IS6110 repeat region
Mycobacterium
tuberculosis complex
PCRmax Ltd qPCR test
TM
150 tests
For general laboratory and research use only
1
Mycobacterium tuberculosis complex is a group of bacterial pathogens responsible for
causing tuberculosis (TB) in humans. They are Gram-positive, non-motile, obligate
aerobes that are related to Actinomycetes. The Mycobacteria species within this group are
characterised by 99.9% similarity at the nucleotide level and identical 16S rRNA
sequences but they may differ widely in terms of their host tropisms, phenotypes, and
pathogenicity. Some of the members of the complex infect only one host species while
others such as Mycobacterium bovis have a wide host spectrum.
Transmission generally occurs via inhalation of infected aerosols with the bacteria able to
remain in the respiratory tract for long periods of time. In the alveoli, macrophages target
the infection but some bacteria manage to grow within the macrophage, avoiding the
immune response, and subsequently causing macrophage cell death allowing bacterial
spread via the blood to the kidneys, brain, bones and lymph nodes. Bacteria within this
group divide every 15 to 20 hours which is extremely slow compared to other bacteria.
They can survive in a dry state for weeks and withstand weak disinfectants but only grow
within a host organism.
Tuberculosis is most commonly seen as an infection of the lungs resulting in pulmonary TB
which has symptoms including weight loss, fever, and loss of appetite. Extrapulmonary TB
results from the bacterial spread from the lungs and causes variable symptoms depending
on the site of infection.
Species within this group include: Mycobacterium tuberculosis, the most common cause of
tuberculosis (TB); M. africanu, a species commonly found in West Africa which causes TB
in humans; M. Canettii which also causes TB and has been found in the Horn of Africa; M.
bovis which causes tuberculosis in bovine and humans and M. microti which causes
tuberculosis in voles but has also recently been reported to infect humans.
Introduction to Mycobacterium tuberculosis complex
2
MIN
MAX
The PCR Max qPCR Kit for Mycobacterium tuberculosis complex (M.
tuberculosis_complex) genomes is designed for the in vitro quantification of M.
tuberculosis_complex genomes. The kit is designed to have the broadest detection profile
possible whilst remaining specific to the M.tuberculosis_complex genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
M.tuberculosis_complex 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 help@pcrmax.com and our bioinformatics team will
answer your question.
Specificity
3
Kit Contents
• M.tuberculosis_complex specific primer/probe mix (150 reactions BROWN)
FAM labelled
• M.tuberculosis_complex positive control template (for Standard curve RED)
• Internal extraction control primer/probe mix (150 reactions BROWN)
VIC labelled as standard
• Internal extraction control DNA (150 reactions BLUE)
• Endogenous control primer/probe mix (150 reactions BROWN)
FAM labelled
• RNAse/DNAse free water (WHITE)
for resuspension of primer/probe mixes and internal extraction control DNA
• Template preparation buffer (YELLOW)
for resuspension of positive control template and standard curve preparation
Reagents and equipment to be supplied by the user
Real-Time PCR Instrument
DNA extraction kit
This kit designed to work well with all processes that yield high quality DNA with minimal
PCR inhibitors.
Lyophilised 2x qPCR Mastermix
This kit is designed to be compatible with all commercially available Mastermixes that run
with standard cycling conditions.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
4
Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the
lyophilised components have been resuspended they should not be exposed to
temperatures above -20ºC for longer than 30 minutes 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.
PCRmax 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 PCRmax M.tuberculosis_complex 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. During the warranty period PCRmax 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. 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.
Trademarks
PCRmax™ is a trademark of PCRmax 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. 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 PCRmax reagents cannot be construed as an authorization or implicit license to practice PCR under any patents held by
Hoffmann-LaRoche Inc.
5
Principles of the test
Real-time PCR
A M.tuberculosis_complex 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 M.tuberculosis_complex 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 M.
tuberculosis_complex 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 M.
tuberculosis_complex 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 crosscontamination 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.
6
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 M.tuberculosis_complex 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 M.
tuberculosis_complex 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. 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. PCRmax 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.
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Component - resuspend in water
Volume
M.tuberculosis_complex primer/probe mix (BROWN)
165 µl
Internal extraction control DNA (BLUE)
600 µl
Internal extraction control primer/probe mix (BROWN)
Endogenous control primer/probe mix (BROWN)
Pre-PCR pack
Pre-PCR heat-sealed foil
165 µl
165 µl
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.
* 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.
DNA extraction
The internal extraction control DNA can be added either to the DNA lysis/extraction buffer
or to the DNA sample once it has been resuspended in lysis buffer.
