title: Evaluation of MolYsis™ Complete5 DNA extraction method for detecting Staphylococcus aureus DNA from whole blood in a sepsis model using PCR/pyrosequencing.
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.
authors: George 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.
authors: Chase D
authors: CD
authors: McCann
authors: George 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.
authors: Jeanne A
authors: JA
authors: Jordan
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004195:Disease Models, Animal; D004285:Dogs; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D018805:Sepsis / Q000175:diagnosis* / Q000382:microbiology; D017422:Sequence Analysis, DNA / Q000379:methods; D013203:Staphylococcal Infections / Q000175:diagnosis* / Q000382:microbiology; D013211:Staphylococcus aureus / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial
keywords: Bacterial bloodstream infection; DNA extraction; Molecular diagnostics; Sepsis; Whole blood