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

[摘要] 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 β2-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 × 103 colony-forming units (CFU)/L for S. epidermidis and 22 × 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.

[学科分类] 过敏症与临床免疫学