title: High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components.
abstract: BACKGROUND
  Nucleic acid isolation, the most technically demanding and laborious procedure performed in molecular diagnostics, harbors the potential for improvements in automation. A recent development is the use of magnetic beads covered with nucleic acid-binding matrices. We adapted this technology with a broad-range 23S rRNA real-time reverse transcription (RT)-PCR assay for fast and sensitive detection of bacterial contamination of blood products.
  
  
  METHODS
  We investigated different protocols for an automated high-volume extraction method based on magnetic-separation technology for the extraction of bacterial nucleic acids from platelet concentrates (PCs). We added 2 model bacteria, Staphylococcus epidermidis and Escherichia coli, to a single pool of apheresis-derived, single-donor platelets and assayed the PCs by real-time RT-PCR analysis with an improved primer-probe system and locked nucleic acid technology. Co-amplification of human beta(2)-microglobulin mRNA served as an internal control (IC). We used probit analysis to calculate the minimum concentration of bacteria that would be detected with 95% confidence.
  
  
  RESULTS
  For automated magnetic bead-based extraction technology with the real-time RT-PCR, the 95% detection limit was 29 x 10(3) colony-forming units (CFU)/L for S. epidermidis and 22 x 10(3) CFU/L for E. coli. No false-positive results occurred, either due to nucleic acid contamination of reagents or externally during testing of 1030 PCs.
  
  
  CONCLUSIONS
  High-volume nucleic acid extraction improved the detection limit of the assay. The improvement of the primer-probe system and the integration of an IC make the RT-PCR assay appropriate for bacteria screening of platelets.
authors.affiliation: Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Störmer
authors.forename: Knut
authors.initials: K
authors.lastname: Kleesiek
authors.forename: Jens
authors.initials: J
authors.lastname: Dreier
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods; D001769:Blood / Q000382:microbiology*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008280:Magnetics; D012329:RNA, Bacterial / Q000032:analysis*; D012338:RNA, Ribosomal, 23S / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013212:Staphylococcus epidermidis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S