Molecular characterization of mutation associated with rifampicin and isoniazid resistance in <i style="">Mycobacterium tuberculosis</i> isolates

Abstract

Nucleotide changes in catalase peroxidase (<i style="">Kat G</i>) gene and gene encoding the beta subunit of RNA polymerase (<i style="">rpo B</i>), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of <i style="">Mycobacterium tuberculosis</i> by PCR-RFLP, Line probe assay and DNA sequencing.<b style=""> </b>PCR-RFLP test was performed by HapII cleavage of an amplified fragment of <i style="">Kat G</i> gene to detect the transversion<b style=""> </b>315AGC→ACC(Ser→Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of <i style="">rpo B</i> gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test.<b style=""> </b><i style="">Kat G</i> S 315 T mutation was found in 74.19% strains of <i style="">M. tuberculosis</i> from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit<b style=""> </b>and DNA sequencing identified 18<b style=""> </b>isolates as RMP<b style=""> </b>resistant<b style=""> </b>with specific mutation,<b style=""> </b>while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the<b style=""> </b>phenotypic drug susceptibility result.<b style=""> </b>Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of<b style=""> </b><i style="">rpo B</i> gene.<b style=""> </b>Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of <i style="">M. tuberculosis</i>.