Coxiella burnetii

Coxiella burnetii (C. burnetii), an obligate intracellular gram-negative bacteriu m, is the causative agent of Q fever. C. burnetii multiplies only within the phagolysosomal vacuoles , particularly the macrophages of the host. During natural infections, the organism g rows to high numbers in placental tissues of animal s such as goats, sheep, and cows. The Center for Dise ase Control and Prevention (CDC) has classified C. burnetii as a category B biological terrorist agent because it consistently causes disability, can be manufactured on a large scale, remains stable under production, storage, and transportation conditions , can be efficiently disseminated and remains viable for years after dissemination.  Q fever, a zoonotic disease found worldwide, may ma nifest as acute or chronic disease. The acute form is generally not fatal and manifests as self-controlle d febrile illness. Chronic Q fever is usually chara cterized by endocarditis. Many animal models, including huma ns, have been studied for Q fever infection through  various exposure routes.


Specificity MIN MAX
The Primerdesign™ genesig ® Kit for Coxiella burnetii (C.burnetii) genomes is designed for the in vitro quantification of C.burnetii genomes.The kit is designed to have the broadest detection profile possible whilst remaining specific to the C.burnetii genome.
The primers and probe sequences in this kit have 100% homology with a broad range of C. burnetii sequences based on a comprehensive bioinformatics analysis.
If you require further information, or have a specific question about the detection profile of this kit then please send an e.mail to enquiry@primerdesign.co.uk and our bioinformatics team will answer your question.
3  This kit is stable at room temperature but should be stored at -20ºC on arrival.Once the lyophilised components have been re-suspended they should not be exposed to temperatures above -20°C for longer than 30 minutes at a time and unnecessary repeated freeze/thawing should be avoided.The kit is stable for six months from the date of resuspension under these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended period.
If you see any degradation in this serial dilution a fresh standard curve can be prepared from the positive control.
Primerdesign does not recommend using the kit after the expiry date stated on the pack.

Suitable sample material
All kinds of sample material suited for PCR amplification can be used.Please ensure the samples are suitable in terms of purity, concentration, and DNA integrity (An internal PCR control is supplied to test for non specific PCR inhibitors).Always run at least one negative control with the samples.To prepare a negative-control, replace the template DNA sample with RNAse/DNAse free water.

Dynamic range of test
Under optimal PCR conditions genesig ® C.burnetii detection kits have very high priming efficiencies of >95% and can detect less than 100 copies of target template.

Notices and disclaimers
This product is developed, designed and sold for research purposes only.It is not intended for human diagnostic or drug purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug Administration in the USA or the appropriate regulatory authorities in the country of use.

Trademarks
Primerdesign™ is a trademark of Primerdesign Ltd.Principles of the test

Real-time PCR
A C.burnetii specific primer and probe mix is provided and this can be detected through the FAM channel.
The primer and probe mix provided exploits the so-called TaqMan® principle.During PCR amplification, forward and reverse primers hybridize to the C.burnetii DNA.A fluorogenic probe is included in the same reaction mixture which consists of a DNA probe labeled with a 5`-dye and a 3`-quencher.During PCR amplification, the probe is cleaved and the reporter dye and quencher are separated.The resulting increase in fluorescence can be detected on a range of real-time PCR platforms.

Positive control
For copy number determination and as a positive control for the PCR set up, the kit contains a positive control template.This can be used to generate a standard curve of C.burnetii copy number / Cq value.Alternatively the positive control can be used at a single dilution where full quantitative analysis of the samples is not required.Each time the kit is used, at least one positive control reaction must be included in the run.A positive result indicates that the primers and probes for detecting the target C.burnetii gene worked properly in that particular experimental scenario.If a negative result is obtained the test results are invalid and must be repeated.Care should be taken to ensure that the positive control does not contaminate any other kit component which would lead to false-positive results.This can be achieved by handling this component in a Post PCR environment.Care should also be taken to avoid cross-contamination of other samples when adding the positive control to the run.This can be avoided by sealing all other samples and negative controls before pipetting the positive control into the positive control well.

Negative control
To validate any positive findings a negative control reaction should be included every time the kit is used.For this reaction the RNAse/DNAse free water should be used instead of template.A negative result indicates that the reagents have not become contaminated while setting up the run.
C.burnetii DNA is known to be highly prevalent within the air and environment generally and the negative control may therefore give a late positive signal due to environmental contamination.The interpretation of results section of this handbook gives guidance on how to interpret results where environmental contamination is evident.

