A comparison of methods for extracting DNA fromCoxiella burnetiias measured by a duplex qPCR assay


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Abstract

Aims:To determine the optimal DNA extraction method for the detection of Coxiella burnetii including the small-cell variant (SCV) by real-time PCR (qPCR) in clinical samples.Methods and Results:A duplex qPCR detecting two Coxiella burnetii gene targets (com1 and IS1111a genes) was developed. Each target in this PCR had a sensitivity of one copy number per reaction. DNA extraction methods were compared on spiked negative samples and included a silica column kit, a chloroform separation prior to a silica column method and a chloroform/phenol separation and DNA precipitation method.Conclusions:The silica column extraction method was more efficient at recovering C. burnetii DNA, from large-cell and small-cell variants, than a chloroform or chloroform/phenol method. The silica column method was useful on spiked human samples including serum, buffy coat and bone marrow samples.Significance and impact of study:This study demonstrated that a simple column kit method is efficient to use for the detection of C. burnetii in clinical samples including the SCV.

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