Abstract
Burkholderia pseudomallei, the etiologic agent of melioidosis, is endemic in northern Australia and Southeast Asia and can cause severe septicemia that may lead to death in 20% to 50% of cases. Rapid detection of B. pseudomallei infection is crucial for timely treatment of septic patients. This study evaluated seven commercially available DNA extraction kits to determine the relative recovery of B. pseudomallei DNA from spiked EDTA-containing human whole blood. The evaluation included three manual kits: the QIAamp DNA Mini kit, the QIAamp DNA Blood Mini kit, and the High Pure PCR Template Preparation kit; and four automated systems: the MagNAPure LC using the DNA Isolation Kit I, the MagNAPure Compact using the Nucleic Acid Isolation Kit I, and the QIAcube using the QIAamp DNA Mini kit and the QIAamp DNA Blood Mini kit. Detection of B. pseudomallei DNA extracted by each kit was performed using the B. pseudomallei specific type III secretion real-time PCR (TTS1) assay. Crossing threshold (CT) values were used to compare the limit of detection and reproducibility of each kit. This study also compared the DNA concentrations and DNA purity yielded for each kit. The following kits consistently yielded DNA that produced a detectable signal from blood spiked with 5.5×104 colony forming units per mL: the High Pure PCR Template Preparation, QIAamp DNA Mini, MagNA Pure Compact, and the QIAcube running the QIAamp DNA Mini and QIAamp DNA Blood Mini kits. The High Pure PCR Template Preparation kit yielded the lowest limit of detection with spiked blood, but when this kit was used with blood from patients with confirmed cases of melioidosis, the bacteria was not reliably detected indicating blood may not be an optimal specimen.
Highlights
Burkholderia pseudomallei, a Gram-negative bacterium, is the etiologic agent of melioidosis; a disease with varying severity that can affect both human and animal populations
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 polymerase chain reaction (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
There was no observable correlation in DNA concentration with the addition of bacteria, which is not unexpected due to the relatively large mass of DNA provided by human cells compared to the bacteria added
Summary
Burkholderia pseudomallei, a Gram-negative bacterium, is the etiologic agent of melioidosis; a disease with varying severity that can affect both human and animal populations. B. pseudomallei is recognized as a Select Agent by the Centers for Disease Control and Prevention based on the criteria of infectivity, severity, and environmental prevalence [1]. Melioidosis is endemic in Southeast Asia and northern Australia but may occur in other tropical regions of the world [2]. In its acute form, melioidosis can manifest as a severe septicemia, which, without prompt diagnosis is often fatal [3,4]. B. pseudomallei is resistant to many of the broad spectrum antibiotics often used for treatment of sepsis [4]. Rapid diagnosis of melioidosis allows for prompt treatment with appropriate antibiotics and reduces mortality [4]
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