Search for genetic markers for PCR-RV indication of the causative agent of viral hemorrhagic septicemia in fish

Viral hemorrhagic septicemia is one of the most dangerous fish diseases, causing significant damage to the economic development of aquaculture. In the Russian Federation, diagnostic measures to identify this pathogen are usually based on serological approaches. However, the fastest and the most accurate methods are molecular genetic diagnostic methods based on the polymerase chain reaction. The aim of this work was to develop a combination of primers and probe suitable for specific PCR indication of hemorrhagic septicemia virus in real time. At the first stage of the work, a bioinformatic analysis of the currently available nucleotide sequences of the genomes of the viral hemorrhagic septicemia causative agent was carried out. After analyzing the sequences of this virus, a marker locus of 180 bp was determined inside of the N gene encoding the nucleoprotein. Inside of this locus, primers and a probe suitable for detecting isolates of viral hemorrhagic septicemia in the real time polymerase chain reaction were developed. As a result of a series of experiments conducted on cDNAs isolated from VHSV CF 1.2 and VHSV 22-ARM virus cultures, the 1.2 and VHSV 22-ARM virus cultures, the amplification program and the composition of the reaction mixture were determined. It has been established that the developed primers have 100% specificity, and sensitivity can reach 12 copies of virus particles per microliter. Thus, the nucleotide markers selected as part of that work and the primers and probe constructed on their basis have all the characteristics necessary for their use as the basis of a test system that allows for rapid PCR indication of the virus to detect and identify the causative agent of viral hemorrhagic septicemia.