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BioMed Research International
Volume 2013 (2013), Article ID 264651, 8 pages
http://dx.doi.org/10.1155/2013/264651
Research Article

Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

1Operative Unit of Clinical Microbiology, St. Orsola-Malpighi University Hospital, 40138 Bologna, Italy
2Department of Haematology and Oncology “L. and A. Seragnoli”, Unit of Clinical Microbiology, Regional Reference Centre for Microbiological Emergencies (CRREM), St. Orsola-Malpighi Hospital, University of Bologna, 9 Via G. Massarenti, 40138 Bologna, Italy
3DIVET, University of Milan, 20100 Milan, Italy
4Fondazione IRCCS Policlinico San Matteo, 27100 Pavia, Italy

Received 17 October 2012; Revised 15 January 2013; Accepted 29 January 2013

Academic Editor: Arun K. Bhunia

Copyright © 2013 Paolo Gaibani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.