Для тестирования и диагностики обычных и новых вирусов в клинических вирусологических лабораториях требуется экономичный, точный и быстрый анализ. Нами подобраны и сконструированы олигонуклеотиды для выявления вируса гриппа типа А и коронавируса SARS-CoV-2 с применением ОТ-ПЦР-анализа. Создание отечественных тест-систем для диагностики вируса гриппа А и SARS-CoV-2 является острой задачей и обусловлено необходимостью ранней диагностики заболеваний. Целью данного исследования является подбор праймеров и зондов для диагностики гриппа А и SARS-CoV-2 методом ОТ-ПЦР в реальном времени (ОТ-ПЦР РВ). В работе представлены результаты конструирования праймеров и зондов для постановки ОТ-ПЦР РВ для идентификации РНК вирусов гриппа А и SARS-CoV-2. В наших исследованиях по подбору специфических праймеров и зондов в качестве гена мишени для обнаружения вируса гриппа А был выбран М-ген, вируса SARS-CoV-2 — RdRp. Были подобраны и синтезированы пары олигонуклеотидных праймеров для гриппа А: InfM2 F и InfM2 R, а также зонд InfM2 Probe, а для SARS-CoV-2 — RdRp-1 F и RdRp-2 R, зонд RdRp-2 Probe, которые при постановке ОТ-ПЦР РВ с рабочей концентрацией 20 пмоль показали высокую эффективность при выявлении вируса гриппа А и SARS-CoV-2. Подбор праймеров проведен с использованием компьютерных программ PrimerBlast и VectorNTI. Конструированные праймеры и зонды в дальнейшем будут использованы при создании отечественной мультиплексной ОТ-ПЦР РВ тест-системы.

Background: Brucellosis remains a significant zoonotic infection affecting approximately 500,000 people worldwide annually, with no licensed human vaccine available. Recombinant influenza virus vectors expressing Brucella spp. antigens represent a promising vaccine platform. However, transitioning from laboratory constructs to clinical candidates requires validation of scalable purification methods compliant with Good Manufacturing Practice (GMP) standards. This study aimed to develop and optimize a pilot-scale purification protocol for these vectors. Methods: Recombinant influenza A viruses (H5N1) expressing Brucella spp. antigens (Omp16, Omp19, L7/L12, Cu-Zn SOD) were propagated in MDCK cell culture. The optimized purification process included: (1) clarification; (2) ultrafiltration/diafiltration (100 kDa MWCO); (3) two-step chromatography (anion-exchange Q-Sepharose^® Fast Flow and multimodal Capto™ Core 700); and (4) sterile filtration. Process validation was performed across three independent pilot-scale batches (20 L each). Results: The purification process demonstrated high reproducibility for all constructs. Final preparations met established quality criteria: infectious titer ≥ 5.2 log₁₀ TCID₅₀/mL, hemagglutination activity 7.33 ± 0.58–8.33 ± 0.58 log₂, total protein content 157–305 μg/mL, residual host cell DNA < 10 ng/dose, and bacterial endotoxin levels ≤ 0.15 IU/mL. The overall recovery of infectious virus was 20–24%, an optimal value for multi-stage bioprocessing. Preservation of the target genetic insert was confirmed in all final preparations by PCR and sequencing. Conclusions: The developed integrated purification protocol yields vectors with high purification efficiency, preserving biological activity and meeting regulatory quality requirements for residual host cell DNA and endotoxins. The technological platform demonstrated versatility, robustness (inter-batch coefficient of variation for yield did not exceed 10–12%), and scalability, establishing a foundation for preclinical and clinical studies of candidate brucellosis vaccines.

