PCR-SSCP analysis in detecting point mutations targeting rpoB, katG and inhA genes for determining multi-drug resistance in Mycobacterium bovis and Mycobacterium tuberculosis strains

摘要

The usefulness of polymerase chain reaction-single stranded confirmation polymorphism (PCR-SSCP) for determination of rifampicin and isoniazid resistance in Mycobacterium tuberculosis and M. bovis cultures from human and animal origin was investigated. Mycobacteria (81) in the study included, were 12 MDR-TB samples, 35 sputum samples, 3 lung and lymph node tissues from bovines and 11 M. tuberculosis and 18 M. bovis culture, M. tuberculosis H37Rv and M. bovis BCG strain. All the mycobacterial cultures were characterized on growth characteristics, biochemical test pattern, MTB complex specific IS6110 PCR and species specific 12.7 kb multiplex PCR. PCR-SSCP was used to determine resistance against rifiampin by targeting rpoB gene (305bp) and isoniazid by targeting katG (237bp) and inhA (261 bp). Rifampicin resistance was detected by PCR-SSCP in 1 out of 12 MDR-TB samples (8.3%), while isoniazid resistance was detected in 66.7% of MDR-TB samples using PCR-SSCP of katG and 75% of MDR-TB samples using inhA SSCP analysis.