Amplification of M. tuberculosis–specific 200-bp DNA
was done in two rounds. First step of amplification was done in a
50-μl reaction mixture, which consisted of 5 μl of 10× buffer (500
mM potassium chloride, 100 mM Tris chloride, 15 mM magnesium chloride,
gelatin 0.1%, pH 8.3), 100 ng each of primers, 200 μM of each
deoxyribonucleotide triphosphate, 1 U Taq DNA polymerase
(Bangalore Genei Pvt.), and 5 μl of DNA template. Distilled water was
added to make the volume to 50 μl. The reaction mixture was overlaid
with 50 μl of sterile mineral oil. PCR was performed in a
Perkin–Elmer automatic thermal cycler (model 480; Cetus,
Norwalk, CT). Amplification was done by using a three step profile
(i.e., denaturation for 1 minute at 94°C, annealing for 1 minute at
55°C, and extension for 1 minute at 72°C for a total of 35 cycles).
Subsequently, a second step amplification was performed using 5 μl
each of the first-round products. A reaction mixture was constructed in
the same way, except that the outer sets of primers were replaced by
the inner sets. A cycle count of 25 cycles (i.e., total of 60 cycles)
was adopted. Each set of amplification was done in the presence of two
negative controls—one for sample extraction and another as a reagent
control—and a positive control, which consisted of 5 μl M.
tuberculosis H37Rv DNA. The nPCR for each specimen was repeated
at least twice to confirm the reproducibility of the results.