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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.

SUBMITTER: Panning M

PROVIDER: S-EPMC2397412 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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

Panning Marcus M 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

BMC microbiology 20080519

<h4>Background</h4>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 g ...[more]

PMID: 18489780

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Project description:BACKGROUND: 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 10(7) 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.

Project description:BackgroundQ fever, a zoonosis caused by Coxiella burnetii, has adverse effects on public health. Ticks are vectors of C. burnetii and they contribute to the transmission of the pathogen. A tool for rapid, sensitive, and accurate detection of C. burnetii from ticks is important for the prevention of Q fever.MethodsUltra-rapid real-time PCR (UR-qPCR) as a chip-based real-time PCR system was developed for the detection of C. burnetii from ticks. The UR-qPCR system was established and evaluated for the rapidity, sensitivity, and specificity of C. burnetii detection.ResultsC. burnetii was detected using UR-qPCR from 5644 larval, nymphal, and adult ticks from 408 pools collected from livestock and epidemiologically linked environments in two provinces, Gangwon and Jeju, in Korea. Ticks from three species were identified; Haemaphysalis longicornis accounted for the highest number, present in 333 of 408 pools (81.62%), followed by Haemaphysalis flava in 62 pools (15.19%) and Ixodes nipponensis in 13 pools (3.19%). The rapidity and sensitivity of PCR detection was demonstrated with the sufficient amplification and detection of approximately 56 copies of C. burnetii DNA with only 20 min of PCR amplification. The kappa value for the diagnostic agreement between UR-qPCR and stationary qPCR was in perfect agreement (κ = 1). PCR detection and sequencing indicated that C. burnetii was present in 5 of the 408 pools (1.23%), in which four pools contained H. longicornis and one pool contained H. flava. The infection rates of C. burnetii in the tick pools collected from Gangwon and Jeju Provinces were 1.70% and 0.58%, respectively. Phylogenetic analysis indicated a close relationship between the detected C. burnetii and those originating from goats, humans, and ticks in different countries, such as the USA, France, Germany, and Serbia.ConclusionsThe methods described in this study could be important for the prevention and control of Q fever in the two provinces. The UR-qPCR, with its features of mobility, sensitivity, and rapidity, is helpful for constructing early alert systems in the field for C. burnetii in ticks and could help alleviate the transmission of and economic damage due to Q fever.

Project description:Introduction:Query (Q) fever is an important zoonosis and a cause of concern for humans, due to the potential bioterrorism threat posed by the causative agent, Coxiella burnetii. Because of the danger of contracting the illness, isolation attempts are seldom made. Serological and molecular diagnostic tests are the main option. Aim:To study the prevalence of acute Q fever in Puducherry and surrounding districts of Tamil Nadu, India, employing a new commercial Real-Time Polymerase Chain Reaction (RT-PCR) kit and confirming it by the gold standard Immunofluorescence Assay (IFA). Materials and Methods:Acute phase blood samples from 72 consecutive febrile patients and 24 healthy individuals were included in this prospective study. DNA was extracted from the buffy coats and preserved at -80°C. Detection of C. burnetii was carried out employing a commercial Real-Time PCR kit. Serum samples were tested for IgM (Phase I+II) and IgG (Phase I+II) by QM-120 and QG-120, Coxiella burnetii IFA Fuller Laboratories, California, USA. Sensitivity, Specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were calculated keeping IFA as the reference. Results:Presumptive diagnosis of acute Q fever was made in two febrile patients by the Genesig Easy kit (2.78%). In addition to these two PCR positive cases, one more patient was positive for both Phase II IgM and Phase II IgG antibodies by the gold standard IFA. All 24 healthy controls were negative for Q fever by both PCR and IFA. The sensitivity, specificity, NPV and PPV for Genesig Easy kit PCR were: 66.67%, 100%, 100% and 98.57 % respectively against IFA as the reference. Conclusion:The true prevalence of Q fever in India and other developing countries is poorly understood, owing to the difficulties in the diagnosis of this infection. Since molecular diagnostic tests have good specificity and are mandated for confirmation of single acute samples, validation of commercial Q fever PCR kits is the need of the hour. Genesig Easy kit in our hands was found to be reliable with the moderate sensitivity and high specificity. Performing both PCR (with acute specimens) and IFA (with paired sera) would be ideal for Q fever diagnosis.

Dataset Information

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

Publications

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

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