Mutational analysis of the rpoB gene of Mycobacterium Tuberculosis Strains

Tuberculosis is an endemic. The menace of tuberculosis has affected one third of human population. Pakistan ranks 6th among high burden TB countries and 4th among the countries with highest rate of MDR-TB. Mycobacterium tuberculosis, the etiological agent of tuberculosis, has an incubation time of 8 weeks. Its fast and rapid diagnosis is required for its effective control. Molecular methods have been developed for the rapid diagnosis of TB yet the importance of conventional methods cannot be denied. LJ Culturing is considered gold standard. In the present study, a total of 90 sputum samples were taken from different cities of Punjab, Pakistan. After decontamination, the samples were subjected to LJ culturing and fluorescent microscopy. GeneXpert MTB/RIF Assay of the samples was also performed. The sensitivity of GeneXpert in comparison to culturing and microscopy was calculated. PCR of 20 rifampicin resistant samples was also performed using the primers for rpoB gene and the purified amplicons were sequenced. The sequences were analysed through various bioinformatics tools. Phylogenetic tree was constructed in comparison with wild type H37Rv MTB strain and mutations leading to rifampicin resistance were determined. The results exhibited greater sensitivity of GeneXpert MTB/RIF Assay over gold standard LJ culturing as well as fluorescent microscopy. The 49 suspected samples were found to be MTB positive with GeneXpert, 23 with microscopy and 20 with LJ culturing. Out of the 49 MTB positive samples, 23 were rifampicin resistant. Mutational analysis showed mutations in the 445th codon of RRDR of rpoB gene which inhibits the activity of RNA polymerase beta subunit. GeneXpert was found to be more sensitive than both LJ Culturing and smear microscopy. Mutational analysis showed mutations most frequently at codon 531. Mutations were also reported in other codons such as 533, 526 and 516 but these mutations were less frequent.