PCR assay based on DNA coding for 16S rRNA for detection and identification of mycobacteria in clinical samples

Abstract

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 species-specific sequences within the genes coding for 16S rRNA. The PCR 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. scrofulaceum complex (pKan1), M. xenopi (pXen1), M. fortuitum (pFor1), M. smegmatis (pSme1), and Mycobacterium spp. (pMyc5a). The PCR assay can detect 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, pInt5, pInt7, pKan1, pXen1, and pMyc5a were specific. With probes pTub1, pFor1, and pSme1, slight cross hybridization occurred. However, the mycobacterial strains from which the cross-hybridizing PCR products were derived belonged to nonpathogenic or nonopportunistic species which do not occur in clinical samples. The test was used on 31 different clinical specimens obtained from patients suspected of having mycobacterial disease, including a patient with a double mycobacterial infection. The samples included sputum, bronchoalveolar lavage, tissue biopsy samples, cerebrospinal fluid, pus, peritoneal fluid, pleural fluid, and blood. The results of the PCR assay agreed with those of conventional identification methods or with clinical data, showing that the test can be used for the direct and rapid detection and identification of mycobacteria in clinical samples.

Occurrence and community composition of fast-growing Mycobacterium in soils contaminated with polycyclic aromatic hydrocarbons

“…The newly designed MYCO66f and MYCO600r primer set is the first primer set that is specifically developed for the detection of fast-growing Mycobacterium species. All previously described primer combinations were specific for the total Mycobacterium genus [22,[24][25][26][27][28][29][30][31] or exclusively for pathogenic and facultative pathogenic slow-growing mycobacteria [23,28]. DGGE analysis of gene fragments amplified with the MYCO primer set could differentiate between most fastgrowing Mycobacterium species, including all important PAH-degrading species.…”

Atypical cutaneous mycobacteriosis diagnosed by polymerase chain reaction

“…Primers and probes have been described elsewhere. 2,3 The fidelity and efficiency of DNA extraction was confirmed for each sample by amplification of a portion of intron 8-exon 8 of human genomic p53, which spans 650 bp. To control for environmental contamination by ubiquitous saprophytic mycobacteria, a negative control was used for each technical section starting from DNA extraction.…”

“…To control for environmental contamination by ubiquitous saprophytic mycobacteria, a negative control was used for each technical section starting from DNA extraction. The internal probes used for enzyme-linked immunosorbent assay (ELISA) detection were chosen according to Kox et al 2 and were linked with streptavidin-coated microplates. Four microlitres of amplicons were hybridized at 50°C for 1 h and stringently washed at 50°C with 2 · trisodium citrate-NaCl, 0AE1% sodium dodecyl sulphate.…”

Multiplex PCR assay for simultaneous detection and differentiation ofMycobacterium tuberculosis,Mycobacterium aviumcomplexes and other Mycobacterial species directly from clinical specimens

“…Not infrequently, such patients will also have multiple infections with Mycobacterium tuberculosis, M. avium and others. Several reports of isolating M. avium concomitantly with M. tuberculosis as mixed infection in HIV-positive patients have recently been published from all parts of the world including the Netherlands (Kox et al 1995), Colombia (Murcia-Aranguren et al 2001), Thailand (Mahaisavariya et al 2003), Brazil (Ruiz et al 2001;Rosas et al 2003) and India . Prophylaxis and management of disease caused by nontubercular mycobacteria differ greatly from that of M. tuberculosis.…”