title: 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.
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 10(3) 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.
authors.affiliation: Operative Unit of Clinical Microbiology, St Orsola-Malpighi University Hospital, 40138 Bologna, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Gaibani
authors.forename: Mara
authors.initials: M
authors.lastname: Mariconti
authors.forename: Gloria
authors.initials: G
authors.lastname: Bua
authors.forename: Sonia
authors.initials: S
authors.lastname: Bonora
authors.forename: Davide
authors.initials: D
authors.lastname: Sassera
authors.forename: Maria Paola
authors.initials: MP
authors.lastname: Landini
authors.forename: Patrizia
authors.initials: P
authors.lastname: Mulatto
authors.forename: Stefano
authors.initials: S
authors.lastname: Novati
authors.forename: Claudio
authors.initials: C
authors.lastname: Bandi
authors.forename: Vittorio
authors.initials: V
authors.lastname: Sambri
mesh_terms: D000818:Animals; D001424:Bacterial Infections / Q000175:diagnosis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D006090:Gram-Negative Bacteria / Q000235:genetics / Q000302:isolation & purification; D006094:Gram-Positive Bacteria / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D012338:RNA, Ribosomal, 23S / Q000097:blood / Q000235:genetics*; D012339:RNA, Ribosomal, 28S / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification
chemical_list: D012338:RNA, Ribosomal, 23S; D012339:RNA, Ribosomal, 28S