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Central European Journal of Medicine

, Volume 3, Issue 2, pp 157–162 | Cite as

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

  • Koray Ergunay
  • Pinar Yurdakul
  • Burcin Sener
  • Ugur Ozcelik
  • Erdem Karabulut
  • Nural Kiper
Research Article
  • 69 Downloads

Abstract

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.

Keywords

DNA extraction PCR Burkholderia Burkholderia cepacia complex Cystic fibrosis 

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Copyright information

© © Versita Warsaw and Springer-Verlag Berlin Heidelberg 2008

Authors and Affiliations

  • Koray Ergunay
    • 1
  • Pinar Yurdakul
    • 1
  • Burcin Sener
    • 1
  • Ugur Ozcelik
    • 2
  • Erdem Karabulut
    • 3
  • Nural Kiper
    • 2
  1. 1.Department of Microbiology and Clinical MicrobiologyHacettepe University Faculty of MedicineSihhiye, AnkaraTurkey
  2. 2.Department of Pediatrics, Division of Chest Diseases; Ihsan Dogramaci Childrens’ HospitalHacettepe University Faculty of MedicineSihhiye, AnkaraTurkey
  3. 3.Department of BiostatisticsHacettepe University Faculty of MedicineSihhiye, AnkaraTurkey

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