Volume 13 Supplement 4

Sepsis 2009

Open Access

Comparison of commercial DNA extraction kits for the detection of bacterial genomic DNA from whole-blood samples using a broad-range PCR

  • B Krulova1,
  • E Nemcova1,
  • B Zaloudikova1,
  • P Nemec1 and
  • T Freiberger1
Critical Care200913(Suppl 4):P13

https://doi.org/10.1186/cc8069

Published: 11 November 2009

Introduction

Blood culture is still considered a gold standard for diagnosis of bloodstream infections. Early pathogen detection is a prerequisite for the successful treatment. Nucleic acid based techniques offer a rapid and sensitive option particularly in blood culture negative samples. Efficient bacterial DNA extraction is crucial for following PCR assays. The aim of this study was to determine the detection limit of bacterial genomic DNA using different extraction protocols.

Methods

We evaluated five commercially available kits for the extraction of bacterial genomic DNA from whole-blood samples (QIAamp DNA Blood Mini kit, UltraClean DNA BloodSpin Kit, Chemagic DNA Blood Kit, ZR Genomic DNAII Kit, NucliSens miniMAG). Whole-blood samples were spiked with Escherichia coli CCM 3988 and Staphylococcus aureus CCM 7111. Tenfold dilution series containing concentrations from 107 to 100 CFU/ml were prepared under sterile conditions and immediately used. A broad-range 16S rDNA end-point PCR was performed for the detection of E. coli and S. aureus DNA.

Results

The sensitivity of each kit was determined as a minimum rate of CFU providing the positive result in the PCR assay. Our results showed the extraction by the QIAamp DNA Blood Mini Kit (supplemented with enzymatic pre-treatment) as the most efficient and sensitive method. This extraction protocol allowed the reproducible detection of E. coli and S. aureus at concentrations of 103 CFU/ml. All kits showed positive results in samples at concentrations from 107 to 105 CFU/ml.

Conclusion

Extraction kits should be capable to recover nucleic acids and remove inhibitors from diverse clinical materials simultaneously. All of the tested kits were able to recover bacterial genomic DNA from whole-blood samples, but the sensitivity of PCR-based detection depends on the DNA extraction protocol used.

Authors’ Affiliations

(1)
Centre for Cardiovascular Surgery and Transplantation, Molecular genetic laboratory

Copyright

© BioMed Central Ltd 2009

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