02278nas a2200361   4500000000100000008004100001260001300042653001800055653002300073653001900096653001100115653001200126653002400138653002500162653002500187653002800212653001000240653002800250653002500278653003000303653003200333653000900365100001500374700001600389700001400405700001500419700001600434245013500450300001200585490000700597520129800604022001401902       1993                            d  c1993 Oct10aBase Sequence10aBlotting, Southern10aDNA, Bacterial10aHumans10aleprosy10aLeprosy, Borderline10aLeprosy, lepromatous10aLeprosy, Tuberculoid10aLeukocytes, Mononuclear10aLymph10aMolecular Sequence Data10aMycobacterium leprae10apolymerase chain reaction10aSensitivity and Specificity10aSkin1 aSantos A R1 aMiranda A B1 aSarno E N1 aSuffys P N1 aDegrave W M00aUse of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy.  a298-3040 v393 a<p>DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.</p>  a0022-2615