, poyavlyayushchikhsya zanovo ili povtorno) v stranakh Vostochnoy Yevropy i Tsentral'noy Azii: materialy soveshchaniya, Stambul, 29 iyunya – 1 iyulya 2009 g. [Elektronnyy resurs]. – Rezhim dostupa: http://www.fao.org/docs/eims/upload/267122/ak698r00.pdf

4. Gorbunova A. S., Pysina T. V. Gripp zhivotnykh. – M., 1973.

5. Syurin V. N., Osidze N. G. Gripp ptits // Maloizvestnye zaraznye bolezni zhivotnykh. – M., 1973.

6. Adlhoch C., Gossner C., Koch G., Brown I., Bouwstra R., Verdonck F. et al. Comparing introduction to Europe of highly pathogenic avian influenza viruses A(H5N8) in 2014 and A(H5N1) in 2005 // Euro Surveill. – 2014. – Vol. 19, № 50. – P. 20996.

7. Control of Communicable Diseases Manual. – 20th Edition. – Washington DC: APHA Press, 2015. – ISBN 978-0-87553-018-5.

8. Li Q. et al. Epidemiology of Human Infections with Avian Influenza A(H7N9) Virus in China // New England Journal of Medicine. – 2014. – Vol. 370. – P. 520–532.

9. Fotina T. I. The role of the microbial monitoring in the prevention of poultry // Proceedings of XXV World Poultry Congress. – China, 2016. – P. 250–251.

10. Jhung M. A., Nelson D. I., Centers for Disease C., Prevention. Outbreaks of avian influenza A (H5N2), (H5N8), and (H5N1) among birds – United States, December 2014 – January 2015 // MMWR Morb Mortal Wkly Rep. – 2015. – Vol. 64, № 4. – P. 111.

11. World Organisation for Animal Health (OIE). Update on highly pathogenic avian influenza in animals (type H5 and H7), 2015–2016 [Elektronnyy resurs]. – Rezhim dostupa: http://www.oie.int/animal-health-in-the-world/update-on-avian-influenza/2015/

12. Geraci J. R., St. Aubin D. J., Barker I. K., Webster R. G., Hinshaw V. S., Bean W. J. et al. Mass mortality of harbor seals: pneumonia associated with influenza A virus // Science. – 1982. – Vol. 215. – P. 1129–1131.

13. CDC. Seasonal Influenza (Flu) [Elektronnyy resurs]. – Rezhim dostupa: http://www.cdc.gov/flu/

14. Lin Y. P., Shaw M., Gregory V., Cameron K., Lim W., Klimov A. et al. Avian-to-human transmission of H9N2 subtype influenza A viruses: Relationship between H9N2 and H5N1 human isolates // PNAS. – 2000. – Vol. 97, № 17. – P. 9654–9658.

15. Ito T., Gorman O. T., Kawaoka Y., Bean W. J., Webster R. G. Evolutionary analysis of the influenza A virus M gene with comparison of the M1 and M2 proteins // Journal of Virology. – 1991. – Vol. 65, № 10. – P. 5491–5498.