JCM Accepts, published online ahead of print on 21 October 2009

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J. Clin. Microbiol. doi:10.1128/JCM.01288-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Evaluation of automated and manual commercial DNA extraction methods for the recovery of Brucella spp. DNA from suspensions and spiked swabs

Leslie A. Dauphin^*, Rebecca J. Hutchins, Liberty A. Bost, and Michael D. Bowen

Bioterrorism Rapid Response and Advanced Technology (BRRAT) Laboratory, Division of Bioterrorism Preparedness and Response (DBPR), National Center for Preparedness, Detection, and Control of Infectious Diseases (NCPDCID), Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA

^* To whom correspondence should be addressed. Email: Ldauphin{at}CDC.GOV.

This study evaluated automated and manual commercial DNA extraction methods for their ability to recover DNA from Brucella species in 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 varying 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 and the MagNA Pure Compact kits 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.