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High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation.

Abstract：

BACKGROUND: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 weapon attack 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 copies that are detectable at 95% chance probit analysis was used. Limit of detection in blood was 2,881 copies/ml [95%CI, 2,188-4,745 copies/ml] with a manual extraction procedure and 4,235 copies/ml [95%CI, 3,143-7,428 copies/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.

journal_name

BMC Microbiol

journal_title

BMC microbiology

authors

Panning M,Kilwinski J,Greiner-Fischer S,Peters M,Kramme S,Frangoulidis D,Meyer H,Henning K,Drosten C

doi

10.1186/1471-2180-8-77

subject

Has Abstract

pub_date

2008-05-19 00:00:00

pages

issn

1471-2180

pii

1471-2180-8-77

journal_volume

pub_type

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High throughput detection of Coxiella burnetii by real-time PCR with internal control system and automated DNA preparation.