Rabies remains one of the most significant zoonotic infections, characterized by high mortality. In Kazakhstan, the virus circulates among both domestic and wild animals, necessitating ongoing molecular genetic monitoring and improved laboratory diagnostic methods. The aim of this study was to investigate the genetic characteristics of the rabies virus circulating among animals in Kazakhstan using nucleoprotein (N) gene analysis and to develop a regionally adapted real-time RT-PCR method for its detection. Kazakhstani rabies virus isolates deposited in GenBank revealed that all belong to a cosmopolitan lineage and form a single phylogenetic cluster, regardless of host species. Isolates from neighboring countries, including China and Russia, were also included in this cluster, demonstrating the regional relationship of circulating virus variants. The obtained data were used to develop a real-time PCR method based on the genetic characteristics of the rabies virus circulating in Kazakhstan. The diagnostic performance of the method was evaluated using 144 samples of various origins obtained from domestic and wild animals, including bats. The developed assay demonstrated a sensitivity of 86.4%, a specificity of 98.4%, and a diagnostic efficiency of 96.5%. The proposed method ensures reliable detection of rabies virus variants circulating in Kazakhstan and can be used for laboratory diagnostics and epidemiological monitoring.

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.

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