Identification of Isolates within the
            <i>Burkholderia cepacia</i>
            Complex by a Multiplex
            <i>recA</i>
            and 16S rRNA Gene Real-Time PCR Assay

Pimentel, Jason D.; Dubedat, Sally; Dodds, Evan L. N.; Benn, Richard

doi:10.1128/jcm.01606-07

Cited by 6 publications

(6 citation statements)

References 12 publications

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“…Amplification by PCR followed by cloning must be reserved for specific cases, especially when it is impossible to isolate and identify a known pathogen from sputa among polymicrobial oral flora in a patient with clinical degradation associated with fever, an inflammatory syndrome, decreased respiratory function, and/or following lung transplantation. By contrast, it would be interesting to develop specific real-time PCR methods, using probes for the identification of several pathogens (including P. aeruginosa [119], B. cepacia [73] and I. limosus [13]), in specific situations.…”

Section: Discussionmentioning

confidence: 99%

“…Some of these assays target a specific bacterial species using real‐time PCR (RT‐PCR) for identification of isolates, e.g. of the B. cepacia complex by multiplex recA and 16S rRNA gene RT‐PCR [73], of I. limosus by 16S rRNA gene RT‐PCR [13] and of P. aeruginosa by duplex ecfX and the gyrB gene RT‐PCR [34]. Fluorescence in situ hybridization has also been used for A. xylosoxidans , Alcaligenes faecalis [74] and B. cepacia complex identification [75].…”

Section: Molecular Means For the Correct Identification Of Misidentifmentioning

confidence: 99%

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Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients

Bittar

Rolain

2010

Clinical Microbiology and Infection

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Respiratory infections remain a major threat to cystic fibrosis (CF) patients. The detection and correct identification of the bacteria implicated in these infections is critical for the therapeutic management of patients. The traditional methods of culture and phenotypic identification of bacteria lack both sensitivity and specificity because many bacteria can be missed and/or misidentified. Molecular analyses have recently emerged as useful means to resolve these problems, including molecular methods for accurate identification or detection of bacteria and molecular methods for evaluation of microbial diversity. These recent molecular technologies have increased the list of new and/or emerging pathogens and epidemic strains associated with CF patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of intact cells has also emerged recently as a powerful and rapid method for the routine identification of bacteria in clinical microbiology laboratories and will certainly represent the method of choice also for the routine identification of bacteria in the context of CF. Finally, recent data derived from molecular culture-independent analyses indicate the presence of a previously underestimated, complex microbial community in sputa from CF patients. Interestingly, full genome sequencing of some bacteria frequently recovered from CF patients has highlighted the fact that the lungs of CF patients are hotspots for lateral gene transfer and the adaptation of these ecosystems to a specific chronic condition.

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Section: Discussionmentioning

confidence: 99%

Section: Molecular Means For the Correct Identification Of Misidentifmentioning

confidence: 99%

Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients

Bittar

Rolain

2010

Clinical Microbiology and Infection

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“…Due to a report of person-to-person spread (11), some centers have introduced segregation of patients colonized with Pandoraea species just as is carried out for those colonized with the B. cepacia complex (3,11). None of the other patients included in a recent molecular survey of the patients in the RPAH CF unit were found to harbor P. sputorum or any of the other Pandoraea species (15).…”

Section: Case Reportmentioning

confidence: 99%

“…An aliquot of the DNA was used for the PCR, and the remainder was stored at Ϫ80°C. In December 2006, a multiplex, real-time PCR to detect both the 16S rRNA gene and the recA gene was performed on the stored DNA (15). The real-time PCR product was used to generate a 16S rRNA gene sequence, as previously described (14), which was later used to identify these organisms by a nucleotide BLAST search (2).…”

Section: Case Reportmentioning

confidence: 99%

Misidentification of Pandoraea sputorum Isolated from Sputum of a Patient with Cystic Fibrosis and Review of Pandoraea Species Infections in Transplant Patients

Pimentel

MacLeod

2008

J Clin Microbiol

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Pandoraea species are considered emerging pathogens in cystic fibrosis (CF) patients, but few data exist regarding outcomes of patients colonized with these organisms. We report a case of Pandoraea sputorum colonization in a CF patient under consideration for lung transplantation and review five cases of lung transplantation involving Pandoraea species.

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“…In a study by Vonberg et al, recA gene was targetted by a novel rapid-cycle PCR and genomovar-specific Flourescence Resonance Energy Transfer (FRET) probes, offering another molecular method for BCC identification and typing [20]. Pyrosequencing and recA real-time PCR had also been employed for BCC typing [21,22]. Recently, sensitivity of an optimized commercial fluorescent in situ hybridization (FISH) assay performed directly on sputum samples was determined as 8 x 10 5 cfu/ml, whereas a novel rRNA-based PCR assay with 100% sensitivity and specificity for all BCC species could detect 10 4 cfu/ml of bacteria in sputa [23].…”

Section: Discussionmentioning

confidence: 99%

Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

et al. 2008

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AbstractDirect detection of Burkholderia cepacia complex (BCC) and its genomovars from sputum by molecular tests emerges as a method for rapid identification. In this study, four DNA extraction methods were evaluated for the identification for BCC from sputum of CF patients. Sputa from 28 CF patients were aliquoted and spiked with BCC reference strain. Boiling, phenol-chloroform, CTAB methods and a commercial spin column kit was used for DNA extraction. Total DNA yields were determined by spectrophotometry and single-round recA PCR was used for detection of BCC. No significant difference was observed in DNA yields from different extraction methods. Lower limit of detection for recA PCR was determined as 106 cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for CTAB and 13/16 (81.2%) of sputa for phenol-chloroform method and spin column kit in the assay sensitivity range determined in the study. Phenol-chloroform and commercial spin column kit were found to be better suited for DNA purification from sputum of CF patients for BCC identification. Diagnostic impact of single-round recA PCR directly from sputum was limited to chronically-infected patients.

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Identification of Isolates within the Burkholderia cepacia Complex by a Multiplex recA and 16S rRNA Gene Real-Time PCR Assay

Cited by 6 publications

References 12 publications

Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients

Detection and accurate identification of new or emerging bacteria in cystic fibrosis patients

Misidentification of Pandoraea sputorum Isolated from Sputum of a Patient with Cystic Fibrosis and Review of Pandoraea Species Infections in Transplant Patients

Comparason of extraction methods for PCR detection of Burkholderia cepacia complex (BCC) from cystic fibrosis patients

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