Genotyping of Mycobacterium tuberculosis complex based on restriction enzyme analysis of the hsp65 gene

Abstract

Several genotypes of Mycobacterium with varied degree of virulence and drug resistance exist globally. Grouping of the genotypes into principal genetic groups is critical for tuberculosis (TB) control. This study determined the suitability of polymerase chain reaction- restriction enzyme analysis (PCR-REA) technique as an epidemiological typing tool and a technique for drug resistance surveillance of TB. Seventy-five Mycobacterium tuberculosis isolates from National TB Reference Laboratory, Nigeria Institute of Medical Research (NIMR) Yaba, Lagos, Nigeria were identified and their susceptibility to isoniazid and rifampicin determined using Genotype MTBDRplus technique. Isolates were further confirmed by polymerase chain reaction (PCR) with primer specific to 65 kDa heat shock protein (hsp65) of Mycobacterium species, Fingerprinting was done by restriction enzyme (Bst Ell) analysis. Of the 75 isolates studied, 5(6.7%) were multi-drug resistant (MDR)- TB, 11(14.7%) and 7(9.3%) were mono-resistant to rifampicin and isoniazid respectively. PCR-REA profile revealed 3 different patterns. Majority (96.2%) of the susceptible strains and 100% each of mono-resistant and MDR -TB yielded 2 fragments of 250 and 120 bp. Our findings demonstrated that hsp65 kDa based molecular genotyping technique is a rapid, simple and easy-to-interpret method and may be used as a predictor of M. tuberculosis genetic variation. Key words: Genotyping, Mycobacterium tuberculosis, heat shock protein, restriction enzyme analysis, drug susceptibility.