| High-volume extraction of nucleic acids by magnetic bead technology for ultrasensitive detection of bacteria in blood components. | |
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MedLine Citation:
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PMID: 17110475 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Melanie Störmer; Knut Kleesiek; Jens Dreier |
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Publication Detail:
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Type: Journal Article Date: 2006-11-16 |
Journal Detail:
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Title: Clinical chemistry Volume: 53 ISSN: 0009-9147 ISO Abbreviation: Clin. Chem. Publication Date: 2007 Jan |
Date Detail:
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Created Date: 2007-01-04 Completed Date: 2007-02-06 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9421549 Medline TA: Clin Chem Country: United States |
Other Details:
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Languages: eng Pagination: 104-10 Citation Subset: IM |
Affiliation:
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Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany. |
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| MeSH Terms | |
Descriptor/Qualifier:
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Bacteriological Techniques
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methods Blood / microbiology* Colony Count, Microbial DNA, Bacterial / analysis* Escherichia coli / genetics, isolation & purification Humans Magnetics* RNA, Bacterial / analysis* RNA, Ribosomal, 23S / analysis Reverse Transcriptase Polymerase Chain Reaction Sensitivity and Specificity Staphylococcus epidermidis / genetics, isolation & purification |
| Chemical | |
Reg. No./Substance:
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0/DNA, Bacterial; 0/RNA, Bacterial; 0/RNA, Ribosomal, 23S |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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