Detection of Mycobacterium leprae DNA Using Nested - PCR from Paraffin - embedded Tissue Samples / 대한피부과학회지

ABSTRACT

BACKGROUND: Diagnosis of paucibacillary leprosy is difficult owing to lack of sensitive diagnostic tools. Recently, several investigators have studied the use of polymerase chain reaction(PCR) to detect Mycobacterium leprae. Using nested-PCR the sensitivity and specificity of DNA amplification is considerably improved. OBJECTIVE: The purpose of investigation is to assess the efficacy to nested-PCR which is applied to formalin-fixed, paraffin-embedded biopsies material of patients with leprosy. METHODS: Biopsy samples were taken from patients with lepromatous(11 patients) and tuberculoid (10 patients) leprosy, fixed in formalin, and embedded in paraffin. The DNA from samples was extracted and amplified through 25 cycles by using the outside pairs of primer(L1 and L2). The second amplification was allowed to proceed through 15 cycles using inside pairs of primer(L3 and L4). RESULTS: All twenty one samples showed 347-base-pair products. To confirm that the 347-bp product did correspond to the expected portion of the M. leprae groEL gene, the amplified product was digested with Pst I. Pst I digestion yielded 254-and 93-bp fragments, as predicted from the sequence of the M. leprae gene. The sensitivity was that a single organism was identified by nested-PCR. CONCLUSION: The nested-PCR is sensitive, specific, and simple diagnostic tool for leprosy.