Heat-killed culture isolates were used for polymerase chain reaction- (PCR-) based deletion typing. The existence or absence of RD9 was used to distinguish MTB from other MTBC species using the following primers: RD9 flank F (5′-GTG TAG GTC AGC CCC ATC C-3′), RD9 intR (5′-CTG GAC CTC GAT GAC CAC TC-3′), and RD9 flank R (5′-GCC CAA CAG CTC GAC ATC-3′). PCR amplification was performed in a thermal cycler following standard procedure [18 (link)]. The cycling conditions were 10 minutes of enzyme activation at 95°C followed by 1 minute of denaturation at 95°C, 0.5 min of annealing at 55°C, 2 min of extension at 72°C, involving a total of 35 cycles, and final elongation at 72°C for 10 min. The product was electrophoresed in a 1.5 percent agarose gel with 1× TAE (Tris-acetate-EDTA) buffer. For gel electrophoresis, a 1 : 10 ratio of ethidium bromide, a 100 base pair DNA ladder, and orange 6× loading dye were used, and the results were visualized using a transilluminator (Bio-Rad Laboratories Inc.). A band size of 396 base pairs was considered positive for MTB detection [18 (link), 19 (link)].
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