We developed the most efficient methods for DNA preparation and PCR analysis of
Mycobacterium leprae-infected tissues. From bacterial suspension of the granuloma in
M. leprae-infected nude mouse footpad, DNA was prepared with various methods :
freezing-thawing, sonication, enzymatic digestion with proteinase K and detergent(Tween
20, SDS, Triton X-100), P/C/I extraction. Physical methods such as freezing-thawing
and sonication produced the most intense 360-bp band in agarose gel analysis following
PCR amplification.
To improve the sensitivity of PCR analysis, we applied hot-start PCR and tried to find
out the best condition of PCR, changing the temperature for denaturation and annealing
and the number of amplification cycles. The 360-bp fragment of 18-kDa protein gene,
the target sequence of PCR amplification, was known specific for M. leprae. The optimal
conditions of PCR reveals that denaturation at 90℃ annealing at 60℃, and 40 cycles of
amplifications was most effective in out lab.
The optimized method of DNA preparation and PCR analysis was very sensitive with
the lower detection limit to 101 M. leprae in routine skin biopsies taken
from leprosy patients, 28 biopsies throughout the clinical spectrum were tested. Of 13
lepromatous leprosy patients which had AFB microscopically, 92.3% were PCR-positive.
One of indeterminate leprosy patients demonstrated PCR-positive although AFB could
not be detected microscopically.
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