Patent RU-2734636-C1 (RU)
Title: Method for indication and identification of strains of the plague pathogen by their belonging to the species yersinia pestis, to subspecies, biovars, phylogenetic branches and by the presence of genes of pathogenicity main factors by the dna-chip method
Inventor: Nikiforov Konstantin Alekseevich
IPC: C12Q1/68
Filed: 2020-02-12
Granted: 2020-10-21
Abstract: FIELD: biotechnology. 
     SUBSTANCE: invention relates to biotechnology, namely to indication and identification of plague agent strains by their belonging to the species Yersinia pestis, to subspecies, biovars, phylogenetic branches by presence of genes of pathogenicity main factors by DNA-chip method. Method involves recovering a DNA strain, carrying out multiplex PCR in one step in six reaction mixtures on target DNA: ‘pla’, ‘3a’, ‘cafl’, ‘yihN’ in first; ‘irp2’, ‘lcrV’ in second; ‘glpD’, ‘Pro’, ‘Med24’, ‘45’ in third; ‘Med70’, ‘Phage’ in fourth; ‘Alt’, ‘His’, ‘U1’ in fifth; ‘Caucas’, ‘Tal’, ‘Micr’ in sixth with subsequent hybridisation with immobilized probes, indication and identification of strains based on the presence and absence of fluorescence signals. Fluorescence on targets "yihN" and "3a" will be observed in all strains of Y. pestis. If differentiating subspecies, there will be no fluorescence signal in the Y. pestis strains of the main subspecies on target "45", in strains of Altaic biovar of Central Asian sub-species by target "Alt", in Gissar biovar of Central Asian subspecies by "His" target, in Talas biovar of Central Asian sub-species by target "Ta1", in microtias biovar of Central Asian subspecies by target "Micr", in ulegeica subspecies strains by target "U1", strains of Caucasian subspecies by "Caucas" target. If differentiating the Y. pestis strains of the main subspecies as belonging to the biovars, there will be no fluorescence signal in the strains of Oriental biovar (1.ORI) on the "glpD" target, in all strains of medieval biovar except 2.MED0 on target "Med24" and fluorescence signals in strains of branch 2.MED0 on target "pCKP", in strains of antique biovar of the main subspecies on targets "glpD" and "Med24" will be noted. Phylogenetic branch "1" (1.ANT and 1.ORI) strains will be characterized by presence of fluorescent signal on "Phage" target, phylogenetic branch "2" (2.ANT and 2.MED) strains will be characterized by absence of fluorescence signal on "Med70" target. When detecting the main pathogenicity genes, there will be a fluorescence signal in Y. pestis strains containing an HPI high pathogenicity island in the chromosome pigmentation region of the target "irp2", in strains carrying pla gene of plasmid pPst on target "pla", in strains with gene lcrV of plasmid pCad on target "lcrV", in strains containing cafl gene of plasmid pFra on target "caf". 
     EFFECT: invention makes it possible to determine affiliation to species, subspecies, biovars, phylogenetic branches and presence of main genes of pathogenicity by DNA-chip method, which provides isolation of DNA-strain, conducting six multiplex PCR on target DNA ‘yihN’, ‘3a’, ‘45’, ‘Alt’, ‘His’, ‘U1’, ‘Caucas’, ‘Tal’, ‘Micr’, ‘glpD’, ‘Med24’, ‘Pro’, ‘Med70’, ‘Phage’, ‘pla’, ‘cafl’, ‘irp2’, ‘lcrV’, with subsequent hybridisation with immobilized probes, indication and identification of strains based on the presence and absence of fluorescence signals in accordance with the tables. 
     1 cl, 6 tbl, 9 ex