Abstract
The development of viral vector vaccines against brucellosis requires strict control of the authenticity of recombinant strains used as vaccine candidates. One of the key stages in the molecular characterization of such strains is confirmation of the presence of heterologous genetic inserts encoding Brucella antigens. The aim of this study was to assess the authenticity of recombinant influenza strains by detecting brucellosis-related genetic inserts using reverse transcription polymerase chain reaction (RT-PCR). The study objects were recombinant influenza virus strains Flu-NS1-80-Omp16, Flu-NS1-80-L7/L12, Flu-NS1-80-Omp19, and Flu-NS1-80-Cu-Zn SOD, containing genes encoding immunodominant Brucella proteins. Analytical sensitivity was evaluated using serial tenfold dilutions of a positive control with a known RNA concentration. The specificity of the method was confirmed by the presence of amplicons of the expected size, approximately 1250–1300 bp for recombinant constructs and approximately 890 bp for the control virus, as well as by the absence of nonspecific amplification in negative controls. During the analysis of recombinant strains Flu-NS1-80-OMP16, Flu-NS1-80-OMP19, Flu-NS1-80-SOD, and Flu-NS1-80-L7/L12, target fragments were detected in all samples, indicating successful integration of Brucella spp. inserts into the viral genome. Reproducibility assessment showed 100% concordance of results in 10 independent replicates for all tested samples. The positive and negative controls demonstrated the expected results, confirming the correct performance of the reaction and the absence of contamination. The obtained results confirm the authenticity of the recombinant influenza strains and the presence of brucellosis-related genetic inserts in their genome, indicating the correctness of the viral vector constructs and their potential for further use in the development of vaccine preparations against brucellosis.