QIAamp DNA Blood Mini QIAcube Kit

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Publication protocol

Kit Selection: The DNA extraction formats tested include glass or silica filter columns and magnetic bead automated systems which appear to be the most common technologies used in commercial kits. These kits also are considered less hazardous than older methods such as the use of phenol/chloroform extractions which may expose staff to carcinogens. Kits were also chosen based on both availability in areas where melioidosis is endemic or where it poses a potential biothreat concern. In addition some kits were not assessed based on higher costs compared to those that were selected for assessment. The following manual kits were evaluated: QIAamp DNA Mini Kit (QIAamp Mini) (QIAGEN, Valencia, CA), QIAamp DNA Blood Mini Kit (QIAamp Blood) (QIAGEN, Valencia, CA), and the High Pure PCR Template Preparation Kit (High Pure) (Roche Diagnostics, Indianapolis, IN). These kits were compared with four automated systems which provide greater throughput than manual kits: the MagNA Pure LC (Roche Diagnostics, Indianapolis, IN) with the DNA Isolation Kit I (MagNA LC) which can process 32 specimens per run, the MagNA Pure Compact (Roche Diagnostics, Indianapolis, IN) with the Nucleic Acid Isolation Kit I (MagNA Compact) which can process eight specimens per run and the QIAcube (QIAGEN, Valencia, CA) using both the QIAamp DNA Mini kit (QIAcube Mini) and QIAamp DNA Blood Mini kit (QIAcube Blood) which has a capacity of 12 specimens per run.
DNA Extractions: The serial diluted spiked blood samples, as well as the negative blood and negative water controls were extracted in triplicate using seven different DNA extraction kits as follows. The two manual QIAGEN kits tested in this study, the QIAamp Mini and the QIAamp Blood, were used following the manufacturer's instructions for the blood and body fluid spin protocol. A 200 μl blood sample was extracted, the optional spin at 20,000×g for 1 min prior to incubation and elution with Buffer AE was performed, and 95% ethanol was used instead of the manufacturer's recommended 96%–100% ethanol. A separate short study indicated no significant difference in using 95% ethanol compared to 99.5% ethanol (data not shown). Additionally, these two QIAGEN kits were used with the QIAcube automated system following the manufacturer's instructions for sample setup of the QIAcube Mini and QIAcube Blood kits. The High Pure kit was used following the manufacturer's instructions for 200 μl of mammalian blood. The MagNA Compact utilized the Nucleic Acid Isolation Kit I (Roche Applied Science, Indianapolis, IN), which contains all necessary reagents and disposables. To optimize DNA recovery and enhance cell deactivation, an optional external lysis protocol was utilized prior to the automated MagNA Compact extraction using the DNA Blood External Lysis Purification protocol. This included combining 200 µl of the blood sample with 300 µl of the MagNA Pure LC DNA Isolation Kit I – Lysis/Binding Buffer, mixing, and incubating at room temperature for 30 min. The MagNA LC utilized the MagNA Pure LC DNA Isolation Kit I and the same external lysis was completed, as described above, and DNA extracted using the DNA Blood External Lysis Purification protocol, as described above. The positive control, for the real-time PCR detection, was a whole-cell lysate of B. pseudomallei (ATCC 23343) produced as described previously by Hoffmaster et al. [24], which provided crossing threshold (CT) values ranging between 23 and 28 cycles. As a precaution all DNA extracts were filtered using 0.22-µm centrifugal filter units (Millipore, Corporation, Billerica, MA) and then an aliquot was plated to assess removal of viable cells. All extracted DNA samples were stored at −20°C in their provided elution buffers, until analyzed by real-time PCR. Both water and unspiked blood were processed alongside spiked blood and served as extraction controls to determine if cross contamination occurred.

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Manufacturer protocol

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