Internal DNA extraction control
When performing DNA extraction, it is often advantageous to have an exogenous source of DNA template that is spiked into the lysis buffer.This control DNA is then co-purified with the sample DNA and can be detected as a positive control for the extraction process.Successful co-purification and real-time PCR for the control DNA also indicates that PCR inhibitors are not present at a high concentration.
A separate primer and probe mix are supplied with this kit to detect the exogenous DNA using real-time PCR.The primers are present at PCR limiting concentrations which allows multiplexing with the target sequence primers.Amplification of the control DNA does not interfere with detection of the C.burnetii target DNA even when present at low copy number.The Internal control is detected through the VIC channel and gives a Cq value of 28+/-3.

Endogenous control
To confirm extraction of a valid biological template, a primer and probe mix is included to detect an endogenous gene.Detection of the endogenous control is through the FAM channel and it is NOT therefore possible to perform a multiplex with the C.burnetii primers.A poor endogenous control signal may indicate that the sample did not contain sufficient biological material.

Carry-over prevention using UNG (optional)
Carry over contamination between PCR reactions can be prevented by including uracil-Nglycosylase (UNG) in the reaction mix.

Reconstitution Protocol
To minimize the risk of contamination with foreign DNA, we recommend that all pipetting be performed in a PCR clean environment.Ideally this would be a designated PCR lab or PCR cabinet.Filter tips are recommended for all pipetting steps.
1. Pulse-spin each tube in a centrifuge before opening.This will ensure lyophilised primer and probe mix is in the base of the tube and is not spilt upon opening the tube.
2. Reconstitute the kit components in the RNAse/DNAse free water supplied, according to the table below: To ensure complete resuspension, vortex each tube thoroughly.
genesig® is a registered trademark of Primerdesign Ltd.The PCR process is covered byUS Patents 4,683,195, and 4,683,202  and foreign equivalents owned by Hoffmann-La Roche AG.BI, ABI PRISM® GeneAmp® and MicroAmp® are registered trademarks of the Applera Genomics (Applied Biosystems Corporation).BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCycler™ is a registered trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of Corbett Research.LightCycler™ is a registered trademark of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular Systems, Inc., The purchase of the Primerdesign ™ reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by Hoffmann-LaRoche Inc.

oasig TM Lyophilised or PrecisionPLUS TM 2x qPCR Mastermix This
• Template preparation buffer (YELLOW)for resuspension of positive control template and standard curve preparation Reagents and equipment to be supplied by the userReal-Time PCR InstrumentDNA extraction kitThis kit is recommended for use with genesig ® Easy DNA/RNA extraction kit.However, it is designed to work well with all processes that yield high quality DNA with minimal PCR inhibitors.kit is designed to work well with all commercially available Mastermixes.However, we recommend the

use of oasig TM or PrecisionPLUS TM 2x qPCR Mastermix. Pipettors and Tips Vortex and centrifuge Thin walled 1.5 ml PCR reaction tubes 4
Additional information on purchasing licenses to practice the PCR process may be obtained by contacting the Director of Licensing at Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501 or Applied Biosystems business group of the Applera Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404.In addition, the 5' nuclease assay and other homogeneous amplification methods used in connection with the PCR process may be covered by U.S. Patents 5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc, and by U.S. Patent 5,538,848, owned by The Perkin-Elmer Corporation.
During the warranty period Primerdesign genesig ® detection kits allow precise and reproducible data recovery combined with excellent sensitivity.For data obtained by violation to the general GLP guidelines and the manufacturer's recommendations the right to claim under guarantee is expired.PCR is a proprietary technology covered by several US and foreign patents.These patents are owned by Roche Molecular Systems Inc. and have been sub-licensed by PE Corporation in certain fields.Depending on your specific application you may need a license from Roche or PE to practice PCR.
Some commercial mastermix preparations contain UNG or alternatively it can be added as a separate component.UNG can only prevent carry over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the original PCR reaction.Primerdesign recommend the application of 0.2U UNG per assay with a 15 minute incubation step at 37°C prior to amplification.The heat-labile UNG is then inactivated during the Taq polymerase activation step.