Scientists from the Federal State Budgetary Institution "ARRIAH" have developed an RT-PCR method for detecting the classical swine fever virus

Despite the fact that in a number of countries CSF has been successfully eliminated, and effective live vaccines are used to prevent infection, including in the Russian Federation, the risk of emergent transboundary introduction of infection is quite high, especially in areas bordering disadvantaged territories (for example, Primorsky Krai) . Conducting operational passive monitoring of CSF in the wild boar population using molecular genetic analysis methods will allow a timely response to potential threats to the pig farming industry in the region.

Modern test systems based on RT-PCR have a maximum analytical sensitivity of 1.5±0.5 lg CCID50/ml. Higher levels of primer affinity, target cDNA, and biostability are achieved by modifying oligonucleotides in LNA (closed nucleic acid). The relevance of the development of this technique is determined by the use of LNA primers modified to a fragment of the CSF virus genome, which made it possible to achieve high sensitivity.

To select primers, molecular genetic and phylogenetic analyzes were carried out to determine conserved regions of the genome. TaqMan probes to the target CSF fragment were matched to the green fluorescent channel (FAM fluorophore), and to the internal control sample – to crimson (Cy5.5 fluorophore).

The novelty of the methodological recommendations lies in the use of the real-time RT-PCR method with the addition of an internal control sample to detect CSF virus RNA in various biological materials and products of pork origin, in the blood of domestic pigs and wild boars, as well as the optimal selection of temperature-time reaction parameters and component composition of the reaction mixture based on domestic reagents for the purpose of effective detection of the infectious agent.