PCR Rotocol

Makerere University College of Health SciencesSchool of Biomedical Sciences
Molecular Diagnostics manual I

PCR Protocol for identification of Mycobacterium tuberculosis Complex
(MTC) from sputum, cultures or any suspect specimen
[Standardized for the lab conditions]
The Molecular biology laboratory, dept of Medical Microbiology, Makerere University College of Health
Sciences. All Rights Reserved.
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Prepared by:
David P. Kateete B. Vet. Med., MSC., and Paul R. Odong, 2006
Last reviewed: August 27, 2009
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9/25/2009
To be periodically reviewed by:
1. Dr Moses L. Joloba, PhD., Principal Investigator
2. Dr Benon B. Asiimwe, PhD., in charge, Quality Assurance
3. Fred Ashaba Katabazi, BBLT., Laboratory Manager
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Principle:
IS6110 is an insertional sequence that is present in the genomes of all
Mycobacterium tuberculosis Complex (MTC) species, the causative agents of
tuberculosis (TB) in humans. Mycobacterium tuberculosis complex consists of the
following subspecies: M. tuberculosis, M. bovis, M. bovis BCG, M. africanum, M.
carneti and M. microti.
The detection of IS6110 by the Polymerase Chain reaction (PCR) accurately
identifies MTC complex and the procedure can be of invaluable help in the
diagnosis of TB.
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Specimen handling prior to PCR:

At the Molecular Diagnostics laboratory, most of the samples we receive for
MTC identification come from the Joint Clinical Research Centre, JCRC
(approx 60%). A number research programmes also bring samples to the
laboratory for identification of MTC. Occasionally, clinicians and health
workers bring suspect specimen to rule out MTC infection from their
patients. So we deal with a diverse source of specimen and samples.
Rigorous steps and utmost care are taken to ensure quality of results
disseminated.
At the Molecular Biology laboratory (Makerere University College of Health Sciences)
Observe the following:
The samples are transported from JCRC (The Joint Clinical Research
Centre, Mengo), or any other source, in sealed biohazard bags in a safely
closed container e.g. ice box.
MTC samples are contained in tightly closed cryovials and there should be
no leakages of the sample; if so reject the samples.
You are handling potentially infective Mycobacteria! Ensure that you have
worn protective clothing (i.e. laboratory coats, gloves, face and mouth masks
etc)
Observe all the safety regulations in the laboratory (see Safety Manual)
Sample processing prior to PCR
1. After receiving the samples, heat-kill the bacilli immediately in the
hybridization oven at 80oC for 2 hours (in case you cannot do this
immediately store samples at 20C till use, but this is very risky and should
be discouraged).
2. After heat killing, allow samples to cool at RT (room temp) for 0.5h. After
this the samples can be kept frozen at -20 C till use.
3. Check that the corresponding list is brought along with the samples and tally
the information. Incase a sample is absent and yet recorded on the list,
communicate immediately to JCRC.
4. Check again to be sure that the cryovial caps are tightly closed.
5. Record the samples in the PCR sample reception book (kept on the lab
shelves).
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6. Carefully dispense the contents into 1.5 ml microfuge tubes correctly labeled
with a similar sample ID (Identification number).
7. Centrifuge the samples at maximum speed (13.2 rpm) for 10 min at RT in an
eppendorf microfuge.
8. Carefully discard the supernatant into a disposable container, leaving a little
volume (~20-50ul) covering the cell pellet.
9. Vortex for 10 sec to suspend the cell pellet.
10. Do a quick/short spin and store at 4oC (fridge #3) in case you cannot
proceed immediately; otherwise the samples are now ready for PCR (storage
of samples this way should not exceed 24h).
Materials for PCR
1. 10X Master mix from ABgene, stable for 1 year at 20oC. Kept in freezer
#4, rack 1, box 2
2. Forward Primer (P1) 43 [5- TCA GCC GCG TCC ACG CCG CCA -3], stable for
1 year at 20oC. Kept in freezer #3, in a box named Primers. Position no 4
3. Reverse Primer (P2) 53 [5- CCG ACC GCT CCG ACC GAC GGT -3], stable for 1
year at 20oC. Kept in freezer #3, in a box named Primers. Position no 4
4. Taq Polymerase (ThermoFisher, formally ABgene, UK). Stable for 1 year at
20oC. Kept in freezer #4, in a box #3, position no 5 or PCR Reagents
Position # 5
5. Nuclease free water, stable for 2 years at 4oC. Bottle in use is kept in fridge
#4. chamber..
The stock of PCR-grade Nuclease free water is kept in the store, RT.
In the Pre-Amp PCR room:
Before starting a PCR:
Disinfected all hoods using 70% ethanol before and after each use
Sterilize the hood further by turning on the UV light for at least 20 min
(however avoid contact of eyes or skin with UV light).
You will reconstitute PCR reagents and make your reactions in the PreAmp room in a sterilized hood
You will add the template DNA as cell fraction (obtained above) in the PostAmp room in a sterilized hood
No DNA or cell products should be introduced into the pre-amp room.
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Used pipette tips and PCR tubes should not be left in the hood.
Equipment designated for PCR such as pipettes and pipette aids should not
be taken out and brought back into the Pre-Amp room as this encourages
contamination.
When preparing for an experiment, prepare your own working solutions
(PCR water and other PCR reagents) and clearly label the tubes. Store your
solutions away from the stocks. NEVER pipette from the Stocks during an
experiment.
.
PCR Procedure
For one reaction, add the following reagents in a PCR tube
Reagent
Stock Concentration
Working
Concentration
Volume (l)
Nuclease free water

