title: A sample extraction method for faster, more sensitive PCR-based detection of pathogens in blood culture.
abstract: Three mechanistically different sample extraction methodologies, namely, silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA (via an Automate Express instrument), were compared for their abilities to purify nucleic acids from blood culture fluids for use in TaqMan assays for detection of Staphylococcus aureus. The extracts from silica columns required 100- to 1000-fold dilutions to sufficiently reduce the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity. Analysis of 60 blood culture bottles indicated that these latter two methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected 2 hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples. These findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples.
authors.affiliation: Applied Molecular Testing, Life Technologies, Foster City, California, USA.
authors.forename: John F
authors.initials: JF
authors.lastname: Regan
authors.forename: Manohar R
authors.initials: MR
authors.lastname: Furtado
authors.forename: Maxim G
authors.initials: MG
authors.lastname: Brevnov
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Jordan
mesh_terms: D001331:Automation; D016470:Bacteremia / Q000097:blood / Q000235:genetics / Q000382:microbiology; D001426:Bacterial Proteins / Q000235:genetics*; D001800:Blood Specimen Collection; D004269:DNA, Bacterial / Q000097:blood* / Q000235:genetics*; D006801:Humans; D008297:Male; D016106:Methicillin Resistance / Q000235:genetics; D046915:Penicillin-Binding Proteins; D060888:Real-Time Polymerase Chain Reaction; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000235:genetics* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification* / Q000472:pathogenicity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D046915:Penicillin-Binding Proteins; C487789:mecA protein, Staphylococcus aureus