Mycoplasma pneumoniae Chlamydophila pneumoniae - Mikrogen

Ref.: Dia-MCpn-050.Vs1
Mycoplasma pneumoniae
Chlamydophila pneumoniae
Real-Time PCR
User Manual
Available on:
ABI 7000 / 7300 / 7500 / 7900
Roche LightCycler 480 / 2.0
Biorad Icycler / IQ5 / CFX96
Qiagen Rotor-Gene 6000
Stratagene MX3000P / 3005P
In combination with:
DIA-EIC/DNA(DR)-050
DIA-EIC/DNA(TR)-050
DIA-EIC/DNA(Cy5)-050
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Contents
General information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Pathogen information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Product description . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Warnings and precautions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Material required, not provided . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6
Human samples used / Claims of the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Human samples Collection, Storage and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
1. Samples' Collection .............................................................7
2. Samples' Storage ...............................................................7
3. Samples' Transport .............................................................7
Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8
A . Procedure to be followed
A.1. Extraction procedure ..........................................................8
A.2. PCR procedure ...............................................................8
A.3. Amplification and real-time system procedures ....................................9
A.4. Interpretation of results .......................................................10
B . Annexes of the procedure (points I-II-III-IV-V-VI)
I. DNA extraction & inhibition control (optional) .......................................11
II. Sample Extraction/DNA Isolation .................................................12
III. PCR Reaction Set-up ..........................................................14
IV. PCR profile ...................................................................16
V. Selection of the appropriate dyes .................................................17
ABI 7000-7300-7500-7900 .......................................................17
Roche Lc480-Lc2.0 ............................................................17
Biorad iCycler-IQ5-CFX96 .......................................................17
Stratagene MX3000P-3005P .....................................................18
Qiagen Rotor-Gene ............................................................18
VI. Interpretation of results ........................................................19
Troubleshooting guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
Sensitivity ......................................................................21
Specificity ......................................................................21
Product Use Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22
Explanation of symbols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Notice to purchaser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
General Information
Diagenode’s Mycoplasma/Chlamydophila pneumoniae Real Time PCR is an in vitro diagnostic
assay used by a clinical laboratory and by trained laboratory scientists.
Diagenode’s Mycoplasma/Chlamydophila pneumoniae Real Time PCR has been validated on:
• ABI (7000 / 7300 / 7500 /7900)
• Roche (Lc2.0 / Lc480)
• Biorad (iCycler / IQ5 / CFX96)
• Qiagen Rotor-Gene (3000 / 6000)
• Stratagene (MX3000P / 3005P)
• Cepheid (SmartCycler II) (depending on software)
Pathogen Information
Mycoplasma pneumoniae & Chlamydophila pneumoniae are the organisms responsible for most
of the cases of atypical pneumonia in children.
Atypical pneumonia due to these bacteria usually causes milder forms of pneumonia and are
characterized by a more protracted course of symptoms unlike other forms of pneumonia which
can come on more quickly with more severe early symptoms.
Mycoplasma pneumoniae can give rise to mild upper respiratory tract infection, bronchitis,
bronchiolitis and bronchopneumonia. The disease usually starts as influenza like syndrome with
the predominant symptoms being fever, illness, headache, scratchy sore throat and cough.
Chlamydophila pneumoniae has been associated with pharyngitis, sinusitis and bronchitis
Product Description
The product contains primers, probes and a positive control. The master mix is not included.
Mycoplasma/Chlamydophila pneumoniae Real-Time PCR: 50 PCRs (50 μl PCR volume), 2
pockets.
I. Primers & probe’s pocket:
1. Mycoplasma and Chlamydophila pneumoniae primers & double-dye probes
(Mycoplasma pneumoniae, FAM, emission 520 nm & Chlamydophila pneumoniae,
Yellow Dye, emission 548 nm): 250 μl, mallow tube.
2. ddH2O: 1500 μl, dark blue tube.
