High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation
Coxiella burnetii is the causative agent of Q-fever, a widespread zoonosis. Due to its high environmental stability and infectivity it is regarded as a category B biological weapon agent.
In domestic animals infection remains either asymptomatic or presents as infertility or abortion. Clinical presentation in humans can range from mild flu-like illness to acute pneumonia and hepatitis.
Endocarditis represents the most common form of chronic Q-fever. In humans serology is the gold standard for diagnosis but is inadequate for early case detection.
In order to serve as a diagnostic tool in an eventual biological weaponattack or in local epidemics we developed a real-time 5'nuclease based PCR assay with an internal control system. To facilitate high-throughput an automated extraction procedure was evaluated.
Results: To determine the minimum number of genome equivalents (geq) that are detectable at 95% chance probit analysis was used.
Limit of detection in blood was 2,881 geq/ml [95%CI, 2,188-4,745] with a manual extraction procedure and 4,235 geq/ml (95%CI, 3,143-7,428 geq/ml) with a fully automated extraction procedure, respectively. To demonstrate clinical application a total of 72 specimens of animal origin were compared with respect to manual and automated extraction.
A strong correlation between both methods was observed rendering both methods suitable. Testing of 247 follow up specimens of animal origin from a local Q-fever epidemic rendered real-time PCR more sensitive than conventional PCR.
Conclusion: A sensitive and thoroughly evaluated real-time PCR was established.
Its high-throughput mode may show a useful approach to rapidly screen samples in local outbreaks for other organisms relevant for humans or animals. Compared to a conventional PCR assay sensitivity of real-time PCR was higher after testing samples from a local Q-fever outbreak.
Author: Marcus Panning, Jochen Kilwinski, Susanne Greiner-Fischer, Martin Peters, Stefanie Kramme, Dimitrios Frangoulidis, Hermann Meyer, Klaus Henning and Christian Drosten
Credits/Source: BMC Microbiology 2008, 8:77
Published on: 2008-05-19
Copyright by the authors listed above - made available via BioMedCentral (Open Access). Please
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