Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR.
BACKGROUND
The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA.


METHODS
Five kits were evaluated using clinical specimens: QIAamp DNA Mini Kit (QIAGEN), peqGold Tissue DNA Mini Kit (PeqLab), UltraClean Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS711 that included an internal amplification control.


RESULTS
Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared.


CONCLUSIONS
We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.
Tomaso
Herbert
H
Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburgerstrasse 96a, 07743 Jena, Germany. herbert.tomaso@web.de
Kattar
Mireille
M
Eickhoff
Meike
M
Wernery
Ulrich
U
Al Dahouk
Sascha
S
Straube
Eberhard
E
Neubauer
Heinrich
H
Scholz
Holger C
HC
D001431:Bacteriological Techniques / Q000379:methods*; D002002:Brucella / Q000235:genetics / Q000302:isolation & purification*; D002006:Brucellosis / Q000175:diagnosis*; D004269:DNA, Bacterial / Q000302:isolation & purification*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*
D004269:DNA, Bacterial
Eur J Clin Microbiol Infect Dis. 2008 Feb;27(2):109-14
17973130
J Clin Microbiol. 1994 Nov;32(11):2660-6
7852552
FEMS Immunol Med Microbiol. 2003 Sep 22;38(2):117-26
13129646
Vet Microbiol. 2002 Dec 20;90(1-4):435-46
12414163
J Clin Microbiol. 1995 Dec;33(12):3087-90
8586678
Clin Chem Lab Med. 2007;45(11):1464-70
17970716
J Clin Microbiol. 2001 Apr;39(4):1661-4
11283112
Clin Lab. 2003;49(9-10):487-505
14572205
Res Vet Sci. 2006 Oct;81(2):170-6
16545848
J Vet Med B Infect Dis Vet Public Health. 2000 Oct;47(8):573-80
11075545