Extraction Methods Compared for Molecular Detection of Pathogens

Three mechanistically different sample extraction methodologies were evaluated for their abilities to purify nucleic acids from blood culture fluids.

The three methods were silica spin columns, phenol-chloroform, and an automated magnetic capture of polymer-complexed DNA via an Automate Express instrument, and were followed by polymerase chain reaction (PCR) assays for the detection of Staphylococcus aureus.

Scientists at Applied Molecular Testing, (Life Technologies, Carlsbad, CA, USA) analyzed 60 blood cultures spiked with bacterial pathogens. On separate days, three replicates from each of the sample aliquots were thawed and processed using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany), phenol-chloroform, or an ABI Automate Express forensic DNA extraction system.

The extracts from silica columns required 100- to 1,000-fold dilutions to reduce sufficiently the powerful PCR inhibitory effects of the anticoagulant sodium polyanetholsulfonate, a common additive in blood culture media. In contrast, samples extracted by either phenol-chloroform or the Automate Express instrument required little or no dilution, respectively, allowing for an approximate 100-fold improvement in assay sensitivity.

Analysis of 60 blood culture bottles indicated that the phenol-chloroform (Invitrogen, Carlsbad, CA)and the ABI extraction (Foster City, CA, USA) methodologies could be used to detect lower numbers of pathogens and that a growing S. aureus culture could be detected two hours earlier than when using silica columns. Of the three tested methodologies, the Automate Express instrument had the shortest time to result, requiring only approximately 80 minutes to process 12 samples.

The authors concluded that their findings highlight the importance of considering the mechanism when selecting a DNA extraction methodology, given that certain PCR inhibitors act in a similar fashion to DNA in certain chemical environments, resulting in copurification, whereas other methodologies use different chemistries that have advantages during the DNA purification of certain types of samples. The study was published in March 2012, in the Journal of Molecular Diagnostics.

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