Carry-over prevention using UNG (optional)
Carry over contamination between PCR reactions can be prevented by including uracil-N-
glycosylase (UNG) in the reaction mix. 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 (95°C for 10 minutes).
7 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Brucella genus (all species)
Quantification of
For general laboratory and research use only
L-glutamine: 2-deoxy-scyllo-inosose amino-transferase
PrimerDesign   Ltd TM
150 tests
Standard kit
1 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05The Brucella genus is a genus of Gram-negative, non-motile bacteria that are rod shaped.
Species within this genus cause Brucellosis in many species of mammals. Brucella species have
a genome of around 3.2M nucleotides in length split over two circular chromosomes.
All Brucella species cause infections in specific animal groups that lead to similar diseases.
Differences between the bacterium within this genus can be found in genes coding for surface
exposed proteins. B. suis infects swine, whereas B. melitensis infects goats and camels B. canis
infects dogs. Cattle are mainly infected by B. abortis, with B. ovis infecting sheep and B.
pinnipediae recently though to infect marine mammals. These bacterial species cause male
infertility resulting from orchitis and epididymitis and may also cause abortion in females due
to andometritis and placentitis.
These bacteria are also capable of infecting humans and can be transmitted via inhalation of
infected animal waste products, direct contact with open wounds or ingestion of
contaminated food subatnaces such as un-pasteurised milk. In humans, Brucella infection can
result in headache, fatigue, fever, sweating, weight loss and muscle pain. The human infection
can be treated by a course of antibiotics in combination to prevent symptoms.
Introduction to Brucella genus (all species)
2 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Specificity
The primers have 100% homology with all Brucella reference sequences included in the
NCBI database and therefore have a very broad detection profile (CP001488.1,
AE008917.1, P000887.1, CP000911.1, CP000872.1, CP000708.1, AE014291.4AF047478.1,
AM040264.1, AE017223.1).
The PrimerDesign™ genesig Kit for Brucella genus (all species) (Brucella_spp) Genomes is
designed for the in vitro quantification of Brucella_spp genomes. The kit is designed to have
the broadest detection profile possible whilst remaining specific to the Brucella_spp genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
clinically relevant reference 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 enquiries@primerdesign.co.uk and our bioinformatics
team will answer your question.
3 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Kit Contents
• Brucella_spp specific primer/probe mix (150 reactions BROWN)
   FAM labeled, BHuenched
• Brucella_spp positive control template (for Standard curve RED)
• RNAse/DNAse free water
Reagents and equipment to be supplied by the user
Real-Time PCR Instrument
DNA extraction kit
This kit is designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.
oasig
TM
 Lyophilised 2 x qPCR Mastermix
This kit is designed to work well with all commercially available Mastermixes. However, we
recommend the use of oasig
TM
 2 x qPCR MasterMix.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
4 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival.
PrimerDesign does not recommend using the kit after the expiry date stated on the pack.
Once the lyophilized components have been re-suspended, unnecessary repeated
freeze/thawing should be avoided. The kit is stable for six months from the date of
resuspension under these circumstances.
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 RNA/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 RNA
sample with RNAse/DNAse free water.
Dynamic range of test
Under optimal PCR conditions PrimerDesign Brucella_spp detection kits have very high
priming efficiencies of >95% and can detect less than 100 copies of target template.
Notices and disclaimers
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. Black Hole Quencher”, “BHQ”, “CAL Fluor, “Quasar” and “Pulsar” are registered trademarks of Biosearch
Technologies, Inc., Novato, CA. This technology is protected by U.S. and World-wide patents either issued or in application and is licensed
and sold under agreement with Biosearch Technologies, Inc. These products are sold exclusively for R&D use by the purchaser. They may
not be used for human or veterinary in vitro diagnostic (IVD) applications and they may not be re-sold, distributed or re-packaged without
express written authorization from Biosearch Technologies Inc. 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. 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.
The purchase of Biosearch Technologies products does not, either expressly or by implication, provide a license to use this or other patented
technology. Licensing information can be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive,
Foster City, CA 94404 or the Licensing Department at Roche Molecular Systems Inc., 1145 Atlantic Avenue, Alameda, CA 94501.”
Trademarks
PrimerDesign ™ is a trademark of PrimerDesign Ltd.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La Roche AG.
ABI, 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.
5 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Principles of the test
Real-time PCR
A Brucella_spp 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 Brucella_spp DNA/cDNA. 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 Brucella_spp
copy number / CT 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 Brucella_spp 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 confirm the absence of contamination, 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. If a positive result is obtained the results should be
ignored and the test samples repeated. Possible sources of contamination should first be
explored and removed.
6 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Component Volume
Brucella_spp Primer/Probe mix (BROWN) 165 µl
Positive Control Template (RED) * 500 µl
Pre-PCR transparent envelope
Post-PCR heat-sealed foil
Bench side 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.
* This component contains high copy number template and is a VERY significant contamination risk. It must be
opened and handled in a separate laboratory environment, away from the other components.
8 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Component Volume
oasig
TM
 2 x qPCR MasterMix
1 µl Brucella_spp Primer/Probe mix (BROWN)
Final Volume 15 µl
10 µl
RNAse/DNAse free water (WHITE) 4 µl
Real-time PCR detection
1. Prepare a reaction mix according to the tables below:
Include sufficient reactions for the standard curve wells (6 samples in duplicate) and also
the negative control.
Brucella_spp detection mix
2. Pipette 15µl of this mix into each well according to your real-time PCR experimental
plate set up.
3. Prepare sample DNA templates for each of your samples (suggested concentration
5ng/µl) in RNAse/DNAse free water.
If the concentration of DNA is not known, then dilute your DNA sample reactions  1:20
(10µl of sample DNA and 190µl of water).
4. Pipette 5µl of diluted DNA template into each well, according to your experimental
plate set up.
For negative control wells use 5µl of RNAse/DNAse free water. The final volume in
each well is 20µl.
5. Preparation of standard curve dilution series.
1) Pipette 900µl of RNAse/DNAse free water into 5 tubes and label 2-6
2) Pipette 100µl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 100µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
9 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05Standard Curve Copy Number
Tube 1 Positive control (RED) 2 x 10
5
 per µl
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
2 x 10
4
 per µl
2 x 10
3
 per µl
2 x 10
2
 per µl
20 per µl
2 per µl
Standard Curve Step
UNG treatment (if required) **
Enzyme activation (if required)
Denaturation
DATA COLLECTION *
Time Temp
15 mins
15 mins
10s
60s
37 
o
C
95 
o
C
95 
o
C
60 
o
C
50 Cycles
6. Pipette 5µl of standard template into each well, according to your experimental
plate set up.
The final volume in each well is 20µl.
Amplification Protocol
Amplification conditions using oasig
TM
 2x qPCR MasterMix.
* Fluorogenic data for the control DNA should be collected during this step through the FAM and VIC channels
** Required if your Mastermix includes UNG to prevent PCR carryover contamination
10 Quantification of Brucella genus (all species) genomes.
genesig Standard kit handbook HB10.04.05