II. Positive control’s pocket:
1. Mycoplasma and Chlamydophila pneumoniae positive control: 250 μl, green tube.
The Mycoplasma/Chlamydophila pneumoniae Real-Time PCR (DIA-MCpn-050) is used in
combination with the DNA extraction & inhibition control Real-Time PCR (DIA-EIC/DNA-050).
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

DNA extraction & PCR inhibition control Real-Time PCR: 50 PCRs (50 μl PCR volume), 1 box.
Storage
Mycoplasma/Chlamydophila pneumoniae Real Time PCR kit must be stored at -20°C, before and
after opening.
NB:
If using DIA-EIC/DNA-050, the kit and the DNA virus culture must be stored at -20°C.
Please check production date mentioned on the pocket and tube labels. The product has a shelf
life of 24 months at -20°C starting from the production date. Opened kits are stable for 12 months
at -20°C.
NB:
Please make aliquots to avoid freeze/ thaw cycles and during use all components should be kept
on ice.
STORE IN DARK PLACE
Warnings and Precautions
General recommendations
• Read all instruction before performing the experiment.
• After use, material-reagents-waste must be handled as potentially infectious and must be
thrown in a specific waste bin for biological substances.
• Do not substitute reagents from the kit with different batch numbers or from other
manufacturers.
Extraction/PCR
• Diagenode’s real-time PCR kits must be used by scientists/laboratory technicians with
a strong know-how of molecular biology and more particularly with real-time PCR
platform.
• To obtain optimal results, Diagenode suggests some appropriate extraction methods
to avoid as much as possible PCR inhibitors (see point II. Sample Extraction/ DNA
Isolation).
• Extraction and/or inhibition control must be used for each reaction of PCR.
Caution
I. (DNA virus) primers & double-dye probe:
If Orange Dye (DIA-EIC/DNA(DR)-050), emission 575 nm: 250 μl, blue tube.
If Texas red (DIA-EIC/DNA(TR)-050), emission 603 nm: 250 μl, red tube
If Cy5 (DIA-EIC/DNA(Cy5)-050, emission 662 nm: 250 μl, dark blue tube.
II. DNA virus culture: 500 μl, yellow tube.
Diagenode commercializes a qualitative extraction and/or inhibition control . It is the responsible
of the user to optimize the extraction procedure according to the result obtained for this
extraction and/or inhibition control in order to get an appropriate result .
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
• Virus control extraction must be manipulated using a safety cabinet for a manual extraction
procedure or in a specific area for automated extraction procedures; as suggested by the
manufacturer.
Contamination
• Perform nucleic acid release, isolation and real time amplification in separate laboratory
areas.
• Keep all tubes and vials closed when not in use.
• The PCR laboratory (safety cabinet, DNA/RNA extraction platform, bench coat, etc.) must
be cleaned after each PCR experiment with appropriate solutions.
• The contaminated kits must be thrown in a specific waste bin for biological substances.
• The kit should not be used by somebody displaying symptoms of the disease being detected
by this kit.
• The positive and negative PCR control must be performed with each reaction of PCR.
• Gloves must be worn.
• Aerosol resistant tips must be used for all PCR mixtures.
• Tubes must be harvested before opening.
• Do not pipette any of the materials by mouth. Do not smoke, eat or drink in areas which
specimens or kit reagents are handled.
• The experiment should not be carried out during pregnancy due to risks to the newborn
baby.
CAUTION
Storage of the kit at room temperature can lead to the degradation of the primers / probes /
positive control . This will lead to a decreased sensitivity and we would strongly advise a new kit .
Material Required, Not Provided
• Pipettes
• Sterile pipette tips with filters
• Powder-free gloves
• Vortex mixer
• Desktop centrifuge
• RNase/Dnase-free microcentrifuge tubes (1,5 and/or 2 ml)
• RNase/DNase free water
• DNA isolation kit (see point II. Sample Extraction/DNA Isolation)
• Consumables and accessories (reaction tubes, microcentrifuge, reaction plates, adhesive
seal) for an ABI / Roche / Biorad / Qiagen / Stratagene or Cepheid system
• Master mix (see point III. PCR Reaction Set-up)
• Real time PCR instrument: ABI / Roche / Biorad / Qiagen / Stratagene or Cepheid.
