Life science for better life Cat. No. BS7M1-2 BioSewoom Real-Q TM M. tuberculosis User Manual (ver. 1.1) For Rotor-Gene TM 2000/3000 BioSewoom Wooyoung Technocenter 2F #273-15 Sungsu 2-ga 3-dong Sungdong-gu 133-831 Seoul, Korea www.biosewoom.com
Sep 27, 2015
Life science for better life
Cat. No. BS7M1-2
BioSewoom Real-QTM M. tuberculosis
User Manual (ver. 1.1)
For Rotor-GeneTM 2000/3000
BioSewoom Wooyoung Technocenter 2F #273-15 Sungsu 2-ga 3-dong Sungdong-gu 133-831 Seoul, Korea
www.biosewoom.com
Index 1. Preface
1.1 Kit contents 1.2 Required but not supplied materials and equipments
2. Introduction
2.1 Overview 2.2 Application 2.3 Storage of kit 2.4 Number of tests 2.5 Sensitivity and dynamic range 2.6 Location of TaqMan probe and primer 2.7 Internal control 2.8 Principle of RQ-PCR
3. Procedure
3.1 Sample material 3.2 Additional reagent recommended 3.3 Schematic workflow 3.4 Preparation of PCR mixture and template set-up 3.5 PCR condition
4. Data Analysis
4.1 Positive control 4.2 Interpretation of results
5. Sensitivity and wide dynamic range 6. Typical results
6.1 Examples of TB standard DNA amplification in Rotor-GeneTM 3000 6.2 Examples of TB sample DNA amplification in Rotor-GeneTM 3000
7. Trouble shooting 8. Procedures of nucleic acid isolation using the QIAamp DNA Mini Kit
8.1 Flow Chart 9. References 10. Explanation of symbols
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1. Preface 1.1 Kit contents
Tube/Cap Label Contents
1 / Red 2X PCR reaction mixture 1.25 ml ready-to-use PCR reaction mixture for TB and IC amplification
2 / Green
TB probe & primer mixture 300 l Primer and TaqMan probe mixture, specific for TB genome
3 / Yellow
IC probe & primer mixture 300 l Primer and TaqMan probe mixture, specific for internal control gene
4 / Blue Positive control 50 l
200 l 5 / White Internal control (IC)
6 / Neutral Water, sterile, DNase/RNase free 1 ml
Note 1 : Protect the probe & primer mixture (tube 2, tube 3) from light.
Note 2 : TB standards should be store in aliquots for one use.
1.2 Required but not supplied materials and equipments
- Rotor-GeneTM 2000/3000
- Pipette man
- Powder-free gloves
- 36-well rotor/72-well rotor
- 72 well loading block
- 36 well loading block
- Table top centrifuge - 0.2 ml tubes, 0.1 ml strip tubes
- Nucleic acid isolation kit - 36 and 72 well locking rings
(see the 3.2 Additional reagent recommended)
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2. Introduction 2.1 Overview
The Real-QTM M. tuberculosis kit is optimized for use with the Rotor-GeneTM 2000/3000 instrument.
The kit is designed for detection of M. tuberculosis complex genome in purified DNA samples via the
gene coding for the IS6110 region. We used the TB TaqMan probe (VIC is attached to 5 end & TAMRA
is attached to 3 end) for quantification of M. tuberculosis genomes and internal control TaqMan probe
(FAM is attached to 5 end & TAMRA is attached to 3 end) for amplification of internal control DNA.
The Internal control can be used to check for nucleic acid isolation and possible PCR inhibition. For this
application, add 2 l of the IC per reaction directly in the isolation procedure. The Real-QTM M.
tuberculosis kit contains all reagents for RQ-PCR, such as PCR reaction mixture, TB primer and probe
mixture, internal control primer and probe mixture, positive control DNA, internal control DNA and water.
Also, this kit contains UNG (Uracil-N-glycosylase) in PCR reaction mixture to prevent carry over
contamination.
2.2 Application
a. The Real-QTM M. tuberculosis kit is designed to be used in life science research studies.
b. The kit is designed to specifically detect M. tuberculosis complex (M. tuberculosis, M. bovis, M.
microti) genome.
2.3 Storage of kit
a. Store the kit at -15 C ~ -20 C. The kit will remain until the expiration date indicated.
b. The kit is shipped on dry ice.
c. Thaw all components thoroughly at room temperature before starting a test. When thawed, mix the
components and centrifuge briefly.
d. Work on ice or in a cooling block.
e. Avoid repeated freezing and thawing.
2.4 Number of tests
This kit is designed for 100 reactions with a final volume of 25 l each.
2.5 Sensitivity and dynamic range
The dynamic detection range capable of detecting TB standard DNA between 5 copies and 5x1010
copies, allows the sensitivity to detect down to 5 copies of TB standard DNA per reaction.
