FIELD: biotechnology.
SUBSTANCE: invention provides method of simultaneous (multiplex) amplification and fluorescent marking of DNA of several segments of the genome of mycobacteria of tuberculosis complex (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum and M. microti). Then the hybridisation or electrophoretic analysis of the sequences of these segments is carried out. The multiplex PCR is carried out in a single reaction volume with use of specific and adapter primers, the fluorescent substrate embedded into the growing DNA chain during PCR, and genomic DNA as a matrix. The multiplex PCR comprises two consecutive amplification profiles, different in annealing temperatures of specific and adapter primers by not less than 10°C. The invention enables to carry out the analysis of sequences of these genes to identify mutations associated with resistance to anti-tuberculosis preparations.
EFFECT: method according to this invention reduces the number of stages of amplifications to obtain single stranded fluorescent-labelled PCR-products, eliminates the transfer of a DNA-matrix from one reaction volume to another, which increases the stability of the procedure to contamination with PCR-products and reduces significantly the time and complexity of the analysis.
4 dwg, 1 tbl, 1 ex