DO NOT add the internal extraction control DNA directly to the unprocessed biological
sample as this will lead to degradation and a loss in signal.
1. Add 4µl of the Internal extraction control DNA (BLUE) to each sample in DNA
lysis/extraction buffer per sample.
2. Complete DNA extraction according to the manufacturers protocols.
3. Reconstitute the positive control template in the template preparation buffer
supplied, according to the table below:
To ensure complete resuspension, vortex the tube thoroughly.
500 µl
Post-PCR heat-sealed foil
Component - resuspend in template preparation buffer
Volume
M.tuberculosis_complex Positive Control Template (RED) *
8
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
M.tuberculosis_complex primer/probe mix (BROWN)
Final Volume
1 µl
15 µl
10 µl
Internal extraction control primer/probe mix (BROWN)
RNAse/DNAse free water (WHITE)
3 µl
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
Endogenous control primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl
Real-time PCR detection protocol
1. For each DNA sample prepare a reaction mix according to the table below:
Include sufficient reactions for positive and negative controls.
2. For each DNA sample prepare an endogenous control reaction according to the
table below (Optional):
This control reaction will provide crucial information regarding the quality of the
biological sample.
3. Pipette 15µl of each mix into individual wells according to your real-time PCR
experimental plate set up.
4. Prepare sample DNA templates for each of your samples.
5. Pipette 5µl of 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.
6. If a standard curve is included for quantitative analysis prepare a reaction mix
according to the table below:
Component
Volume
Lyophilised 2x qPCR Mastermix
1 µl
M.tuberculosis_complex primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl9Standard Curve
Copy Number
Tube 1 Positive control (RED)
2 x 105 per µl
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
2 x 104 per µl
2 x 103 per µl
2 x 102 per µl
20 per µl
2 per µl
Step
UNG treatment (if required) **
Enzyme activation
Denaturation
DATA COLLECTION *
Time
Temp
15 mins
2 mins
10 secs
60 secs
37 oC
95 oC
95 oC
60 oC
Cycling x50
7. Preparation of standard curve dilution series.
1) Pipette 90µl of template preparation buffer into 5 tubes and label 2-6
2) Pipette 10µl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
8. Pipette 5µl of standard template into each well for the standard curve according
to your experimental plate set up.
The final volume in each well is 20µl.
Amplification Protocol
Amplification conditions using Lyophilsed 2x qPCR Mastermix.
* Fluorogenic data should be collected during this step through the FAM and VIC channels
** Required if your Mastermix includes UNG to prevent PCR carryover contamination
10
Interpretation of Results
enviro
commensal
NO
+ / -
+
> 35
+ / -
+
≤ 35
EXPERIMENT FAILED
due to test contamination
*
++-
NEGATIVE RESULT
-+-
SAMPLE PREPARATION FAILED
+ / -
-
+ / -
EXPERIMENT FAILED
POSITIVE QUALITATIVE RESULT
do not report copy number as this
may be due to poor sample extraction
--+
-++
POSITIVE QUANTITATIVE RESULT
calculate copy number
+ / -
-
POSITIVE QUANTITATIVE RESULT
calculate copy number
+
Interpretation
Positive
control
Negative
control
Internal
control
(VIC)
+ / -
≤ 30
Target
(FAM)
> 30
+ / -
> 30
--+ / -
*Where the test sample is positive and the negative control is positive with a Cq > 35, the
sample must be reinterpreted based on the relative signal strength of the two results:
Positive control template (RED) is expected to amplify between Cq 16 and 23. Failure to
satisfy this quality control criterion is a strong indication that the experiment has been
compromised.
If the sample amplifies < 5 Cq earlier than
the negative control then the positive
sample result is invalidated and the result
should be determined inconclusive due to
test contamination. The test for this
sample should be repeated.
Sample
Negative
control
∆Cq$<$5
INCONCLUSIVE
If the sample amplifies > 5 Cq earlier than
the negative control then the sample
should be reinterpreted (via the table
above) with the negative control verified
as negative.
Sample
Negative
control
∆Cq$>$5
SAMPLE$POSITIVE
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Internal PCR control
The Cq value obtained with the internal control will vary significantly depending on the
extraction efficiency, the quantity of DNA added to the PCR reaction and the individual
machine settings. Cq values of 28±3 are within the normal range. When amplifying a M.
tuberculosis_complex sample with a high genome copy number, the internal extraction
control may not produce an amplification plot. This does not invalidate the test and should
be interpreted as a positive experimental result.
Endogenous control
The signal obtained from the endogenous control primer and probe set will vary according
to the amount of biological material present in a given sample. An early signal indicates
the presence of a good yield of biological material. A late signal suggests that little
biological material is present in the sample.
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