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2.6 Location of TaqMan probe and primer
The Real-QTM M. tuberculosis kit contains primer and probe mixture for the specific amplification of a
80 bp of the IS6110 region of the mycobacterial genome. The kit is used the TaqMan probes for detection
of TB genome and internal control
MTB
VIC TAMRA Forward primer Reverse primer
Internal Control
FAM TAMRA Forward primer Reverse primer
Fig. 1 Location of amplification primers and TaqMan probe
2.7 Internal control (IC)
The Internal control can be used to check for nucleic acid isolation and possible PCR inhibition. For
this application, add 2 l of the IC per reaction directly in the isolation procedure.
Although the Ct range of IC is 333 cycles in the normal nucleotide extraction, It can vary depending on the quantity of the TB DNA at the beginning (Refer to 4.2 Interpretation of results on p8). Repeat
the experiment if none of TB genome and IC are detected from the sample.
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2.8 Principle of RQ-PCR
Differently from the conventional PCR, real-time quantitative PCR detects the fluorescent signal, and
generally uses TaqMan chemistry. TaqMan Chemistry is similar to the conventional PCR, but the probe
labeled with fluorescent dye is used in real-time quantitative PCR. The dye such as FAM, VIC, TET or
HEX is attached at the 5end of TaqMan probe, and it is generally called 'Reporter dye'. At the 3end of
the probe is attached with TAMRA dye, and it is generally called Quencher dye. When both dyes are
attached to the probe, emission intensity of Reporter is quenched by the fluorescence resonance energy
transfer (FRET) of Quencher, so no fluorescent signal appears. As the PCR begins, primers and probe
bind to the target sequence specifically, and reporter dye at the 5 end is removed by 5' exo-nuclease
activity of Taq DNA polymerase. As a result, the fluorescent signal begins to appear. As one molecule of
the probe is removed when one target molecule is amplified, the signal from the reporter dye increases
proportionally to the quantity of amplified DNA, and it allows the efficient quantification.
535 3
5353
35
3
5
35
probe
5Forward primer
3 Reverse primer
Reporter: FAM, VIC, TET, HEX
Quencher : TAMRA
Taq DNA polymerase
Emitting reporter
Excited quencher
Fig. 2 Principle of real-time quantitative PCR
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3. Procedure 3.1 Sample material
- Sputum
- BAL (bronchoalveolar lavage)
- Bronchial secretion
- CSF (Cerebro-Spinal Fluid)
- Cultured cells
3.2 Additional reagent recommended
For the use of internal control provided in this Real-QTM M. tuberculosis kit, make sure that you use
the nucleotide extraction kit mentioned below.
Step Manufacturer Kit Cat. #
Nucleic acid
isolation Qiagen QIAamp DNA Mini Kit (50T) 51304
3.3 Schematic workflow
200 l buffer AL
+ 20 l proteinase K
See 8.1 for nucleic acid isolation processing using the QIAamp DNA Mini Kit.
+ 2 l internal control (IC)
200 l sample m
aterial
5 l purified DNA 20 l RQ-PCR reaction mixture
optical reaction tube
Rotor-GeneTM 2000/3000
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3.4 Preparation of PCR mixture and template set-up
a. Prepare the TB RQ-PCR master mixture.
Total reaction number = n sample + 5 standards + 1 negative control + 1
Containing the internal control Component Volume per reaction Volume for total reactions
PCR reaction mixture (2X) : tube 1 12.5 l 12.5 x (n + 3) l
TB probe & primer mixture : tube 2 3 l 3 x (n + 3) l
IC probe & primer mixture : tube 3 3 l 3 x (n + 3) l
Water, sterile : tube 6 1.5 l 1.5 x (n + 3) l
total 20 l 20 x (n + 3) l
Not containing the internal control Component Volume per reaction Volume for total reactions
PCR reaction mixture (2X) : tube 1 12.5 l 12.5 x (n + 3) l
TB probe & primer mixture : tube 2 3 l 3 x (n + 3) l
Water, sterile : tube 6 4.5 l 4.5 x (n + 3) l
total 20 l 20 x (n + 3) l
Note : Mix gently by pipetting or tapping. Do not vortex.
b. Pipet 20 l of the master mixture into each reaction tubes.
c. Add 5 l of the TB standard DNA and corresponding TB DNA samples.
d. Add 5 l of water into well of the negative control to check for contamination.
e. Place the tubes in the rotor of the Rotor-GeneTM 2000/3000 instrument.
