title: Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood.
abstract: PCR detection of microbial pathogens in blood from patients is a promising issue for rapid diagnosis of sepsis and early targeted therapy. However, for PCR assays detecting all bacterial groups, broad range primers, in particular the 16S rDNA targeting primers have to be used. Upcoming false signals and reduced sensitivity are a common problem as a consequence of unspecific amplification reactions with the human DNA background. Here we show that, using total DNA extracts from blood, unspecific signals occurred in general 16S rDNA PCRs as a result of the amplification of human sequences. To address this problem, we developed a protocol by which the human background DNA is removed and bacterial DNA is enriched during sample preparation, a method we termed background-free enrichment method (BFEM). In general, we aimed to exclude false signals due to the human background DNA yielded from 16S rDNA PCR, Real-Time-PCR and IGS-PCR analyses. We applied the BFEM to the analysis of blood samples from 22 patients and obtained results similar to standard blood culture methods. The BFEM allows specific and sensitive detection of pathogens in downstream PCR assays and is easy to handle due to the quick sample preparation procedure. Thus, the BFEM contributes to the generation of replicable and more reliable data in general 16S rDNA PCR assays.
authors.affiliation: Ludwig Boltzmann Institute for Leukemia Research and Hematology, Vienna, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Handschur
authors.forename: Heidrun
authors.initials: H
authors.lastname: Karlic
authors.forename: Christian
authors.initials: C
authors.lastname: Hertel
authors.forename: Michael
authors.initials: M
authors.lastname: Pfeilstöcker
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Haslberger
mesh_terms: D001419:Bacteria / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000097:blood*; D004275:DNA, Ribosomal / Q000097:blood*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D018746:Systemic Inflammatory Response Syndrome / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S