Spoligotyping

For the detection of presence or absence of 43 spacers was done on all isolates as described by Kamerbeek, et al⁶⁹. using a commercially available kit (ISOGEN BIOSCIENCES, BV, Maarsen the Netherlands now Ocimum Biosolutions). Briefly, the direct repeat (DR) region was amplified with primer pair Dra, 5′-GGTTTTGGGTCTGACGAC-3′ (biotinylated 5′ end) and DRb, 5′-CCGAGAGGGGACGGAAAC-3′. The DNA amplification was carried out in GENEAMPPCR system 9700 of Applied Biosystems. The amplified PCR products were hybridized with nitrocellulose membrane having covalently linked 43 spacer oligonucleotides following the standard procedure⁶⁹. The hybridized fragments were detected using an enhanced chemiluminescence system (GE Healthcare, UK Ltd., Buckinghamshire, UK) and subsequent exposure in X-ray film in darkroom⁷⁰. The spoligotypes were initially reported as 43 digits binary representation of 43 spacers; one was scored for positive hybridization and zero for no hybridization.