Detection of Mycobacterium tuberculosis and Mycobacterium bovis from human sputum samples through multiplex PCR

ABSTRACT

Tuberculosis [TB] has a long history and being present even before the start of recording history. It has left detrimental effects on all aspect of the life and geared the developments in the science of health. TB is caused by Mycobacterium tuberculosis complex [MTBC] including five species M. tuberculosis, M. bovis, M. africanum, M. canetti, and M. microti. M. tuberculosis and M. bovis infect both animals and humans. Therefore, differentiation of these two closely related species is very important for epidemiological and management purpose. We undertook the present study to characterize mycobacteria isolated from sputum of known TB patients by conventional methods and further, by multiplex PCR [mPCR] to detect the prevalence of Zoonotic TB [TB caused by M. bovis]. Sputum samples from TB patient were collected from two tertiary care hospitals in Peshawar i.e. Lady Reading Hospital and Hayatabad Medical Complex. All the samples were subjected to Ziehl Neelsen [ZN] stain, culture on Lowenstein Jensen [LJ] and Stone Brink medium, Nitrate reduction test and multiplex PCR. A total of hundred mycobacterial strains were isolated from these samples on the basis of ZN staining, cultural and biochemical methods. Later on, these isolates were subjected to multiplex PCR by using pncATB-1.2 and pncAMT-2 primers specific to M. tuberculosis and JB21, JB22 primers specific to M. bovis. By means of conventional method, these hundred cultures isolates were differentiated into M. tuberculosis [ninety six] and M. bovis [four]. Furthermore, by mPCR, it was determined that out of hundred isolates, ninety-eight were identified as M. tuberculosis and two isolates as M. bovis. This molecular method enables to differentiate M. bovis from M. tuberculosis in human sputum