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

Human sample used / Claims of the kit
Mycoplasma/Chlamydophila pneumoniae Real Time PCR kit is dedicated at the detection of
the Mycoplasma pneumoniae and Chlamydophila pneumoniae in bronchoalveolar lavage,
sputum and nasopharyngeal swabs/washes samples.
The kit is used on samples from potentially infected patients by Mycoplasma pneumoniae
and/or Chlamydophila pneumoniae.
Human samples’ Collection, Storage and Transport
I. Samples’ Collection
Human respiratory samples such as bronchoalveolar lavage, sputum and nasopharyngeal
swabs/washes should be collected.
II. Samples’ Storage
Human sample must be stored at 4°C for up to 24 hours or frozen at -20°C or -80°C for longer
periods of time.
Human samples should be stored in DNase/RNAse free polypropylene tubes. The sensitivity
of the PCR could be reduced if you keep freezing and thawing human samples repeatedly.
Avoid freeze / thaw cycles, which will affect the sensitivity of the assay.
III. Samples’ transport
Human samples must be transported in adapted containers. The samples must be transported
following the local and national regulations for the transport of pathogen material.
Human samples should be transported at -20°C.
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
Protocol
A. Procedures to be followed
A .1 Extraction procedure (see point II . Sample Extraction/DNA Isolation)
Perform this procedure on the appropriate level containment facility.
Please follow the following steps:
• Place sample(s) at room temperature
NB:
It is recommended to manipulate potential infected samples under a safety cabinet.
• Choose the appropriate extraction method validated for your sample. (See point II for
Diagenode validated methods).
Please, follow manufacturer’s instructions to perform extractions .
• (Optional) If using DNA extraction & PCR inhibition control Real-Time PCR, add 10 μl of
the Diagenode DNA extraction & inhibition control (DNA virus culture tube) in the extracted
volume (see point I . DNA Extraction & inhibition control and II . Sample Extraction/DNA
Isolation).
NB:
The Diagenode DNA extraction & PCR inhibition control Real-Time PCR is provided separately,
ref: DIA-EIC/DNA-50. You can select the dye adapted to your real-time PCR system (see point V.
Selection of the appropriate dyes).
• When the extraction procedure is finished, prepare the DNA on ice before using it (or at
-20°C if you don’t use it the same day).
• Place the Mycoplasma pneumoniae & Chlamydophila pneumoniae positive control tube
(for long term storage) on ice.
NB:
If the extracted samples are at room temperature for a long period of time, DNA could be degraded.
If DNA is degraded, the sensitivity of the PCR will decrease!
If the Mycoplasma pneumoniae & Chlamydophila pneumoniae positive control tube is at room
temperature for a long period of time (> 24 hours), the positive control could be degraded and
influence the results obtained!
A .2 PCR procedures
Perform the procedure on the appropriate level containment facility.
Master mix suggested: Quantifast Multiplex + R kit (ref : 204756), Qiagen (Not supplied) (see point
III . PCR Reaction Set-up).
NB:
Rotor-Gene Multiplex PCR Kit (Qiagen) (Not supplied) for Qiagen Rotor-Gene
LightCycler ® TaqMan ® Master (Roche) (Not supplied) for LightCycler 2.0 (Lc2.0)
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

Please follow the following steps:
• Place the Mycoplasma/Chlamydophila pneumoniae Real-Time PCR kit at room
temperature: the mallow tube (Mycoplasma and Chlamydophila pneumoniae primers &
double-dye probe), the dark blue tube (ddH2O) from Primers/probe’s pocket.
• If using the DNA extraction & PCR inhibition control Real-Time PCR, place the tube
containing primers & double-dye probe at room temperature.
