USE OF PCR-MEDIATED AMPLIFICATION OF MYCOBACTERIUM-LEPRAE DNA IN DIFFERENT TYPES OF CLINICAL-SAMPLES FOR THE DIAGNOSIS OF LEPROSY

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic ac id extraction procedure, was used for standardisation of the amplifica tion of an M. leprae-specific repetitive sequence by use of the polyme rase chain reaction (PCR). With pure DNA, M. leprae-specific amplifica tion was obtained with as low as 100 ag (1 ag = 10(-18) g) of target D NA, a quantity equal to about one-tenth of the bacterial genome. Optim al processing of different types of clinical samples such as biopsy ma terial, blood and lymph fluid, from multibacillary leprosy patients, w as studied. Simple freezing-boiling cycles in the presence of Triton X 100, with some additional sample-specific modifications such as pre-tr eatment with NaOH to eliminate PCR inhibitors, was found to be suffici ent to yield amplification of bacterial DNA in samples from paucibacil lary patients. Clinical samples from 27 untreated leprosy patients, co vering the various clinical forms of the disease, and with a bacterial index ranging from 5 + to 0, were collected and processed for PCR ana lysis. After hybridisation of the amplified material with a specific s equence, 25 of 27 patients analysed gave positive results for M. lepra e in at least one of the samples. The potential of PCR for the diagnos is of leprosy is discussed.