PCR Custom Master Mix
Forward primer(P1)
Reverse primer(P2)
Taq polymerase enzyme
Cell sample/DNA
template
N/A (Not applicable)

10X
10molar
10molar
5U/ul
N/A
N/A
1X
10pmoles (~25ng)
10pmoles (~25ng)
0.5U
~0.5 ug total DNA
8
1
0.5
0.5
0.1 (vol negligible)
<2ul
Total volume: 10
(with out template)
Negligible means volume is not considered in the total volume
Note:
1. For more than 3 PCR reactions, you may consider preparing a reaction
Master mix (Mx):
For instance, for 25 PCRs, simply multiply each item in the volume column
by 25(N)(except the Cell sample), and in the pre-amp hood dispense, all the
required volumes in one sterile microfuge tube. i.e. 200 (8 x25) + 25 (1 x25)
+ 12.5 (0.5 x25) + 12.5 (0.5 x25) + 2.5 (0.1 x25)=250/25 (N=sample
number). Mix well by pipetting up and down several times. The Taq and
template vols are negligible. Dispense 10ul Mx into a correctly labeled PCR
tube in the general lab hood add 2ul of cell sample to the correspondingly
labeled PCR tube. Mix well by pippeting up and down several times.
2. Include H37RV DNA as positive control and M. smegmatis/E. coli DNA,
H2O/no DNA as negative controls at the end of the tubes row.
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Quality Assurance & Quality Control
H37Rv
Tests
Negative Controls
The design and lay out of the Molecular laboratory:

The Molecular Diagnostic Laboratory is partitioned in such a way that
contamination is greatly minimized. There three designated areas: Pre-Amp,
Post-Amp and general working area, designed in a manner that strictly
prohibits contamination. Movement in and between these rooms is
unidirectional and highly restricted to only qualified and trained personnel.
Controls:
Positive control: M. tuberculosis H37Rv DNA
Negative Controls:
M. smegmatis DNA
Nuclease free water (sterile)
External quality control and quality assurance (EQA)
MTC samples from external laboratories are brought in our laboratory. We
also send samples outside for external quality assurance. There should be
approx 100% congruence in sample results upon PCR and our laboratory has
been with acceptable range of results.
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In the Post-amp room:

Load the thermocycler and run the PCR reaction using the program named Julius in the
JR Thermocycler.
PCR Program (Julius):