• Place the master mix on ice.
NB:
Spin down all solutions briefly when thawed and pipette up and down to homogenize them.
• Select the appropriate protocol depending of the Real-Time PCR system used (see point
III. PCR Reaction Set-up) and prepare mixes without the sample and/or the Mycoplasma
and Chlamydophila pneumoniae positive control.
NB:
Prepare the same mix for all samples.
To validate the run, you need to perform a negative (water) and a Diagenode positive control
PCR.
If DIA-EIC/DNA-050 is not use, replace Diagenode DNA extraction & PCR inhibition control doubledye
probe & primers by ddH2O.
• Dispense the mixe prepared in appropriate tubes/capillaries or a 96 well plate.
• Add samples and the Diagenode positive control (same volume as samples).
• Close tubes or the plate.
NB:
If the Mycoplasma and Chlamydophila pneumoniae primers & double-dye probe tube (mallow)
is at room temperature for a long period of time, primers and probes could be degraded and
influence (> 24 hours) the results obtained!
A .3 Amplification and real-time system procedures
Perform the procedure on the appropriate level containment facility.
Diagenode designs and validates kits adapted on ABI / Roche / Biorad / Qiagen / Stratagene /
Cepheid Real-Time PCR Systems.
Follow the manufacturer’s instructions before using Real-Time instruments .
Please follow those steps:
• Place the closed tubes/capillaries or plate in the Real-Time system.
• Select the PCR profile (see point IV . PCR profile).
• Select dyes adapted to the Real-Time system (see point V . Selection of the appropriate
dyes):
- Mycoplasma pneumoniae is detected with Fam dye.
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
- Chlamydophila pneumoniae is detected with Yellow dye.
- DNA extraction & inhibition control (DIA-EIC/DNA-050) is detected with Orange Dye or
Texas red or Cy5.
• Enter the name and the position of samples in the software.
• Start the run.
A .4 Interpretation of results (see point VI . Interpretation of results)
Follow manufacturer’s instructions for analyzing results .
Please follow the following steps:
• Analyze the FAM detector (Mycoplasma pneumoniae), the Yellow dye (Chlamydophila
pneumoniae) and the Orange Dye/Texas red/Cy5 separately (DNA extraction and PCR
inhibition control) (see point VI . Interpretation of results):
- Negative control: FAM signal must not be observed.
- Positive control: FAM and Yellow dye signals must be observed, Ct/Cp value around 30
(for Mycoplasma pneumoniae and Chlamydophila pneumoniae, depending of the Real-
Time system used!).
- For Unknown sample:
• FAM signal observed indicates that the sample is positive for Mycoplasma
pneumoniae .
• FAM signal not observed indicates that the sample is negative for Mycoplasma
pneumoniae .
• Yellow dye signal observed indicates that the sample is positive for Chlamydophila
pneumoniae .
• Yellow dye signal not observed indicates that the sample is negative for
Chlamydophila pneumoniae .
If FAM and Yellow dye signals not observed:
• Orange Dye or Texas red or Cy5 signal above the threshold, 27 ≤ Ct/Cp value ≥ 36, must be
considered as positive for the DNA extraction & inhibition control. The sample is valid.
• Orange Dye or Texas red or Cy5 signal below the threshold must be considered as negative
for the DNA extraction & inhibition control. The sample is invalid, start the DNA extraction
procedure and/or the PCR again
NB:
If the Ct value of the DNA extraction and PCR inhibition control (DIA-EIC/DNA-050) is ≥ 36:
I. Firstly we strongly suggest starting the PCR with a 10x dilution of the sample in order to
exclude as many inhibition factors as possible.
II. Secondly, if the problem persists, we recommend starting the extraction procedure again
and/or investigate another more appropriate nucleic acid extraction procedure.
• Select the report document to obtain a summary of Ct/Cp results and curves.
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B. Annexes of the procedure (points I-II-III-IV-V-VI):
I .DNA extraction & inhibition control (optional)
Diagenode’s DNA extraction & inhibition control is a virus culture containing a 1.000
TCID50/ml viral load.
