Highly sensitive real-time PCR for specific detection and quantification of <it>Coxiella burnetii</it>

<p>Abstract</p> <p>Background</p> <p><it>Coxiella burnetii</it>, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect <it>C. burnetii </it>DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular <it>icd </it>(isocitrate dehydrogenase) gene and the transposase of the <it>IS1111a </it>element present in multiple copies in the <it>C. burnetii </it>genome.</p> <p>Results</p> <p>To evaluate the precision of the <it>icd </it>and <it>IS1111 </it>real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the <it>C. burnetii </it>Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the <it>icd </it>marker and 6.5 for the <it>IS </it>marker. Plasmid standards with cloned <it>icd </it>and <it>IS1111 </it>fragments were used to establish standard curves which were linear over a range from 10 to 10⁷starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated <it>Coxiella </it>isolate with a detection limit of 17 <it>C. burnetii </it>particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different <it>C. burnetii </it>isolates originating from all over the world. Using this approach, the number of <it>IS1111 </it>elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of <it>IS1111 </it>elements varied widely (between seven and 110) and seemed to be very high in some isolates.</p> <p>Conclusion</p> <p>We validated TaqMan-based real-time PCR assays targeting the <it>icd </it>and <it>IS1111 </it>markers of <it>C. burnetii</it>. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a <it>C. burnetii </it>isolate were reliably quantified. PCR quantification suggested a high variability of the number of <it>IS1111 </it>elements in different <it>C. burnetii </it>isolates, which may be useful for further phylogenetic studies.</p>