Development of an Integrated System for Molecular-Genetic Identification of Yersinia pestis Strains

The paper describes a developed comprehensive system for molecular-genetic identification of Yersinia pestis strains according to their appurtenance to certain subspecies, biovars, phylo-geographic populations, using realtime PCR (RT-PCR), allele-specific RT-PCR, and multiplex PCR with hybridization fluorescent registration of results on a solid substrate. Application of this system makes it possible to establish the appurtenance of Y. pestis strains to the following phylogenetic branches: 0.ANT1, 0.ANT2, 0.ANT3, 0.ANT5, 3.ANT, 4.ANT of antique biovar of the main subspecies; 2.MED0, 2.MED1, 2.MED2, 2.MED3, 2.MED4 of medieval biovar of the main subspecies; 1.IN1, 1.IN2, 1.IN3 of intermedium biovar of the main subspecies; 1.ORI1, 1.ORI2, 1.ORI3 of oriental biovar of the main subspecies; 0.PE3 (angolica subspecies), 0.PE7 (tibetica subspecies) and 0.PE10 (qinghaica subspecies). The first stage of the studies within the frames of the developed system is indication of plague agent using registered diagnostic drugs. The second stage is the determination of belonging to individual subspecies through RT-PCR or by the method of multiplex PCR system with hybridization-fluorescent registration of results on a solid substrate, which also allows for establishing to which biovars of the main subspecies and the main phylogenetic lines of the ancient biovar the strains belong. The third stage is the identification of strain appurtenance to phylogenetic branches by the AS-RT-PCR method. The designed complex system for molecular-genetic identification of Y. pestis strains can be applied at the regional and federal levels of the laboratory network of the Russian Federation for diagnostics of infectious diseases. Its use will considerably facilitate and increase the efficiency of intraspecific differentiation of Y. pestis strains within the framework of the epidemiological investigation of outbreaks or importation of strains of plague pathogen into the territory of the Russian Federation or during the certification of strains in collection activities.

1. Kutyrev V.V., Eroshenko G.A., Motin V.L., Nosov N.Y., Krasnov J.M., Kukleva L.M., Nikiforov K.A., Al’khova Z.V., Oglodin E.G., Guseva N.P. Phylogeny and classification of Yersinia pestis through the lens of strains from the plague foci of Commonwealth of Independent States. Front. Microbiol. 2018; 9:1106. DOI: 10.3389/fmicb.2018.01106.

2. Achtman M., Zurth K., Morelli G., Torrea G., Guiyoule A., Carniel E. Yersinia pestis, the cause of plague, is a recently emerged clone of Yersinia pseudotuberculosis. Proc. Natl Acad. Sci. USA. 1999; 96(24):14043–8. DOI: 10.1073/pnas.96.24.14043.

3. Morelli G., Song Y., Mazzoni C.J., Eppinger M., Roumagnac P., Wagner D.M., Feldkamp M., Kusecek B., Vogler A.J., Li Y., Cui Y., Thomson N.R., Jombart T., Leblois R., Lichtner P., Rahalison L., Petersen J.M., Balloux F., Keim P., Wirth T., Ravel J., Yang R., Carniel E., Achtman M. Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity. Nat. Genet. 2010; 42(12):1140–3. DOI: 10.1038/ng.705.

4. Cui Y., Yu C., Yan Y., Li D., Li Y., Jombart T., Weinert L.A., Wang Z., Guo Z., Xu L., Zhang Y., Zheng H., Qin N., Xiao X., Wu M., Wang X., Zhou D., Qi Z., Du Z., Wu H., Yang X., Cao H., Wang H., Wang J., Yao S., Rakin A., Li Y., Falush D., Balloux F., Achtman M., Song Y., Wang J., Yang R. Historical variations in mutation rate in an epidemic pathogen, Yersinia pestis. Proc. Natl Acad. Sci. USA. 2013; 110(2):577–82. DOI: 10.1073/pnas.1205750110.

5. Zhang Y., Luo T., Yang C., Yue X., Guo R., Wang X., Buren M., Song Y., Yang R., Cao H., Cui Y., Dai X. Phenotypic and molecular genetic characteristics of Yersinia pestis at an emerging natural plague focus, Junggar Basin, China. Am. J. Trop. Med. Hyg. 2018; 98(1):231–7. DOI: 10.4269/ajtmh.17-0195.

6. Shi L., Qin J., Zheng H., Guo Y., Zhang H., Zhong Y., Yang C., Dong S., Yang F., Wu Y., Zhao G., Song Y., Yang R., Wang P., Cui Y. New genotype of Yersinia pestis found in live rodents in Yunnan Province, China. Front. Microbiol. 2021; 12:628335. DOI: 10.3389/fmicb.2021.628335.

7. Motin V.L., Georgescu A.M., Elliott J.M., Hu P., Worsham P.L., Ott L.L., Slezak T.R., Sokhansanj B.A., Regala W.M., Brubaker R.R., Garcia E. Genetic variability of Yersinia pestis isolates as predicted by PCR-based IS100 genotyping and analysis of structural genes encoding glycerol-3-phosphate dehydrogenase (glpD). J. Bacteriol. 2002; 184(4):1019–27. DOI: 10.1128/jb.184.4.1019-1027.2002.

8. Soleimani V.D., Baum B.R., Johnson D.A. Efficient validation of single nucleotide polymorphisms in plants by allele-specific PCR, with an example from barley. Plant Mol. Biol. Rep. 2003; 21:281–8. DOI: 10.1007/BF02772803.

9. Nikiforov K.A., Oglodin E.G., Kukleva L.M., Eroshenko G.A., Germanchuk V.G., Devdariani Z.L., Kutyrev V.V. [Subspecies differentiation of Yersinia pestis strains by PCR with hybridizationfluorescence detection]. Zhurnal Mikrobiologii, Epidemiologii, Immunobiologii [Journal of Microbiology, Epidemiology, Immunobiology]. 2017; (2):22–7. DOI: 10.36233/0372-9311-2017-2-22-27.

10. Nikiforov K.A., Utkin D.V., Makashova M.A., Kukleva L.M., Eroshenko G.A., Kutyrev V.V. [Designing a system of multiplex PCR with hybridization-fluorescent registration of results, on a solid substrate, for indication and identification of Y. pestis strains]. Biotekhnologiya [Biotechnology]. 2020; 36(3):46–56. DOI: 10.21519/0234-2758-2020-36-3-46-56.

11. Nikiforov K.A., Oglodin E.G., Kukleva L.M., Makashova M.A., Balykova A.N., Eroshenko G.A., Kutyrev V.V. [Construction of a real-time allele-specific PCR system for determining the phylogenetic affiliation of Yersinia pestis strains]. Biotekhnologiya [Biotechnology]. 2022; 38(3):82–91. DOI: 10.56304/S0234275822030073.