Comparason Of Extraction Methods For Pcr Detection Of Burkholderia Cepacia Complex (Bcc) From Cystic Fibrosis Patients

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 forth e 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 10(6) cfu/ml. Amplification was observed in 7/16 (43.7%) of sputa for boiling, 8/16 (50%) of sputa for GTAB 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.