3.5 PCR condition
Step Cycle # 1 50 C 2 min 1 cycle
2 95 C 10 min 1 cycle
3 95 C 20 sec
58 C 30 sec 45 cycles
72 C 30 sec
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4. Data analysis 4.1. Positive control
Firstly, Ct values of TB and IC amplified in the positive control should be checked. Ct values are
recommended to be lower than 303 Cycle, and it should be repeated if it is bigger than 33 cycle. In the
negative control, no signal should be observed for both TB and IC, and it should be repeated if the signal
is observed.
TB (VIC)
Ct
IC (FAM)
Ct Result Comment
Positive control 303 303 Positive Valid Negative control Neg Neg Negative Valid
4.2. Interpretation of sample results
If positive and negative controls are satisfied, result is reported according to contents in the table.
TB (VIC)
Ct
IC (FAM)
Ct
Result Comment
< 40 333 Positive 40 333 Negative Neg 333 Negative < 40 Reduced signal In case of the large amount of TB DNA(VIC signal), signal
of IC(FAM signal) can be reduced or does not appear due to PCR competition.
positive or neg
Neg Neg Invalid Repeat the isolation of nucleic acid and PCR.
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5. Sensitivity and wide dynamic range The TB standard DNA with serial dilution was used to test the detection range and the limit of the
Real-QTM M. tuberculosis kit, and the detection range was proven to be greater than 10 log. Also, the TB
standard DNA was applied for the sensitivity test, and it showed the sensitivity of 5 copies per reaction.
Fig. 3 Wide dynamic range of Real-QTM M. tuberculosis kit
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6. Typical results 6.1 Examples of TB standard amplification in Rotor-GeneTM 3000
Fig. 4 Amplification curve of the TB standard 1~5 by detection of the VIC signal
Fig. 4 Standard curve of the TB standard 1~5
6.2 Examples of TB sample DNA amplification in Rotor-GeneTM 3000
Fig. 5 Amplification curve of the TB sample DNA by detection of the VIC signal
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7. Trouble shooting Problems Causes Recommendations
Error in the PCR condition when
tting up the PCR program se
Verify the PCR cycling
program
Omitted components Verify each component, and repeat
the PCR mixture preparation
If no fluorescent signal is
obtained in all samples
including the standards
Data acquisition not selected when
setting up the PCR program
Make sure that the acquisition
mode is single in the segment 3 of
PCR program 2 (amplification
step)
If the fluorescent signal is
obtained in negative control
Carry-over contamination Be careful not to contaminate
during pipetting
Error in the volume of some
omponent added in the reaction c
Verify each component, and repeat
the PCR mixture preparation
Error in the PCR condition when
setting up the RQ-PCR program
Verify the PCR cycling program
If the fluorescent intensity for
all samples including the
standards are week
Kit stored in the wrong condition
Store the probe and primer
mixtures(tube 2, 3) at -15C ~ -
20C, and protect these tubes
from the light
Poor quality of DNA samples - Shorten the DNA extraction
time, and store the extracted
DNA at -20 - Extract DNA from serum
using the kit recommended
If the fluorescent intensity is
week or does not appear only in
the unknown samples
Not enough volume of DNA
samples added
Repeat the PCR reaction using
the correct volume of DNA
samples
Degradation of standard plasmid
DNA samples
- Store and use the standards after
aliquoting
-Avoid the repeated thawing and
freezing
If the fluorescent intensity is
week or does not appear only in
the standards
Incorrect volume of standard
plasmid DNA added
Repeat the PCR reaction using
the correct volume of standards
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Pipetting error Make sure that the equal
volume of reactants are added
in each tube
If the diverse intensity of
fluorescent signals appears
Contamination in the outer
surface of PCR tubes
Wear gloves during the
experiment
Incomplete centrifugation of
tubes
Centrifuge (3000 rpm x 5 sec)
tubes after all reagents are
added
8. Procedures of nucleic acid isolation using the QIAamp DNA Mini Kit (Qiagen) 8.1 Flow Chart
Sample
Bind
Lysis
Wash buffer AW1
Wash buffer AW2
Elute
Pure genomic DNA
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9. References 1. Rapid and specific detection of Mycobacterium tuberculosis by using the Smart Cycler instrument and
a specific fluorogenic probe. Journal of Clinical Microbiology. 2003 Oct;41(10):4783-6.
2. Rapid diagnosis of mycobacterial infections and quantitation of Mycobacterium tuberculosis load by
two real-time calibrated PCR assays. Journal of Clinical Microbiology. 2003 Oct;41(10):4565-72.
3. Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA
in sputum during treatment of tuberculosis. Journal of Clinical Microbiology. 1998 Jul;36(7):1964-8.
4. Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR.
Journal of Clinical Microbiology. 2004 Nov;42(11):5277-85.
10. Explanation of symbols
Caution Catalog number Batch number
Used by
Temperature limitation
Date of manufacture Sum of reactions
Manufacturer
Keep away from sunlight
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