A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay
Lockhart, M.G., Graves, S.R., Banazis, M.J., Fenwick, S.G. and Stenos, J. (2011) A comparison of methods for extracting DNA from Coxiella burnetii as measured by a duplex qPCR assay. Letters in Applied Microbiology, 52 (5). pp. 514-520.
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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.
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.
|Publication Type:||Journal Article|
|Murdoch Affiliation:||School of Veterinary and Biomedical Sciences|
|Copyright:||2011 The Authors|
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