Selection of optimal PCR conditions for the indication of the causative agent of infectious Hematopoietic necrosis

УДК 639.3.09
DOI 10.33861/2071-8020-2026-1-29-31

Original Empirical Research

Gromova E. A., Osyanin K. A., Gorbunova M. E., Dodonova E. A., Zainullin L. I.

Abstract. The infectious hematopoietic necrosis virus (IHNV) is the causative agent of one of the most dangerous fish diseases, the circulation in aquatic ecosystems of which can lead to significant economic losses in the field of aquaculture. The purpose of this work was to develop the method for identification the virus of infectious necrosis of hematopoietic tissue by PCR-RV. Two combinations of primers and probes were developed, which the targets are the G and N genes encoding the proteins VHSV – glycoprotein and nucleoprotein, respectively. Based on the pET28 plasmid vector the positive control was developed, which integrates the desired nucleotide sequence of two marker regions. As a result of a series of experiments conducted on positive control, the optimal for conducting double-focus PCR-RV the amplification program and the composition of the reaction mixture were determined. Using cDNA from heterologous viral cultures, the designed primers were found to have 100% specificity and the limit of detection of viral particles was 8.42?102 copies/ ml. To identify the causative agent of infectious necrosis of hematopoietic tissue, the use of two-step reverse transcriptase polymerase chain reaction is the most preferable because it has higher efficiency compared to one-step reaction. The developed oligonucleotide primers have all the necessary characteristics required for their use as the basis of a test system for rapid PCR-based identification and detection of IHNV. The diagnostic approach proposed in this work, using two targets – G and N genes of IHNV, will allow to identify the largest number of IHNV strains, taking into account the intraspecific genetic diversity of this virus.

Keywords: fish diseases, infectious hematopoietic necrosis, Rhabdoviridae, indication, RT-PCR-RV, molecular diagnostics, specificity, analytical sensitivity, genetic markers, primers.

Referenсes:

1. Vide supra.

2. Gromova E. A. et al. Genetic markers for PCR detection of infectious hematopoietic necrosis virus in salmonids. 2024; (5): 88-94.

3. Balakhnina K. A., Melnikov V. P. Infectious necrosis of hematopoietic tissue in salmonids (review). Veterinary Science Today. 2024; (13 (2): 124-135.

4-5. Vide supra.

6. Rudakova S. L. Features of the circulation of infectious hematopoietic necrosis virus in the sockeye salmon population of Lake Kurilskoye (Kamchatka). 2021; (63): 89-101.

7. Doronin M. I., Pylnov V. A., Mudrak N. S. Development of a real-time RT-PCR method for the detection of infectious hematopoietic necrosis virus in salmonids. 2015; (8 (10): 1052-1057.

8. Tarasova A. S., Perchun A. V., Melnikov V. P. The use of polymerase chain reaction to identify pathogens of some particularly dangerous viral diseases of fish. Veterinary Science Today. 2020; (1 (32): 11-16.

9. Vide supra.

10. Anisimova E. A. et al. Selection of optimal conditions for PCR for identification of the causative agent of canine brucellosis. 2023; (12 (230): 55-59.

11. Gromova E. A. et al. Development of a multiplex RT-PCR test system for detecting the causative agent of brucellosis. 2024; (3): 34-40.

12. Khammadov N. I. et al. Marker loci of the Brucella genome for differential PCR indication of pathogenic strains. 2018; (3): 88-93.

Author affiliation:

Osyanin Konstantin A., Ph.D. in Biology, Leading Scientific Researcher of the Laboratory of Genotyping and Certification of Strains of the Federal Center for toxicological, radiation, and biological safety; Nauchny gorodok-2, Kazan, Republic of Tatarstan, 420075; e-mail: kostja-2003@mail.ru.

Gorbunova Mariya E., Ph.D. in Biology, Scientific Researcher of the Laboratory of Molecular Genetic Analysis of the Federal Center for toxicological, radiation, and biological safety; Nauchny gorodok-2, Kazan, Republic of Tatarstan, 420075; e-mail: maria.metax@bk.ru.

Dodonova Ekaterina A., Junior Scientific Researcher of the Laboratory of Genotyping and Certification of Strains of the Federal Center for toxicological, radiation, and biological safety; Nauchny gorodok-2, Kazan, Republic of Tatarstan, 420075; e-mail: katya.dodonova.80@mail.ru.

Zajnullin Lenar I., Ph.D. in Biology, Leading Scientific Researcher of the Laboratory of Genotyping and Certification of Strains of the Federal Center for toxicological, radiation, and biological safety; Nauchny gorodok-2, Kazan, Republic of Tatarstan, 420075; e-mail: lenarilgizayn@mail.ru.

Responsible for correspondence with the editorial board: Gromova Elizaveta A., Senior Ph.D. in Biology, Scientific Researcher of the Laboratory of Molecular Genetic Analysis of the Federal Center for toxicological, radiation, and biological safety; Nauchny gorodok-2, Kazan, Republic of Tatarstan, 420075; e-mail: elizaveta-real@mail.ru.

Authors’ Contribution:

Gromova E. A.: conceptualization, formal analysis, investigation, validation, visualization, writing – original draft preparation.

Osyanin K. A.: conceptualization, data curation, writing – review and editing.

Gorbunova M. E.: investigation.

Dodonova E. A.: investigation.

Zainullin L. I.: project administration.

Conflict of Interest Statement: the authors declare no conflict of interest.