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2006-01-19Zeitschriftenartikel DOI: 10.1186/1471-2180-6-2
Highly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii
dc.contributor.authorKlee, Silke
dc.contributor.authorTyczka, Judith
dc.contributor.authorEllerbrok, Heinz
dc.contributor.authorFranz, Tatjana
dc.contributor.authorLinke, Sonja
dc.contributor.authorBaljer, Georg
dc.contributor.authorAppel, Bernd
dc.date.accessioned2018-05-07T13:47:44Z
dc.date.available2018-05-07T13:47:44Z
dc.date.created2010-03-24
dc.date.issued2006-01-19none
dc.identifier.otherhttp://edoc.rki.de/oa/articles/reTTTZjIa1MKY/PDF/20Aq4hZnpxux6.pdf
dc.identifier.urihttp://edoc.rki.de/176904/613
dc.description.abstractBackground: Coxiella burnetii, 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 C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. Results: To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii 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 icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 107 starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 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 IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. Conclusion: We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.ger
dc.language.isoeng
dc.publisherRobert Koch-Institut
dc.subjectPolymerase Chain Reaction/methodseng
dc.subjectSensitivity and Specificityeng
dc.subjectCoxiella burnetii/geneticseng
dc.subjectCoxiella burnetii/isolation & purificationeng
dc.subjectDNA Transposable Elements/geneticseng
dc.subjectIsocitrate Dehydrogenase/geneticseng
dc.subject.ddc610 Medizin
dc.titleHighly sensitive real-time PCR for specific detection and quantification of Coxiella burnetii
dc.typeperiodicalPart
dc.identifier.urnurn:nbn:de:0257-1006249
dc.identifier.doi10.1186/1471-2180-6-2
dc.identifier.doihttp://dx.doi.org/10.25646/538
local.edoc.container-titleBMC Microbiology
local.edoc.fp-subtypeArtikel
local.edoc.type-nameZeitschriftenartikel
local.edoc.container-typeperiodical
local.edoc.container-type-nameZeitschrift
local.edoc.container-urlhttp://www.biomedcentral.com/1471-2180/6/2
local.edoc.container-publisher-nameBioMedCentral
local.edoc.container-volume6
local.edoc.container-issue2
local.edoc.container-year2006

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