High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components.
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.
Störmer
Melanie
M
Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.
Kleesiek
Knut
K
Dreier
Jens
J
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
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S