Evaluation of a Real‐Time PCR Assay to Detect <i>Coxiella burnetii</i>

Klee, Silke R.; Ellerbrok, Heinz; Tyczka, Judith; Franz, Tatjana; Appel, Bernd

doi:10.1196/annals.1374.111

Cited by 24 publications

(19 citation statements)

References 5 publications

(4 reference statements)

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“…in 7 week-old female mice at day 1 (first challenge) and day 21 (second challenge). Spleens were harvested at day 32 and analyzed for the presence of C. burnetii by real-time qPCR as previously described [29]. Only those samples displaying a Ct value <25 were kept for processing in the second step, (ii) which consisted in C. burnetii amplification in seven-day old embryonated chicken eggs obtained by injection in the yolk sac of 100 µl of a 1∶10 dilution of the spleen homogenate in phosphate buffered saline (PBS) from the previous step.…”

Section: Methodsmentioning

confidence: 99%

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

et al. 2013

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Q-fever is a zoonosis caused by the gram-negative obligate intracellular pathogen Coxiella burnetii. Since its discovery, and particularly following the recent outbreaks in the Netherlands, C. burnetii appeared as a clear public health concern. In the present study, the infectious potential displayed by goat and cattle isolates of C. burnetii was compared to a reference strain (Nine Mile) using both in vitro (human HeLa and bovine macrophage cells) and in vivo (BALB/c mice) models. The isolates had distant genomic profiles with one - the goat isolate - being identical to the predominant strain circulating in the Netherlands during the 2007–2010 outbreaks. Infective doses were established with ethidium monoazide-PCR for the first time here applied to C. burnetii. This method allowed for the preparation of reproducible and characterized inocula thanks to its capacity to discriminate between live and dead cells. Globally, the proliferative capacity of the Nine Mile strain in cell lines and mice was higher compared to the newly isolated field strains. In vitro, the bovine C. burnetii isolate multiplied faster in a bovine macrophage cell line, an observation tentatively explained by the preferential specificity of this strain for allogeneic host cells. In the BALB/c mouse model, however, the goat and bovine isolates multiplied at about the same rate indicating no peculiar hypervirulent behavior in this animal model.

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Section: Methodsmentioning

confidence: 99%

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

et al. 2013

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“…Signature sequences most commonly used for the detection of C. burnetii DNA are plasmid sequences (QpH1 or QpRS) or chromosomal genes such as isocitrate-dehydrogenase (icd), the outer membrane protein-coding gene com1, the superoxide dismutase gene (sod), or the transposase gene in insertion element IS1111. The latter target is a preferred target for PCR assays due to its presence in multiple copies within the genome, thereby enhancing the sensitivity of detection (16,35). However, since the number of IS1111 copies in the C. burnetii genome varies between strains, the direct quantification of the number of organisms within a sample using this target is more complicated (17).…”

mentioning

confidence: 99%

“…More recently, other types of PCR assays, like nested PCR (41,44) and quantitative PCR (qPCR) (6,7,16,17), have been developed, sometimes in combination with high-throughput capabilities (26). PCR-based methods target one or more specific sequences in the genome, most often in separate (singleplex) assays.…”

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confidence: 99%

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Bruin

Groot

Heer

et al. 2011

Appl Environ Microbiol

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Q fever, caused by Coxiella burnetii, is a zoonosis with a worldwide distribution. A large rural area in the southeast of the Netherlands was heavily affected by Q fever between 2007 and 2009. This initiated the development of a robust and internally controlled multiplex quantitative PCR (qPCR) assay for the detection of C. burnetii DNA in veterinary and environmental matrices on suspected Q fever-affected farms. The qPCR detects three C. burnetii targets (icd, com1, and IS1111) and one Bacillus thuringiensis internal control target (cry1b). Bacillus thuringiensis spores were added to samples to control both DNA extraction and PCR amplification. The performance of the qPCR assay was investigated and showed a high efficiency; a limit of detection of 13.0, 10.6, and 10.4 copies per reaction for the targets icd, com1, and IS1111, respectively; and no crossreactivity with the nontarget organisms tested. Screening for C. burnetii DNA on 29 suspected Q fever-affected farms during the Q fever epidemic in 2008 showed that swabs from dust-accumulating surfaces contained higher levels of C. burnetii DNA than vaginal swabs from goats or sheep. PCR inhibition by coextracted substances was observed in some environmental samples, and 10-or 100-fold dilutions of samples were sufficient to obtain interpretable signals for both the C. burnetii targets and the internal control. The inclusion of an internal control target and three C. burnetii targets in one multiplex qPCR assay showed that complex veterinary and environmental matrices can be screened reliably for the presence of C. burnetii DNA during an outbreak.

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“…Several PCR-based assays have been developed in the past decade [1,37,40,41]. Although lacking sensitivity, PCR targeting the htpAB -associated repetitive element, which is present in 20 copies in the genome of C. burnetii [10], is routinely used to detect bacteria in cell cultures and clinical samples from both acute and chronic Q fever patients [1].…”

Section: Host-bacteria Interactionsmentioning

confidence: 99%

“…This test, together with serology, is recommended in the first 2 weeks of acute Q fever [37]. Real-time quantitative PCR assay targeting the multicopy insertion sequences IS1111 and IS30a is also highly specific and sensitive [40,41]. Detection of the adaA gene (encoding acute disease antigen A) can be used to confirm acute Q fever [1].…”

Section: Host-bacteria Interactionsmentioning

confidence: 99%

Proteomics paves the way for Q fever diagnostics

2011

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Q fever is a worldwide zoonosis caused by Coxiella burnetii. The disease most frequently manifests clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness that presents mainly as infective endocarditis. The extreme infectivity of the bacterium results in large outbreaks, and the recent outbreak in the Netherlands underlines its impact on public health. Recent studies on the bacterium have included genome sequencing, the investigation of host-bacterium interactions, the development of cellular and animal models of infection, and the comprehensive analysis of different clinical isolates by whole genome and proteomic approaches. Current approaches for diagnosing Q fever are based on serological methods and PCR techniques, but the diagnosis of early stage disease lacks specificity and sensitivity. Consequently, different platforms have been created to explore Q fever biomarkers. Several studies using a combination of proteomics and recombinant protein screening approaches have been undertaken for the development of diagnostics and vaccines. In this review, we highlight advances in the field of C. burnetii proteomics, focusing mainly on the contribution of these technologies to the development and improvement of Q fever diagnostics.

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Evaluation of a Real‐Time PCR Assay to Detect Coxiella burnetii

Cited by 24 publications

References 5 publications

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

In Vitro and In Vivo Infectious Potential of Coxiella burnetii: A Study on Belgian Livestock Isolates

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

Proteomics paves the way for Q fever diagnostics

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