Open Access Highly Accessed Research article

Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

Herbert Tomaso12*, Mireille Kattar3, Meike Eickhoff4, Ulrich Wernery5, Sascha Al Dahouk6, Eberhard Straube7, Heinrich Neubauer1 and Holger C Scholz2

Author Affiliations

1 Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburgerstrasse 96a, 07743 Jena, Germany

2 Department of Bacteriology, Bundeswehr Institute of Microbiology, Munich, Germany

3 Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon

4 Roche Molecular Diagnostics, Rotkreuz, Switzerland

5 Central Veterinary Research Laboratory, Dubai, United Arab Emirates

6 RWTH Aachen University, Department of Internal Medicine III, Aachen, Germany

7 Institute of Medical Microbiology, Friedrich Schiller University of Jena, Jena, Germany

For all author emails, please log on.

BMC Infectious Diseases 2010, 10:100  doi:10.1186/1471-2334-10-100

Published: 20 April 2010

Abstract

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.