title: Rapid diagnosis of bacteremia by universal amplification of 23S ribosomal DNA followed by hybridization to an oligonucleotide array.
abstract: The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.
authors.affiliation: Department of Microbiology, King's College St. Thomas' Campus, St. Thomas' Hospital, London SE1 7EH, United Kingdom.
authors.forename: R M
authors.initials: RM
authors.lastname: Anthony
authors.forename: T J
authors.initials: TJ
authors.lastname: Brown
authors.forename: G L
authors.initials: GL
authors.lastname: French
mesh_terms: D016470:Bacteremia / Q000175:diagnosis* / Q000382:microbiology; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D006801:Humans; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011237:Predictive Value of Tests; D012338:RNA, Ribosomal, 23S / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012338:RNA, Ribosomal, 23S