94.0oC, 5 min
94.0oC, 30 sec
65.0oC, 30 sec
72.0oC, 45 sec
72.0oC, 10 min
x30
4.0oC, forever
From the thermocycler, remove the products and keep at 4oC prior to
electrophoresis, otherwise proceed to electrophoresis immediately.
Agarose Gel electrophoresis of MTC IS 6110 PCR products & Analysis of
Results:
To analyze the PCR reaction, prepare a 1 % agarose mini-gel and stain with
ethidium bromide (avoid contact with ethidium bromide because it is
carcinogenic).
Prepare a 1% agarose gel by weighing and dissolving 1.5g agarose (LEE) in
150 ml of 1X TBE or TAE (see appendix for preparation).
1. Boil thoroughly in a microwave for ~3 min and cool to 40 C.
2. Add 5ul of 5mg/ml Ethidium bromide and mix well by gentle agitation.
Note: Ethidium Br- should not be added to hot or vaporizing boiled
agarose, otherwise you breathe it in.
3. Pour the agarose into an assembled gel tray with a comb attached. Allow
to set at RT (~1h).
4. When the gel sets, transfer to an electrophoretic tank and vertically
remove the inserted combs.
5. Pour 1X TBE or TAE (depending on what you used to prepare the gel) to
just cover the gel.
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6. Add 5l of loading dye to each PCR product and mix well: carefully load
the PCR products into individual wells, excluding the first and last ones.
7. While loading, always begin with the 1Kb DNA ladder and the positive
control (H37Rv), followed by the sample test reactions.
Negative controls (Smegmatis/E.coli and H2O) should always be loaded
last.
8. Perform electrophoresis on the samples at 120V (constant Voltage,
variable current) for 1h. Stop the reaction and carefully transfer the minigel on top of the UV illuminator inside the Bio-imager.
Tightly close the Bio-imager door and turn on the UV.
9. Visualize the DNA bands on the Bio-imager screen and save a picture of
the gel. You can improve the image by focusing; magnify or reduce the
intensity of the bands by pressing the +/- buttons of the bio-imager.
10. Analyze and interpret the results. Record the sample Identification
Numbers on the PCR result sheet based on how the samples were loaded
in the gel.
11. Score the results based on the presence (+) or absence (-) of an approx
500bp band for each sample.
Analysis and interpretation of results following Agarose gel electrophoresis

1. Check for presence of an approx 500bp fragment in the positive control
lane (H37Rv). This is expected; absence of the band implies a failed reaction
or experiment.
2. Check for absence of DNA bands in the negative control lanes (M.
smegamatis and water). Presence of amplification DNA bands in negative
control lanes is indicative of sample contamination or sample spill over
while loading (this is least likely though).
3. Check for the presence or absence of an approx ~500bp band in a test
reactions. Presence of a band of correct size indicates amplification of
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IS6110 amplified from the sample: this implies presence of Mycobacteria

tuberculosis complex (MTC) in the sample specimen.
Absence of a 500bp fragment indicates that IS6110 was not detected in the
sample, implying that MTC is missing in the specimen. This is only true if
ALL the controls (positive and negative) have worked fine. Otherwise failed
reactions will also not show bands in negative control lanes.
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APPENDIX
Primer Sequences for the amplified IS6110 (~500bp)
Fwd: 5- TCA GCC GCG TCC ACG CCG CCA -3
Rev: 5- CCG ACC GCT CCG ACC GAC GGT -3
Oligos are ordered from IDT
Preparation of 10X TBE and 10X TAE:
Please refer to the IS6110-RFLP protocol manual for preparation of thes
solutions
ABgene Contacts- Now Thermo-Fisher scientific (for purchase the PCR Custom
Master Mix, Taq polymerase and PCR tubes).
Contact Ms Geraldine Nalwadda (the administrative assistant) to help with orders.
ABgene House
Blenheim Road
Epsom, Surrey
KT19 9AP
UK
Tel: 44(0)1372 723456
Fax: 44(0)1372 741414
Email: info@abgene.com
Website: www.abgene.com
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References:
J. Muhumuza, B. B. Asiimwe, S. Kayes, P. Mugyenyi, C. Whalen, R. D. Mugerwa, H. Boom,
K. D. Eisenach, and M. L. Joloba. Introduction of an in-house PCR for routine identification
of M. tuberculosis in a low-income country, 2006. INT J TUBERC LUNG DIS 10(11):1262
1267
Example:
Analysis, interpretation and reporting of results for PCR identification of IS6110 in
MTC clinical isolates ------------------- see below
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Makerere University College of Health SciencesSchool of Biomedical Sciences

Molecular Diagnostics manual I

David P. Kateete B. Vet. Med., MSC., and Paul R. Odong, 2006

Last reviewed: August 27, 2009

To be periodically reviewed by:

1. Dr Moses L. Joloba, PhD., Principal Investigator

2. Dr Benon B. Asiimwe, PhD., in charge, Quality Assurance

3. Fred Ashaba Katabazi, BBLT., Laboratory Manager

Specimen handling prior to PCR:

Nuclease free water

N/A (Not applicable)

Negligible means volume is not considered in the total volume

Quality Assurance & Quality Control

The design and lay out of the Molecular laboratory:

In the Post-amp room:

PCR Program (Julius):

Analysis and interpretation of results following Agarose gel electrophoresis

IS6110 amplified from the sample: this implies presence of Mycobacteria

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