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

Herbert Tomaso,Heinrich Neubauer,Holger C Scholz,Mireille Kattar,Sascha Al Dahouk,Meike Eickhoff,Ulrich Wernery,Eberhard Straube

doi:10.1186/1471-2334-10-100

Abstract

BackgroundThe 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.MethodsFive 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.ResultsKits 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.ConclusionsWe 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.

Highlights

The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low
Brucellosis is a 're-emerging' zoonosis with a worldwide distribution caused by small fastidious Gram negative bacteria of the genus Brucella
Comparison of DNA yield obtained with the kits tested in this study showed significant differences as determined by one-way ANOVA (p < 0.05)

Summary

Introduction

The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. The aim of this study was to assess the performance of commercial kits for the extraction of Brucella DNA. Brucellosis is a 're-emerging' zoonosis with a worldwide distribution caused by small fastidious Gram negative bacteria of the genus Brucella. The cultivation and identification of Brucella species with conventional microbiological techniques is time-consuming and hazardous and should be performed under BSL-3 conditions [1]. Real-time PCR assays allow for the rapid and specific detection of Brucella species, but the amount of bacteria in tissues and body fluids is frequently extremely low [2,3].

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Conclusion