DNA Fingerprinting of Mycobacterium tuberculosis Isolates in Nepal Using PCR-Labeled Is 6110 Probe

Abstract

Restriction Fragment Length Polymorphism (RFLP) using IS6110 probe has been taken as gold standard DNA fingerprinting technology and used all over the world quite successfully to characterize My cobacterium tuberculosis strains. The present study has been carried out to study genomic polymorphism among M. tuberculosis isolates collected from patients attending clinics at two different tuberculosis centers of Kathmandu.

M. tuberculosis isolates were collected from pure cultured specimen on Ogawa slants, in sterilized TE buffer pH 8.0, heat killed and brought to MRL, Lele. DNA from these isolates were extracted and purified by physiochemical method, restricted with PvuII enzyme and hybridized with PCR amplified and DIG labeled 245bp IS6110 probe. Fingerprinting patterns were inspected visually. Among 59 isolates analyzed, 4 isolates were observed to have no copy, 3 had single copy, 9 had 2-5 copies, 23 had 6-12 copies and 20 had 13-17 copies of IS6110 in their genome. For the purpose of analysis, the patients were divided in to Aryans and Mongols. The low copy numbered strains (≤5 copies) were more common among Aryans while opposite was true for the Mongols. Excluding isolates with one copy or loss of IS6110, 23 % of isolates were clustered. All together 48 different fingerprinting patterns were observed showing 80 % of genomic variations amongst isolates. The isolates of the largest cluster were found more commonly in the younger age groups, females, and in patients from the central region. DNA fingerprinting using IS6110 probe was found to be quite discriminating for molecular typing of most (73 %) of strains which harboured ≥6 copies of IS6110. A larger number of isolates from defined geographical area need to be studied to understand molecular epidemiology of M. tuberculosis in Nepal.

Key words - RFLP, IS6110, M. tuberculosis, PvuII, Nepal