METHOD FOR DIFFERENTIATION OF YERSINIA PESTIS STRAINS BY MOLECULAR GENETIC TYPING USING IS ELEMENTS AS GENETIC MARKERS Russian patent published in 2022 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of medical biotechnology. The substance of the invention lies in the fact that eight IS markers are detected in the DNA of the strain under study on the basis of mobile genetic elements, for this, during PCR, eight tubes with a volume of 0.6 mcl are prepared, where the reaction mixture is introduced and oligonucleotide primers are introduced into each tube one pair. In this case, after PCR, the reaction mixtures from eight tubes are applied onto a 2% agarose gel and the amplified fragments are separated by electrophoresis, followed by staining with ethidium bromide in the presence of molecular weight markers and when visualized in transmitted UV light, the results are taken into account by a set of fragments with a certain molecular weight for each pair of primers and establish a unique genotype for eight IS-markers, then the obtained results are compared with the identification table and strains of Y. pestis are differentiated according to their genetic groups into main and non-main subspecies. In this case, PCR is carried out in a volume of 25 mcl and the reaction mixture contains: 10 mcl of 2.5-fold buffer, 1.0 mM MgCl₂, 0.25 mM dNTP mixture, 0.5 units. Tag DNA polymerase, 25 ng of the analyzed DNA in a volume of 5 mcl. In addition, PCR is carried out in compliance with the following stages and modes: 95°С denaturation stage - 15 s, 57°С primer annealing stage - 15 s, 72°С elongation stage - 15 s, repeat 2, 3 and 4 stages 35 times, 72°С elongation stage - 5 min, 95°С denaturation stage - 3 min.

EFFECT: invention allows to obtain individual genotypic characteristics of Yersinia pestis strains based on marker IS-loci, making it possible to differentiate strains into major and minor ones.

3 cl, 4 tbl