title: 'Real-time' PCR-based detection of Coxiella burnetii using conventional techniques.
abstract: The diagnosis of Q fever (Coxiella burnetii infection) relies primarily on the serological detection of specific antibodies. Recently, PCR-based methods have been introduced in diagnostic laboratories. Unfortunately, the fastest and most reliable 'real-time' detection method, which employs the 'online' detection of target nucleotide sequences while the amplification process is still in progress, requires expensive devices and consumables. In this study, we present a simple method that combines the simplicity of conventional PCR with new technical and methodical enhancements, resulting in a fast, specific and easy method for the molecular detection of C. burnetii. A collection of C. burnetii reference strains was tested with the modified conventional gel-based PCR approach applying a particluar PCR buffer (QIAGEN(®) Fast Cycling PCR kit) and using a closed ready-to-use gel-cassette-system (FlashGel(®)) for the visualization of specific PCR products. The modified conventional PCR method reached nearly the speed of the LightCycler(®) HybProbe real-time PCR assay (120 vs. 90 min) and showed equal sensitivity and specificity. The general cost per PCR run was 25% less than that for the LightCycler method. These improvements make this method suitable for small laboratories with limited resources and for deployable PCR diagnostics in field laboratories.
authors.affiliation: Bundeswehr Institute of Microbiology, Munich, Germany. dimitriosfrangoulidis@bundeswehr.org
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Frangoulidis
authors.forename: Hermann
authors.initials: H
authors.lastname: Meyer
authors.forename: Claudia
authors.initials: C
authors.lastname: Kahlhofer
authors.forename: Wolf D
authors.initials: WD
authors.lastname: Splettstoesser
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods*; D003365:Costs and Cost Analysis; D016997:Coxiella burnetii / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D011778:Q Fever / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors