For detection of M. tuberculosis using hot-air capillary-thermocycler, FTC-2000 [Daehan Medical System Co, Korea], We amplified 188 base pair IS986 fragment from chromosomal DNA of M. tuberculosis H37Rv strain by two-step nested method, and the
concentrations of each component of polymerase chain reaction(PCR) mixture and the temperatures and times of each PCR stage were indivisually optimized.
When compared to conventional heating-block method, only one tenth of reaction volume and time were required, i. e. 10ul and 10-30 minute, respectively. Final concentrations of each component at optimal condition were 0.5uM each primer, 0.2mM
each
dNTP,
50mM Tris-HCI(pH 8.3), 500ug/ml bovine serum albumin(BSA) 4mM MgCl2 and 0.5-1U Taq polymerase per reaction. Optimal temperatures and times of each stage were 94℃ and 1 sec of predenaturation, 45-55℃ and 1 sec of annealing, 72℃ and 10-20 sec of
post-elongation, and optimal cycles of each nested-PCR were 30-50.
Among various condition of capillary-PCR, BSA, annealing temperature and MgCl2 effect most critically on PCR result. At an annealing temperature of 45℃ with 3.5mM MgCl2 or 55℃ with 3mM MgCl2, two-step nested-PCR gave greatest yield and highest
sensitivity. At these optimal condition of capillary-PCR, 0.5fg of M. tuberculosis H37Rv DNA(same amount of DNA extracted form a single tubercle bacillus) was amplified effectively.
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