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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
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  • 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

    .................2

    .................2

    .................2 .................3 .................3 .................3 .................3 .................3 .................3 .................4 .................4 .................5 .................6 .................6 .................6 .................6 .................7 .................7 .................8 .................8 .................8 .................9 .................10 .................10 .................10 .................11 .................12 .................12 .................13 .................13

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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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