METHOD OF DIAGNOSING SENSITIVITY OF Mycobacterium tuberculosis STRAINS TO FLUOROCHINOLONES BY GENE gyr B

摘要

FIELD: medicine.;SUBSTANCE: method by invention includes cloning if analysed sequence in gene gyrB DNA M. tuberculosis (MBT) by method of polymerase chain reaction (PCR), with further denaturation of PCR-product in presence of denaturing solution for obtaining single-stranded DNA fragments. After that mutations are detected in them by separation of said fragments in polyacrylamide gel. In order to carry out PCR a pair of primers was selected: "external" F1 -5' AGA GTT GGT GCG GCG TAA GA 3', R1 - 5' AAC ACA TGC CCG TTC TCG AT 3' and "internal" F2 - 5' TAA GAG CGC CAC CGA CAT CG 3', R2 -5' GCA TGA ACC GGA ACA ACA AC 3', and amplification modes (1-st stage -94° - 4 min; 2-nd stage - 94° - 30 sec, 59° - 40 sec, 72° - 40 sec (25 cycles); 3-rd stage: 72° - 4 min; 10° - storage), making it possible to clone DNA M. tuberculosis from not only grown cultures, but directly from biological secretions (sputum, BAL). Denaturation of obtained amplicons is carried out in denaturing solution, destabilising double helix at heating. Electrophoretic separation of obtained single-strand fragments of DNA is carried out in 8% polyacrylamide gel with 5% glycerol with voltage 400 V during 5 hours at 8°C. Presence of mutations in examined gene is determined by comparing degree of divergence of denatured DNA strands of analysed and sensitive strains of M. tuberculosis to fluorochinolones.;EFFECT: method makes it possible to determine sensitivity of MBT to fluorochinolones in shorter terms, it is available and safe.;1 dwg