Dataset Information

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

ABSTRACT: Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes

SUBMITTER: Gomez DI

PROVIDER: S-EPMC2854294 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

Gomez Diana I DI Fisher-Hoch Susan P SP Bordt Andrea S AS Quitugua Teresa N TN Robledo Jaime J Alvarez Nataly N Correa Nidia N McCormick Joseph B JB Restrepo Blanca I BI

Diagnostic microbiology and infectious disease 20100312 1

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. Rifampicin (RIF) resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes </=2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-suscep ...[more]

PMID: 20227226

Similar Datasets

Project description:In a previous study, we have evaluated genetic identification by using the rpoB gene, which was recently introduced by Kim et al. (J. Clin. Microbiol. 39:2102-2109, 2001; J. Clin. Microbiol. 37:1714-1720, 1999). In this process, we examined the rpoB gene heterogeneity of clinical isolates identified as Mycobacterium gordonae with the conventional biological and biochemical tests and/or a commercially available DNA probe kit. Sequencing of the rpoB gene of 34 clinical isolates revealed that M. gordonae clinical isolates were classified into four major clusters (A, B, C, and D). Interestingly, organisms belonging to cluster D (15 isolates) did not hybridize with M. gordonae ATCC 14470 and specifically possessed urease activity. Therefore, it could be considered to be a novel mycobacterium. The identification of M. gordonae is known to have ambiguous results sometimes. On the other hand, identification of clinical isolates seems to be inconvenient and unsuitable because of a more than 99% 16S rRNA gene similarity value between clusters. These findings suggest that the existence of M. gordonae-like mycobacteria that share similar biochemical and biological characteristics with the 16S rRNA gene of an M. gordonae type strain but less similarity at the genomic DNA level may have complicated the identification of M. gordonae in many laboratories. Furthermore, compared with hsp65 PCR restriction analysis (PRA), rpoB PRA would have the advantage of producing no ambiguous results because of the intracluster homogeneity of the rpoB gene. In this case, rpoB would provide clearer results than hsp65, even if PRA analysis was used. We demonstrated that these M. gordonae-like mycobacteria were easily distinguished by PRA of the rpoB sequence. Additionally, the significance of this M. gordonae-like cluster may help to establish the comparison between the M. gordonae isolates from a clinical specimen and an infectious process in a given patient and to determine the true incidence of infection with this microorganism.

Project description:Background and aimDifferent polymerase chain reaction (PCR) techniques have and are still being used for the direct detection of Brucella DNA in serum samples of different animal species and humans without being validated or properly validated, resulting in discrepancies. Thus, this study aimed to evaluate the diagnostic performance of the TaqMan Real-Time-PCR (RT-PCR) targeting the bcsp31 gene versus conventional PCR for the accurate diagnosis of brucellosis at the genus level in cattle sera.Materials and methodsOne hundred and eighty-four serum samples were collected from bacteriologically positive and negative cows with ages ranging from 1 to 5 years old at some infected private farms in the Nile Delta under quarantine measures as well as brucellosis free farms. These samples were classified into four groups after serological diagnosis and investigated by TaqMan RT-PCR and conventional PCR targeting the IS711 gene for Brucella DNA detection. The diagnostic performance characteristics of both PCR techniques were estimated considering the bacteriological results as a gold standard.ResultsTaqMan RT-PCR revealed superiority over conventional PCR; it was able to detect Brucella DNA in 95% (67/70) and 89% (25/28) of the cattle sera samples belonging to Group 1 (serologically and bacteriologically positive) and Group 2 (serologically negative but bacteriologically positive), respectively. On evaluating the diagnostic performance, TaqMan RT-PCR showed superior diagnostic sensitivity (93.9%), diagnostic specificity (88.4%), performance index (182.3), almost perfect kappa agreement (0.825±0.042), strong positive correlation (r=0.826), high accuracy based on the receiver operating characteristic (ROC) curve, and area under the ROC curve (0.911) at p<0.05 and CI of 95%.ConclusionA cattle serum sample is not the metric of choice for targeting Brucella genomic DNA by conventional PCR. The time-saving and rapid TaqMan RT-PCR method revealed a better diagnostic performance in the detection of Brucella DNA in cattle sera. Such performance offered by TaqMan RT-PCR may be considered a step toward the possibility of using such technology in the direct differentiation between Brucella-infected and -vaccinated cattle immunized by smooth vaccines from cattle sera using primers specific for such vaccines.

Dataset Information

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

Publications

Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets