title: Comparative evaluation of automated and manual commercial DNA extraction methods for detection of Francisella tularensis DNA from suspensions and spiked swabs by real-time polymerase chain reaction.
abstract: This study evaluated commercial automated and manual DNA extraction methods for the isolation of Francisella tularensis DNA suitable for real-time polymerase chain reaction (PCR) analysis from cell suspensions and spiked cotton, foam, and polyester swabs. Two automated methods, the MagNA Pure Compact and the QIAcube, were compared to 4 manual methods, the IT 1-2-3 DNA sample purification kit, the MasterPure Complete DNA and RNA purification kit, the QIAamp DNA blood mini kit, and the UltraClean Microbial DNA isolation kit. The methods were compared using 6 F. tularensis strains representing the 2 subspecies which cause the majority of reported cases of tularemia in humans. Cell viability testing of the DNA extracts showed that all 6 extraction methods efficiently inactivated F. tularensis at concentrations of ≤10⁶ CFU/mL. Real-time PCR analysis using a multitarget 5' nuclease assay for F. tularensis revealed that the PCR sensitivity was equivalent using DNA extracted by the 2 automated methods and the manual MasterPure and QIAamp methods. These 4 methods resulted in significantly better levels of detection from bacterial suspensions and performed equivalently for spiked swab samples than the remaining 2. This study identifies optimal DNA extraction methods for processing swab specimens for the subsequent detection of F. tularensis DNA using real-time PCR assays. Furthermore, the results provide diagnostic laboratories with the option to select from 2 automated DNA extraction methods as suitable alternatives to manual methods for the isolation of DNA from F. tularensis.
authors.affiliation: Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Laboratory Preparedness and Response Branch (LPRB), Division of Preparedness and Emerging Infections (DPEI), Centers for Disease Control and Prevention (CDC), Atlanta, GA 30333, USA. ldauphin@cdc.gov
authors.forename: Leslie A
authors.initials: LA
authors.lastname: Dauphin
authors.forename: Roblena E
authors.initials: RE
authors.lastname: Walker
authors.forename: Jeannine M
authors.initials: JM
authors.lastname: Petersen
authors.forename: Michael D
authors.initials: MD
authors.lastname: Bowen
mesh_terms: D001431:Bacteriological Techniques / Q000379:methods*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005604:Francisella tularensis / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D014406:Tularemia / Q000175:diagnosis* / Q000382:microbiology
chemical_list: D011933:Reagent Kits, Diagnostic; D004247:DNA