FIELD: medical microbiology and genetic engineering.
SUBSTANCE: invention relates to the field of medical microbiology and genetic engineering. The substance of the invention lies in the fact that in the method for detecting toxigenic strains 01 Vibrio cholerae of the "post-Haitian" line by real-time PCR, two amplification reactions are carried out using the designed two pairs of primers:
0.5 mcl of the 5OX SYBR Green I intercalating dye is added to the incubation mixtures. The results are recorded according to the geometric method by registering Delta Cp reflected on the monitor screen, if the difference between the amplification results of the first pair of primers Rtx01-Rtx02 and the second pair of Rtx03-Rtx04 exceeds 8 cycle units, then it is concluded that the cell of the strain under study contains the rtx A4a gene with a 60 bp deletion, therefore, it is referred to the strains of the "post-Haitian" line of toxigenic strains 01 Vibrio cholerae. In a particular case, PCR is carried out in a volume of 25 mcl and the reaction mixture contains: 2.5 mcl of buffer, 1 U of Taq DNA polymerase, 0.2 mcM dNTP mixture, 0.5 mcl of 50X SYBR Green 1, 1.0 mcM of each primer , 5 mcl DNA - DNA template, the remaining volume - water. In addition, in a particular case, PCR reactions are carried out in compliance with the following modes: denaturation at 95°C - 2 min (1 cycle); then 30 cycles: denaturation at 95°С - 20 s, annealing and accounting via the FAM channel at 67°С - 20 s.
EFFECT: invention can be used in laboratory diagnostics to quickly determine the epidemic potential of each freshly isolated strain, including the identification of the full range of unique genetic markers, allowing to establish the origin of a specific pathogen.
3 cl, 3 dwg, 3 tbl, 3 ex
