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2008 | 3 | 2 | 157-162
Tytuł artykułu

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

Treść / Zawartość
Warianty tytułu
Języki publikacji
EN
Abstrakty
EN
Direct 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.
Wydawca

Czasopismo
Rocznik
Tom
3
Numer
2
Strony
157-162
Opis fizyczny
Daty
wydano
2008-06-01
online
2008-04-09
Twórcy
  • Department of Microbiology and Clinical Microbiology, Hacettepe University Faculty of Medicine, Morphology Building 3rd floor, Sihhiye, Ankara, 06100, Turkey, ekoray@hacettepe.edu.tr
  • Department of Microbiology and Clinical Microbiology, Hacettepe University Faculty of Medicine, Morphology Building 3rd floor, Sihhiye, Ankara, 06100, Turkey
autor
  • Department of Microbiology and Clinical Microbiology, Hacettepe University Faculty of Medicine, Morphology Building 3rd floor, Sihhiye, Ankara, 06100, Turkey
autor
  • Department of Pediatrics, Division of Chest Diseases; Ihsan Dogramaci Childrens’ Hospital, Hacettepe University Faculty of Medicine, Sihhiye, Ankara, 06100, Turkey
  • Department of Biostatistics, Hacettepe University Faculty of Medicine, Sihhiye, Ankara, 06100, Turkey
autor
  • Department of Pediatrics, Division of Chest Diseases; Ihsan Dogramaci Childrens’ Hospital, Hacettepe University Faculty of Medicine, Sihhiye, Ankara, 06100, Turkey
Bibliografia
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  • [11] Lipuma J.J., Spilker T., Gill H.L., Campbell III P.W., Liu L., et al. Disproportionate distribution of Burkholderia cepacia complex species and transmissibility markers in cystic fibrosis. Am. J. Respir. Crit. Care. Med., 2001, 164, 92–96 [Crossref]
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  • [17] Merk S., Neubauer H., Meyer H., Greiseer-Wilke I. Comparison of different methods for the isolation of Burkholderia cepacia DNA from pure cultures and waste water. Int. J. Hyg. Environ. Health., 2001, 204, 127–131 http://dx.doi.org/10.1078/1438-4639-00083[Crossref]
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  • [21] Slinger R., Yan L., Myers R., Ramotar K., St Denis M., Aaron S.D. Pyrosequencing of a recA gene variable region for Burkholderia cepacia complex genomovar identification. Diagn. Microbiol. Infect. Dis., 2007, 58, 379–84 http://dx.doi.org/10.1016/j.diagmicrobio.2007.02.011[Crossref]
  • [22] Pimentel J.D., Dubedat S.M. Identification of isolates within the Burkholderia cepacia Complex by a multiplex recA and 16S rRNA gene real-time PCR assay. J. Clin. Microbiol., 2007, 45, 3853–3854 http://dx.doi.org/10.1128/JCM.01606-07[WoS][Crossref]
  • [23] Brown A.R., Gowan J.R.W. Assessment of Fluorescent In Situ Hybridization and PCR-Based methods for rapid identification of Burkholderia cepacia Complex organisms directly from sputum samples. J. Clin. Microbiol., 2007, 45, 1920–1926 http://dx.doi.org/10.1128/JCM.00147-07[Crossref][WoS]
Typ dokumentu
Bibliografia
Identyfikatory
Identyfikator YADDA
bwmeta1.element.-psjd-doi-10_2478_s11536-007-0069-4
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