Optimisation of RT-PCR for the detection of rabies virus RNA

The relevance of accurate and rapid laboratory diagnosis of rabies is determined by the high epizootic and epidemiological significance of this disease, its widespread prevalence among wild and domestic animals, as well as the need for timely and effective anti-epizootic and preventive measures. The introduction of molecular genetic diagnostic methods, which reduce the time required for laboratory confirmation of infection and increase the sensitivity of virus detection in the early stages of the disease, is of additional importance. The aim of this work was to develop and optimise a one-step reverse transcription polymerase chain reaction (RT-PCR) designed for the reliable detection of rabies virus RNA in biological material. The study used molecular biological methods, including the selection and analysis of specific primers for the N gene of the rabies virus, as well as the optimisation of the main parameters of the one-step RT-PCR. As a result of the work, RabF_N and RabR_N primers were selected, targeting a conserved region of the rabies virus N gene, which ensured high amplification specificity. Key reaction conditions were investigated and analysed, including primer annealing temperature, MgCl₂ concentration, and primer concentration in the reaction mixture. Optimal RT-PCR parameters were established, ensuring stable and reproducible amplification of the target viral RNA fragment. It was shown that the developed protocol allows for the effective detection of rabies virus RNA and can be used for laboratory confirmation of the diagnosis. The results confirm the feasibility of using one-step RT-PCR as a rapid and sensitive method of molecular diagnosis. The practical value of the work lies in the possibility of introducing the developed and optimised RT-PCR method into the routine activities of veterinary diagnostic laboratories and reference centres for the diagnosis of rabies and epizootic monitoring