OLIGONUCLEOTIDE PRIMERS, FLUORESCENT DNA-PROBES AND METHOD FOR REVEALING THE MYCOBACTERIUM TUBERCULOSIS CLONAL COMPLEX 2-W148 OF BEIJING GENOTYPE IN CLINICAL SAMPLES

摘要

The invention relates to medicine, in particular to phthisiology, microbiology, genetics, laboratory and molecular diagnostics and concerns oligonucleotide primers, fluorescent DNA-probes and a method for revealing Mycobacterium tuberculosis of Beijing genotype of clonal complex 2-W148 (CC2-W148) using the proposed primers and probes. The invention can be used for DNA isolated from Mycobacterium tuberculosis strains and for DNA from the sputum, urine sediment, liquor, biopsy and autopsy material. A method is provided for detecting CC2-W148 using the technique of real time PCR (RT-PCR) for mutation 2541_2542delCA in kdpD gene. The method is characterized by identificating CC2-W148 by a unique deletion in kdpD revealed by specific primers kdpDF 5'- GGCGGCACGATTTCGGCTAC-3' and kdpDR 5'-TCGTCGTCAATCACCAAGACGA-3' and fluoescent-labeled probes FAM-5'-GGCGGGCTCA(LNA-C)AG(LNA-T)GGTGATC-3'-RTQ1 and R6G-5'-GGCGGGCTCA(LNA-G)TGGTGATCG(LNA-A)T-3'-BHQ2. Recording is performed by fluorescence of FAM (520 nm) and R6G (557 nm) during RT-PCR. The presence of CC2-W148 causes exponential fluorescence growth by R6G between cycles 18-38, in case of any other genotype fluorescence is recorded by FAM between cycles 18-38. The provided invention allows to reveal CC2-W148 in the test material rapidly, with high specificity.