DIAGNOSTIC TECHNIQUE FOR OF MYCOBACTERIUM TUBERCULOSIS STRAIN SENSITIVITY TO AMINOGLYCOSIDES Russian patent published in 2011 - IPC C12Q1/68 C12Q1/04 C12R1/32 

FIELD: medicine.

SUBSTANCE: diagnostic technique for Mycobacterium tuberculosis (MBT) strain sensitivity to aminoglycosides involves the stages of DNA recovery, polymerase chain reaction (PCR) based on DNA amplification of an analysed and sensitive MBT strains, PCR-product restriction, separation of restriction products in agarose gel and the following analysis of restriction profiles. MBT DNA is recovered from cultures produced from a fluid medium, heated for 20 min at 95°C in TE buffer with 1% triton pH 8.8. For conducting the PCR of the preset sequence, there is used a reaction buffer containing in 50 mcl: 10 mM of Tris-HCl pH 8.8; 50 mM of KC1; 0.5% Twin-20; 5% formamide, 2.5 mM of MgCl₂, 2.5 U Taq polimerase, 200 mcM of each nucleoside triphosphate, 0.5 mcM of each oligonucleotide primer, 3 mcl of a reference. The amplification program includes: the 1st stage - 94° - 5 min; the 2nd stage - 94° - 60 sec, 55° - 60 sec, 72° - 60 sec (35 cycles); the 3rd stage - 72° - 5 min; 10° - storage. The produced PCR-products in amount 5 mcl are processed by three restrictases - BsfDEl, Btrl, Hpy99l, and restriction products are separated in 3% agarose gel coloured with ethidium bromide.

EFFECT: ensured accurate pattern of restriction analysis of detecting mutations in a rrs MBT gene site responsible for resistance to a group of aminoglycoside preparations.

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