Evaluation of automated and manual commercial DNA extraction methods for recovery of Brucella DNA from suspensions and spiked swabs.
This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in phosphate-buffered saline (PBS) suspension and from spiked swab specimens. Six extraction methods, representing several of the methodologies which are commercially available for DNA extraction, as well as representing various throughput capacities, were evaluated: the MagNA Pure Compact and the MagNA Pure LC instruments, 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. These six extraction methods were performed upon three pathogenic Brucella species: B. abortus, B. melitensis, and B. suis. Viability testing of the DNA extracts indicated that all six extraction methods were efficient at inactivating virulent Brucella spp. Real-time PCR analysis using Brucella genus- and species-specific TaqMan assays revealed that use of the MasterPure kit resulted in superior levels of detection from bacterial suspensions, while the MasterPure kit and MagNA Pure Compact performed equally well for extraction of spiked swab samples. This study demonstrated that DNA extraction methodologies differ in their ability to recover Brucella DNA from PBS bacterial suspensions and from swab specimens and, thus, that the extraction method used for a given type of sample matrix can influence the sensitivity of real-time PCR assays for Brucella.
Dauphin
Leslie A
LA
Division of Bioterrorism Preparedness and Response, Bioterrorism Rapid Response and Advanced Technology Laboratory, National Center for Preparedness, Detection, and Control of Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA. Ldauphin@cdc.gov
Hutchins
Rebecca J
RJ
Bost
Liberty A
LA
Bowen
Michael D
MD
D001331:Automation; D001431:Bacteriological Techniques; D002002:Brucella / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D002003:Brucella abortus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D017347:Brucella melitensis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D041021:Brucella suis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D002006:Brucellosis / Q000175:diagnosis / Q000382:microbiology; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013048:Specimen Handling / Q000379:methods
D004269:DNA, Bacterial; D011933:Reagent Kits, Diagnostic