Development of real-time multiplex PCR for simultaneous indication of classical swine fever and Aujeski's disease viruses

Special attention should be paid to the development and implementation of innovative diagnostic methods when carrying out preventive measures, as well as operational actions in the event of outbreaks of classical swine fever (CSF) and Aujesky's disease (AD), which will allow timely detection and localization of infection foci. The purpose of this work was to design a multiplex RT-PCR test system for the diagnosis of classical swine fever and Aujesky's disease. Using bioinformatic analysis of the nucleotide sequences of the CSF virus and the pathogen of AD, the most promising sites for the indication of these pathogens were found – 139 pairs of nucleotides (bp) in size located in the region of the CSF virus genome and 135 bp in the region of the AD genome. Based on the variability of the nucleotide sequences of the selected regions, oligonucleotides were constructed that are suitable for indicating the desired viruses by RT-PCR in one tube using different fluorescence channels. Mono- and multiplex reactions were performed for each of the created combinations of oligonucleotides using a specially developed positive control, which is a genetic construct of plasmid DNA (5827 bp) containing markers for the detection of all detectable viruses and RNA/DNA samples obtained from pig biological material. Based on the results obtained, the amplification program and the optimal composition of the reaction RT-PCR mixture were determined. It was found that an individual fluorescent label corresponded to each biological pathogen. The sensitivity of the developed RTPCR test for the diagnosis of CSF and AD can reach 4 copies of DNA in the reaction mixture. When testing the multiplex RTPCR method using the strains "ShImyn" of the CSF virus and "Arsky" of the causative agent AD, the following result was obtained – an increase in the fluorescence signal along the ROX channel (Ct – 18.3) for CSF and along the R6G channel (Ct – 22.6) for AD. Thus, we have demonstrated the possibility of simultaneous PCR to identify the genomes of the desired pathogens.

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