Intraspecific Differentiation of Francisella tularensis Strains Using Multilocus Real-Time Polymerase Chain Reaction

The aim of the study was to develop a method for intraspecific differentiation of the tularemia microbe: subspecies tularensis (subpopulations AI and AII), holarctica (biovars japonica, EryS/R), mediasiatica, and novicida using multilocus real-time PCR. Materials and methods. We used 48 strains of F. tularensis of various subspecies, biovars, and subpopulations. Intraspecific appurtenance of the strains was carried out on the basis of the analysis of the RD-1 region variability applying PCR, the sdhA gene by Sanger fragment sequencing and by the disk diffusion method using disks with erythromycin. The selection of primers and probes was performed using the software available at www.genscript.com and GeneRunner 6.5.52. Sequence homology was assessed using the BLAST algorithm and the GenBank NCBI database. Results and discussion. New data on the structure and occurrence of the differentiation regions RD-8, RD-12, RD-28 of FTT1122c gene and its homologous sequences in strains of tularemia microbe of various subspecies have been obtained. Novel RDhm 346 bp in size, characteristic of strains of the subsp. mediasiatica, holarctica, which is deleted in subsp. tularensis and absent in subsp. novicida has been detected. Based on the detection of the FTT1670, FTT1122с, FTT1067, FTW_2084 loci, a multilocus real-time PCR has been developed – “F. tularensis 4c”, providing for identification of all subspecies of the tularemia microbe, separately for the biovar japonica of the Holarctic subspecies and subpopulations AI, AII of the subspecies tularensis. The PCR specificity was confirmed in the study of strains of tularemia microbe from the fund of the “State Collection of Pathogenic Bacteria” at the premises of the Russian Reserarch Anti-Plague Institute “Microbe”. The results obtained expand the concept of intraspecific genetic heterogeneity of tularemia microbe and possibilities of identifying the causative agent of tularemia using molecular-genetic methods. They are important for understanding the processes of adaptation of the pathogen to circulation in the host organism and environmental objects, the course of evolution and formation of new species of Francisella.

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