ÓÄÊ 619:636.2:616.98:578.828.11
DOI 10.33861/2071-8020-2026-1-3-6
Original Empirical Research
Petropavlovskiy M. V., Donnik I. M., Isaeva A. G., Martynov N. A., Krivonogova A. S., Chernykh O. Yu.
Abstract. Objective. To develop a highly sensitive real-time polymerase chain reaction (qPCR) test system for the detection of bovine leukemia virus (BLV) proviral DNA.
Materials and methods. Using bioinformatic tools (MEGA, UGENE, Primer3) and an analysis of 200 complete BLV genomic sequences, specific primers and a hydrolysis probe targeting a conserved region of the proviral LTR were designed. System functionality was verified using DNA from an infected FLK cell line. qPCR was performed with an annealing temperature gradient (56-64°C). Amplification specificity was confirmed by agarose gel electrophoresis.
Research results and their discussion. A set of oligonucleotides (forward and reverse primers, probe) was developed, enabling the amplification of a specific 198 bp fragment. Stable amplification curves were obtained across the entire annealing temperature range during qPCR. Electrophoretic analysis confirmed the absence of non-specific products and primer-dimers.
Conclusion. A highly specific PCR system for the direct detection of BLV proviral DNA has been developed. The method is a promising tool 3 for the early diagnosis of infection, particularly in cases of low proviral load, the presence of maternal antibodies in calves, and animal immunotolerance, where serological methods are less informative. Further studies to assess the analytical and diagnostic sensitivity, specificity, and testing on clinical samples are required for complete system validation.
Keywords: bovine leukemia virus, proviral DNA, real-time PCR, LTR region, laboratory diagnosis, virus detection.
Author affiliation:
Petropavlovskiy Maksim V., D.Sc. in Veterinary Medicine, leading scientific researcher of the Ural Federal Agrarian Scientific Research Centre of the Ural Branch of the Russian Academy of Sciences; 112a, Belinskogo st., Ekaterinburg, 620142; e-mail: petropavlovsky_m@mail.ru.
Donnik Irina M., Academician of the Russian Academy of Sciences, D.Sc. in Biology, professor, ñhief scientific researcher of the Ural Federal Agrarian Scientific Research Centre of the Ural Branch of the Russian Academy of Sciences; 112a, Belinskogo st., Ekaterinburg, 620142; e-mail: ktqrjp7@yandex.ru.
Martynov Nikolay A., laboratory assistant of the Ural Federal Agrarian Scientific Research Centre of the Ural Branch of the Russian Academy of Sciences; 112a, Belinskogo st., Ekaterinburg, 620142; e-mail: martynov_kolya98@mail.ru.
Krivonogova Anna S., D.Sc. in Biology, associate professor, director of the University Technology Center of the Ural State Agrarian University; 42, Karla Liebknekhta, Ekaterinburg, 620000; e-mail: tel-89826512934@yandex.ru.
Chernykh Oleg Yu., D.Sc. in Veterinary Medicine, professor of the Department of Microbiology, Epizootology and Virology of the Kuban State Agrarian University named after I.T. Trubilin; 13, Kalinina st., Krasnodar, 350044; e-mail: gukkvl50@kubanvet.ru.
Responsible for correspondence with the editorial board: Isaeva Albina G., D.Sc. in Biology, associate professor, leading scientific researcher of the Ural Federal Agrarian Scientific Research Centre of the Ural Branch of the Russian Academy of Sciences; 112a, Belinskogo st., Ekaterinburg, 620142; e-mail: isaeva.05@bk.ru.
Authors’ Contribution:
Petropavlovskiy M.V.: conceptualization, investigation, formal analysis, methodology, visualization, writing – review & editing.
Donnik I.M.: conceptualization, methodology, supervision.
Isaeva A.G.: data curation, formal analysis, methodology.
Martynov N.A.: investigation, writing – original draft preparation.
Krivonogova A.S.: formal analysis, visualization.
Chernykh O.Yu.: data curation.
Conflict of Interest Statement: the authors declare no conflict of interest.
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