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나균 동정을 위한 Polymerase Chain Reaction의 적용
오광열,송준영 계명대학교 醫科大學 皮膚科學 1999 논문집 Vol.S No.-
The diagnosis of leprosy is often based solely on the observation of acid-fast bacilli in a lesion displaying characteristic histopathologic features. This is due .mainly to inability to cultivate the etiologic agent of leprosy, Mycobacterium leprae(H.leprae), in vitro. Serological assays and skin tests lack the required sensitivity and specificity to serve as diagnostic tools for M.1eprae infection. This study applied po1ymerase chain reaction(PCR) on sections of paraffin-fixed, frozen biopsy samples and periphera1 blood from 1eprosy patients. In agarose ge1 and Southern b10t analysis of PCR-amplified products , amplification of the 530-bp fragment by PCR showed the fragment to be specifica11y amplified from purified genomic H. leprae DNA. The sensitivity of the PCR with primer S13 and S62 was determind by adding to the reaction mixtures DNA extracted from 20μl samp1es of sequentia1 2-fo1d di1ution of a suspension containing 8X101 bacteria μ1?¹. This indicated that the PCR described here is much more sensitive than other methods for the direct detection of H. leprae, such as micriscopic visua1ization and DNA hybridization. The use of primers S13 and S62 . resu1ted in the specific amplification of M. leprae DNA. No dectectab1e amplification occured with DNA from purified M. tuberculosis or λ bacteriophage. The size of the amp1ified fragment was 530-bp, which is in agreement with the position.