Development and testing of the real-time polymerase chain reaction for identification and quantitative determination of the bovine respiratory syncytial virus

Nefedchenko A.V.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Glotov A.G.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Koteneva S.V.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Glotova T.I.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Development and testing of the real-time polymerase chain reaction for identification and quantitative determination of the bovine respiratory syncytial virus

Authors:

Nefedchenko A.V., Glotov A.G., Koteneva S.V., Glotova T.I.

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Journal: Molecular Genetics, Microbiology and Virology. 2020;38(3): 145‑150

DOI: 10.17116/molgen202038031145

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To cite this article:

Nefedchenko AV, Glotov AG, Koteneva SV, Glotova TI. Development and testing of the real-time polymerase chain reaction for identification and quantitative determination of the bovine respiratory syncytial virus. Molecular Genetics, Microbiology and Virology. 2020;38(3):145‑150. (In Russ.)
https://doi.org/10.17116/molgen202038031145

Подписка 2025-2 (Molecular Genetics, Microbiology and Virology)

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INTRODUCTION

Bovine respiratory syncytial virus (BRSV) is one of the most important etiological agents of respiratory diseases in calves. Rapid and reliable methods of detecting and assessing the concentrations of this pathogen are needed.

OBJECTIVE

Development of real-time PCR for detection and quantitative determination RNA of BRSV and studying the amounts it genome in the respiratory tract of sick animals.

MATERIAL AND METHODS

The nucleocapsid (N) protein gene of the virus was selected as the target for amplification. The mRNA bovine GAPDH gene was chosen as a reference gene. A panel of positive control samples of known concentration was used to calculate the amount of virus and GAPDH gene. The concentration of viral RNA in samples was quantified relative to the level of mRNA of the GAPDH gene of cattle.

RESULTS

The analytical sensitivity of PCR was 1·10³ genomic equivalents in 1 cm3 with high specificity and reproducibility. Viral RNA was detected in 19.4% of the samples (total 273). RNA of the virus more often detected in the lungs — 10.61%, less often in the mucous membrane of the trachea and bronchi, as well as in the pulmonary lymph nodes — 0.73% of positive samples. The concentration of the virus in samples was different. Maximal concentrations were determined in the lungs (1.3±0.5—4.8±0.47 log10 copies of BRSV/GAPDH RNA).

CONCLUSION

The developed kit can be used for quantitative determination of the BRSV concentration in calves and for the study of the pathogenesis of the disease, the effectiveness of vaccines or antiviral drugs for animals.

Keywords:

calves

bovine respiratory syncytial virus

quantitative polymerase chain reaction

genome

Authors:

Nefedchenko A.V.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Glotov A.G.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Koteneva S.V.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Glotova T.I.

Siberian Federal Scientific Centre of Agro-Biotechnologies of the Russian Academy of Science Institute of Experimentally Veterinary Medicine of Siberia and Far East

Received:

21.06.2019

Accepted:

15.12.2019

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