A method for the quantitative determination of viral nucleic acids to standardize the production conditions of an intranasal vaccine against respiratory diseases of newborn calves

The main criteria for evaluating vaccines are their immunogenicity, specific activity, and safety. The aim of this work was to develop real-time PCR for the detection and quantification of viral nucleic acids in order to standardize the production conditions for an intranasal vaccine against respiratory diseases in newborn calves. Combinations of primers and probes (for each pathogen individually) were designed to identify a 97 bp region of glycoprotein G of the respiratory syncytial virus (RSV) located in the range from 4849 to 4945 bp; a 130 bp region of the PI-3 pathogen P-gene in the range from 2856 to 2985 bp; a 171 bp locus. In the region from 8271 to 8341 bp, a PKO was created and synthesized, which is a double-stranded DNA molecule (5618 bp) closed in a ring, containing a nucleotide sequence consisting of engineered primers and probes for the indication of IRT, PI-3 and IBR viruses. Using PCO, the optimal conditions for quantitative PCR-RV and the composition of the reaction mixture were determined. The minimum concentration of plasmid DNA at which successful amplification was observed was 5 pg/μL, which corresponded to a dilution of 10-10, with 826 plasmid DNA molecules per mL of solution. According to the results of amplification of the viruscontaining material, the following concentrations of pathogens were established: IBR – 0.7-1.5 million copies of DNA/ml, PI-3 – 0.1 -0.5 million copies of RNA/ml, and RSV – 20-80 thousand copies of RNA/ml. The developed method for determining the quantitative content of viral nucleic acids based on RT-PCR is a promising approach for the production of vaccines for the prevention of respiratory infections in young animals, as it ensures compliance with quality standards.

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