DETECTION AND CHARACTERIZATION OF ATYPICAL MYCOBACTERIA BY THE POLYMERASE CHAIN-REACTION

The purpose of this study was to develop a simple protocol of nested r eamplification polymerase chain reaction (PCR) to detect and character ize diverse mycobacterial species. DNA extracted from 126 pure mycobac terial cultures isolated from clinical specimens was amplified by nest ed PCR with use of a novel set of oligonucleotide primers specific for the 65-kDa antigen gene of mycobacteria. The PCR products were each d igested with three restriction enzymes and electrophoresed on an agaro se gel. The observed DNA fragment sizes of the different species with each enzyme were compiled into a simple algorithm. This method can rap idly detect and characterize a wide variety of mycobacterial species, including the most common pathogens Mycobacterium tuberculosis, Mycoba cterium avium-intracellulare, and Mycobacterium kansasii, without hybr idization to labeled probes. The application of this method to surgica l pathology was demonstrated by amplification and identification of at ypical mycobacteria, including M. kansasii and Mycobacterium leprae, i n formalin-fixed paraffin-embedded tissue. This protocol broadens the diagnostic potential of PCR for rapidly diagnosing mycobacterial infec tion in clinical samples, particularly in paraffin-embedded tissue sec tions.