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


CONCLUSIONS
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.
Panning
Marcus
M
Clinical Virology, Bernhard-Nocht Institute for Tropical Medicine, Bernhard Nocht Str, 74, 20359 Hamburg, Germany. panning@virology-bonn.de
Kilwinski
Jochen
J
Greiner-Fischer
Susanne
S
Peters
Martin
M
Kramme
Stefanie
S
Frangoulidis
Dimitrios
D
Meyer
Hermann
H
Henning
Klaus
K
Drosten
Christian
C
D000820:Animal Diseases / Q000382:microbiology; D000818:Animals; D001331:Automation; D002460:Cell Line; D016997:Coxiella burnetii / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D006801:Humans; D008297:Male; D016133:Polymerase Chain Reaction / Q000379:methods*; D011778:Q Fever / Q000382:microbiology / Q000662:veterinary; D012680:Sensitivity and Specificity; D013048:Specimen Handling
D004269:DNA, Bacterial