Development of a Method for Determination of brucella suis Biovars Using Multilocus Real-time PCR

The aim of the study was to develop a methodological approach to determination of Brucella suis biovars through multilocus PCR with real-time registration of results.Materials and methods. We used 16 strains of B. suis of various biovars, B. neotomae and B. canis – 2 strains of each. Determination of the taxonomic affiliation of Brucella strains was carried out according to the Bruce-ladder, Suis-ladder, BRU-DIF protocols. The selection of primers and probes was performed using the software on the website www.genscript.com and the GeneRanner 6.5.52 program. Fragment sequencing according to Sanger was performed on a 3500 XL genetic analyzer in accordance with the manufacturer’s recommendations. Nucleotide sequence homology was assessed using the BLAST algorithm and the GenBank NCBI database.Results and discussion. An analysis of the structural organization of IncP and GI-3 genomic islands has been carried out in B. suis strains of various biovars. It has been established that in strains of B. suis II, IV biovars and B. canis, the terminal part of the BRA0368 gene, comprising 21 nucleotides (repeated in the BRA0367 gene) and the “TAA” stop codon, as well as almost the entire sequence of the BRA0367 gene were lost, owing to homologous recombination in the IncP genome island. A 21-nucleotide direct repeat and the “TGA” stop codon of the BRA0367 gene replaced the analogous region of the BRA0368 gene which resulted in the deletion the size of 185 bp. No differences have been noted in the structure of GI-3 in biovars. The evidence obtained made it possible to develop the approach (SuisDIF) for differentiating B. suis biovars, based on the amplification of genes located in the IncP and GI-3 genomic islands using real-time PCR. Its specificity was confirmed in the study of B. suis strains from the fund of the State Collection of Pathogenic Bacteria of the Russian Research Anti-Plague Institute “Microbe”. The conducted studies expand and supplement the data on the genetic heterogeneity of Brucella species and biovars. The proposed method for differentiating biovars of B. suis using multilocus PCR with real-time registration of results enhances the capacities for Brucella identification using molecular-genetic methods

Molecular-Genetic Monitoring of SARS-CoV-2 Genovariants in the Territory of the Volga Federal District of the Russian Federation. Communication 1

Emergence of various genovariants of the SARS-CoV-2 virus, which are characterized by a higher ability to spread and a more severe clinical manifestations compared to the initial variants, require molecular-genetic monitoring of strains circulating in the Russian Federation.The aim of the work was to identify the VOC SARS-CoV-2 genovariants in the territory of the Republics of Bashkortostan, Tatarstan, Udmurtia, and Samara, Penza, Saratov, Ulyanovsk, and Orenburg Regions.Materials and methods. The identification of genovariants and the determination of the type of mutations was carried out by the Sanger fragment sequencing method.Results and discussion. The study examined 298 samples of clinical material obtained from the Centers for Hygiene and Epidemiology in the Republics of Bashkortostan, Tatarstan, Udmurtia, Samara, Penza, Saratov, Ulyanovsk, and Orenburg Regions. In 17 % of cases, the variability of the SARS-CoV-2 virus was observed for one or more markers: in three samples, a new coronavirus of the B. 1.1.7 line (“British”) was detected; in a number of cases, only one mutation was detected in the virus found in samples – deletion Y144 or substitution D138Y, E484K, N501Y, and very rarely two mutations – deletion Y144 and substitution E484K. The presence of the L141-G142-V143 deletion localized in the recurrent deletion region RDR2 of the S-gene was shown in 10 % of the cases. The data obtained indicate the heterogeneity in macroorganism of the population of the new coronavirus with the deletion L141-G142-V143, which leads to a change in the antigenic structure of the virus, which probably allows the virus to evade the immune response