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J Microbiol Methods. 2014 Apr;99:1-7. doi: 10.1016/j.mimet.2014.01.013. Epub 2014 Feb 3.

Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.

Author information

  • 1George Washington University, School of Public Health and Health Services, Department of Epidemiology and Biostatistics, 2300 I Street NW, Washington, DC 20037, USA. Electronic address: sphcdm@gwu.edu.
  • 2George Washington University, School of Public Health and Health Services, Department of Epidemiology and Biostatistics, 2300 I Street NW, Washington, DC 20037, USA. Electronic address: jajordan@gwu.edu.

Abstract

Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized Staphylococcus aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P<0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.

Copyright © 2014 Elsevier B.V. All rights reserved.

KEYWORDS:

Bacterial bloodstream infection; DNA extraction; Molecular diagnostics; Sepsis; Whole blood

PMID:
24503182
[PubMed - indexed for MEDLINE]
PMCID:
PMC3976640
Free PMC Article
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