PCR ASSAY BASED ON DNA CODING FOR 16S RIBOSOMAL-RNA FOR DETECTION ANDIDENTIFICATION OF MYCOBACTERIA IN CLINICAL-SAMPLES

A PCR and a reverse cross blot hybridization assay were developed for the detection and identification of mycobacteria in clinical samples. The PCR amplifies a part of the DNA coding for 16S rRNA with a set of primers that is specific for the genus Mycobacterium and that flanks s pecies-specific sequences within the genes coding for 16S rRNA. The PC R product is analyzed in a reverse cross blot hybridization assay with probes specific for M. tuberculosis complex (pTub1), M. avium (pAvi3) , M. intracellulare (pInt5 and pInt7), M. kansasii complex-M. scrofula ceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smeg matis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can dete ct 10 fg of DNA, the equivalent of two mycobacteria. The specificities of the probes were tested with 108 mycobacterial strains (33 species) and 31 nonmycobacterial strains (of 17 genera). The probes pAvi3, pIn t5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the my cobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which d o not occur in clinical samples. The test was used on 31 different cli nical specimens obtained from patients suspected of having mycobacteri al disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy sa mples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed,vith those of conventional identification methods or with clinical data, showing that the test ca n be used for the direct and rapid detection and identification of myc obacteria in clinical samples.