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Clinical Chemistry 53: 104-110, 2007. First published November 16, 2006; 10.1373/clinchem.2006.070987
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(Clinical Chemistry. 2007;53:104-110.)
© 2007 American Association for Clinical Chemistry, Inc.

Automation and Analytical Techniques

High-Volume Extraction of Nucleic Acids by Magnetic Bead Technology for Ultrasensitive Detection of Bacteria in Blood Components

Melanie Störmer, Knut Kleesiek and Jens Dreiera

Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany.

aAddress correspondence to this author at: Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Georgstr. 11, D-32545 Bad Oeynhausen, Germany. Fax 49-5731-97-2307; e-mail jdreier{at}hdz-nrw.de.

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. Coamplification of human ß2-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 103 colony-forming units (CFU)/L for S. epidermidis and 22 x 103 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.




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