Evaluation of nonradioactive DNA probes for identification of mycobacteria

Lebrun, L; Espinasse, F.; Poveda, Jean-Dominique; Vincent-Lévy-Frébault, Véronique

doi:10.1128/jcm.30.9.2476-2478.1992

Cited by 145 publications

(56 citation statements)

References 13 publications

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“…By conventional biochemical methods (52,57), the identification of the species to which the cultured mycobacterial strain belongs may require an additional 2 to 4 weeks. More rapid methods for the identification of cultured mycobacteria are the analysis of lipid composition (7,11,33), the use of species-specific antibodies (44,56), and species-specific DNA or RNA probes (14,16,32).…”

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confidence: 99%

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

et al. 1995

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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.

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confidence: 99%

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

et al. 1995

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“…The resulting bacterial suspension, with or without the test drug, was cultured on an agitating plate at 37ЊC for up to 5 days. At 0-, 1-, 3-, and 5-day intervals, each sample was removed and subjected to the HPA protocol with an acridinium ester (AE)-labeled DNA probe (AccuProbe; Gen-Probe, Inc., San Diego, Calif.) (5,11). Briefly, 50 l of each sample was sonicated in a tube containing glass beads and lysing agents in a sonication bath (Bransonic ultrasonic cleaner, model B2200; Emerson, Tokyo, Japan) for 20 min, followed by heating of the mixture at 95ЊC for 10 min.…”

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confidence: 99%

New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay

et al. 1996

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We developed a novel method for early detection of drug-resistant strains of Mycobacterium tuberculosis by using the hybridization protection assay (HPA). The number of viable bacteria during the incubation period correlated well with the number of relative light units measured by the HPA. In addition, the relative light unit values of susceptible strains on the first, third and fifth days of incubation were significantly different from those of resistant strains for both isoniazid and rifampin. Our results suggest that after isolation of the organism from clinical specimens, drug-resistant strains of M. tuberculosis are accurately detected by the HPA even after 1 day of incubation with the drug.

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“…Plates were incubated at 37°C in the presence of 5% CO 2 for 3 weeks. (ii) They were used to perform the hybridization method by the commercially available AccuProbe M. tuberculosis complex test (Gen Probe, Inc., San Diego, Calif.) (6,8,13) according to the instructions of the manufacturer. The test consists of the following four basic steps: sample preparation, hybridization, selective chemical degradation, and chemiluminescence.…”

Section: Methodsmentioning

confidence: 99%

Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes

Martín-Casabona¹,

Mimó²,

Gonzalez³

et al. 1997

J Clin Microbiol

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The increasing number of multidrug-resistant Mycobacterium tuberculosis strains has stimulated the interest of investigators in finding a rapid method for susceptibility testing. We used commercially available rRNA DNA-bioluminescence-labelled probes (Accu-Probe, Gen Probe, Inc. San Diego, Calif.) for this purpose. The study was performed in three chronological steps. (i) We studied the correlation between the photometric light units (PLUs) given by the hybridization method, the numbers of CFU per milliliter, and turbidity as nephelometric units for six different inocula of an M. tuberculosis strain over 14 days. A good correlation (c > 0.9; P < 0.05) was found from the third day for all concentrations used. (ii) Over a period of 14 days we studied the evolution of the PLUs for 20 strains growing in medium with 0.2 l of isoniazid (H) per ml and 18 strains in medium with 1 l of rifampin (R) per ml to standardize the method. Susceptible and resistant strains were used according to the reference proportions method in Middlebrook 7H10, and the MICs were determined in solid and liquid media. The final inoculum of a 10 ؊2 dilution from a McFarland no. 1 standard and reading at 3 and 5 days provided the best results. A quotient was established to find a cutoff point between resistant and susceptible strains. (iii) We used the standardized parameters in 117 tests with H and R. On day 3, the sensitivity, specificity, positive predictive value, and negative predictive value for detecting resistant strains were 86.8, 100, 100, and 90.1%, respectively, and on day 5 they were 96.2, 100, 100, and 94%, respectively. We concluded that the method is readily available, is easy to perform, and could be useful for screening resistant M. tuberculosis strains.

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Evaluation of nonradioactive DNA probes for identification of mycobacteria

Cited by 145 publications

References 13 publications

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

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

New drug susceptibility test for Mycobacterium tuberculosis using the hybridization protection assay

Rapid method for testing susceptibility of Mycobacterium tuberculosis by using DNA probes

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