Mhase, P. P.Dalvi, S. A.2017-10-122017-10-122012http://krishikosh.egranth.ac.in/handle/1/5810032960The present investigation dealt with isolation of mycobacteria from human and animal specimens and their characterization by conventional and molecular techniques. Processing of 165 human and animal specimens resulted in recovery of 23 mycobacteria of which 2 (8.7%) were rapidly growing while 21 (91.3 %) were slow growing. On characterization by conventional biochemical tests 13 (56.5 %) isolates were found to be Mycobacterium tuberculosis whereas remaining 10 (43.5 %) were categorized as nontuberculous mycobacteria (NTM). A total of 12 isolates were recovered from 132 animal specimens with isolation rate of 9.09 % whereas, 11 isolates were recovered from 33 human sputa with an isolation rate of 33.33 %. Out of the 33 sputum samples 13 (39.39 %) were found positive for presence of acid-fast bacilli by direct microscopy. The cultural isolation was found have lower sensitivity as compared to direct microscopy. Characterization of 12 isolates from animal specimens by conventional tests revealed their species-wise distribution as; M. avium (8), M. fortuitum (2) and M. tuberculosis (2) whereas that of 11 isolates from human samples confirmed their identity as M. tuberculosis. All the 23 isolates and reference strains M. tuberculosis H37Rv, M. bovis 3/86 and M. avium were analyzed by PRA of hsp65 gene. In PCR using primers Tb11 and Tb12 amplification of 439 bp products was found in all the isolates. Restriction digestion of the amplicons with BstEII and HaeIII in separate reactions and analysis of RE digests by 3% agarose gel electrophoresis revealed distinct RFLP patterns characteristic for each species. Based on the results of PRA, 12 isolates recovered from animal specimens were identified as M. avium I (8) and M. fortuitum II and MTBC (2 each). The PRA of hsp65 could identify M. avium and M. fortuitum to subspecies level whereas it could not distinguish between M. tuberculosis and M. bovis. IS6110 PCR assay was applied on 13 isolates identified as MTBC on the basis of results of PRA of hsp65 and reference strains M. tuberculosis H37Rv and M. bovis 3/86. All the 13 isolates and reference strains yielded specific amplification product of 445 bp confirming their identity as the members of MTBC. In order to confirm species level identity of 13 MTBC isolates a single tube multiplex PCR targeting 12.7 Kb fragment was performed. All the 13 clinical isolates and Mycobacterium tuberculosis H37Rv yielded specific amplification product of 389 bp confirming their identity as M. tuberculosis. M. bovis 3/86 on the contrary revealed amplification of 823 bp product.ennullThesis