Создание отечественной тест-системы для диагностики вируса бешенства является актуальной задачей и обусловлено необходимостью ранней диагностики заболевания. Целью данного исследования является подбор праймеров и зонда для диагностики вируса бешенства с помощью метода обратно-транскриптазной полимеразной цепной реакции в режиме реального времени (ОТ-ПЦР РВ). В работе представлены результаты конструирования и синтеза праймеров и зонда для постановки ОТ-ПЦР РВ по идентификации вируса бешенства. В ходе экспериментов были подобраны и синтезированы пара олигонуклеотидных праймеров N-RabF и N-RabR, также зонд N-Rab. При постановке ОТ-ПЦР РВ олигонуклeотидныe прaймeры N-Rab F и N-Rab R с рабочей концентрацией 20 пмоль показали высокую эффективность при выявлении вируса бешенства. Подбор прaймeров проведен с использованием компьютерных программ Primer Blast и Vector NTI. Праймeры были использованы для амплификации фрагментa гена N, кодирующего нуклеопротеин генома вируса бешенства. Конструированные прaймeры и зонд в дальнейшем будут использованы при создании отечественной ОТ-ПЦР РВ тест-системы.

Based on the results of an epizootological survey of the area and microbiological studies of pathological material from saigas brought from the West Kazakhstan region during the epizootics of 2010 and 2011, the cause of the mass death of saigas was established. Gram-positive bacteria with spores, located predominantly subterminally, were found in fingerprint smears from the internal organs of a fallen saiga. Bacteriological nutrient media show the growth of bacterial colonies in the form of intense turbidity (MPA), uniform turbidity (Kitt-Tarozzi) with abundant gas formation, colonies of small, round, smooth, colorless (blood agar). Microscopic examination of colonies on Kitt-Tarozzi medium revealed gram-positive, thick, short bacteria. White mice infected with the bacterial culture died after 16-18 hours with signs of plethora and edema. Gram-positive bacteria with rounded ends were found in fingerprint smears from the internal organs of dead mice.Based on the research, it is argued that the saiga died from anaerobic clostridiosis. The species identity of the isolated bacteria (Clostridium histolyticum and Clostridium perfringens) was determined based on the results of studying the biochemical, molecular and genetic properties of the cultures.

Rabies remains an important public health problem worldwide. The rabies epidemic situation in Kazakhstan has remained tense throughout the entire period of official registration. This pattern is typical not only for Kazakhstan, but also for many European and Asian countries.The development of a domestic test system for rabies virus diagnostics is animportant task due to the need for early detection of the disease. Determination of sensitivity and specificity, selection of optimal concentrations of each component in the reaction mixture is an important parameter in the development of the RT PCR real time test system. Optimization of the melting annealing temperature of the N-RabF and N-RabR primer pair was 55 ° C, the concentration of magnesium ions MgCl22.5 mM, the probe concentration 150-250 nM, the optimal concentration of the Enzyme mix enzyme is developed from 0.25 units. and above, the working concentration of oligonucleotide primers N-Rab F and N-Rab R is 20 pmol. The aim of the study is to improve the efficiency of rabies laboratory diagnostics, and therefore a reagent kit was developed for the detection of rabies virus RNA using the reverse transcriptase polymerase chain reaction method in real time. Such parameters of the reaction mixture as concentrations and ratio of primers, Mg2+ ions, Enzyme mix and probe were optimized

Ticks are important vectors of pathogens affecting humans and animals, posing a serious threat to health. For the first time, we studied the metagenomic profile of the microbial composition of Hyalomma scupense and Hyalomma asiaticum ticks in Kazakhstan. For microbiome analysis, 94 adult H. asiaticum and H. scupense ticks collected from cattle in Kazakhstan in 2023 were randomly selected. 16S rRNA gene sequencing was performed using the Ion Torrent NGS platform. Taxonomic classification carried out in the BV-BRC platform with the Kraken2 database. Metagenomic analysis revealed 26 bacterial genera, including both pathogenic and symbiotic taxa. In H. scupense, the dominant groups were Francisella (89.0%), Staphylococcus (76.0%), and Candidatus Midichloria (61.0%), while in H. asiaticum they were Francisella (99.0% and 95.0%) and Helcococcus (65.0%). In male H. scupense, the proportion of Francisella reached 89%, whereas in females it varied from 2% to 28%. In H. asiaticum, Helcococcus accounted for 65% in males compared to 11% in females. This is the first report on the metagenomic profile of the microbiota of H. scupense and H. asiaticum in Kazakhstan. The detection of pathogens indicates a risk of their transmission to humans and animals and highlights the need to develop new tick control strategies.