Universal extraction & inhibition DNA control is a complete virus, having preserved
its infectious power . This virus is listed in the Ea1* class established by the European
Federation of Biotechnologies (EFP) .
This virus is not reported to be dangerous for human beings but it must be used
under safety cabinet .
*(more details at the end of this manual) .
The following results were obtained when using Nuclisens easyMAG ® System (Biomerieux).
Procedure:
• 10 μl of each dilution extracted in 500
μl of water.
• Nuclisens easyMAG ® System on
board extraction.
• Elution in 60 μl.
• 5 μl used for PCR.
• TaqMan ® Universal PCR mastermix
(Applied Biosystems).
• Experiments were performed on
the ABI Prism ® 7000 instruments
(Applied Biosystems).
The Ct value for 1.000 TCID50/ml is +/-28,5. The picture also shows results for other dilutions (100
TCID50/ml is +/-31,5 and 10 TCID50/ml is +/-35).
For Sample Extraction/DNA Isolation, add 10μl of the IC DNA virus culture into the sample just
before the beginning of the extraction procedure .
(The concentration can be modified in order to adapt multiplex PCRs with the extraction method
used.)
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
II . Sample Extraction/DNA Isolation
Diagenode suggests some appropriate extraction methods:
> Nuclisens easyMAG ® System (Biomerieux)
Clinical samples: nasal swab, whole sputum, bronchoalveolar lavage samples.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 250 μl (+ 10 μl of the IC DNA virus culture)
DNA isolation volume: 60 μl
The following pre-extraction procedure is recommended:
Bronchoalveolar lavage (BAL):
• 250 μl whole sputum
• Add 250 μl proteinase K buffer (1mg/ml proteinase K, 0,5% SDS, 20 mM Tris-
HCl, pH 8.3), vortex
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
• Incubate 60 minutes at 55°C
• Use the complete 500 μl manipulated sputum for downstream extraction
procedure (Nuclisens Lysis Buffer)
> MagNa pure LC system, Roche: MagNa pure LC Total Nucleic Acid Isolation Kit
(Ref. 03038505001)
Clinical samples: nasal swab, whole sputum, bronchoalveolar lavage samples.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 200 μl (+ 10 μl of the IC DNA virus culture)
DNA elution volume: 100 μl
The following pre-extraction procedure is recommended:
Bronchoalveolar lavage (BAL):
• Add 3 volume of sputolysin (1:10) with 1 volume of the BAL sample, vortex
NB:
Proteinase K (100 mg), Merck, ref: 1.24568.0100
Bronchoalveolar lavage (BAL):
• Add 3 volume of sputolysin (1:10) with 1 volume of the BAL sample, vortex
NB:
Sputolysin, Calbiochem, ref: 560000
Before using sputolysin, the solution must be diluted 10X with DNA/RNAse water.
• Incubate 15 minutes at room temperature
• Centrifuge 5 minutes at 1500 rpm
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• Completely remove and discard the supernatant
• The pellet is completely resuspended with 200 μl of sterile TE buffer
> QIAamp DNA Mini Kit (50) (Blood and Body Fluid Spin Protocol)
(Quiagen Ref. 51304)
Clinical samples: nasal swab, whole sputum, bronchoalveolar lavage samples.
Follow manufacturer’s instructions with the following:
Human sample extracted volume: 200 μl (+ 10 μl of the IC DNA virus culture)
DNA isolation volume: 60 μl
General extraction information:
If you perform the PCR on the same day, the DNA extraction can be conserved on ice . For a
longer conservation period, DNA must be stored at -20°C .
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
III . PCR Reaction Set-up
(ABI 7000-7300-7500-7900/ Roche Lc480-Lc2.0 / Biorad iCycler-IQ5-CFX96 / Stratagene MX3000P-
3500P / Qiagen Rotor-Gene / Cepheid SmartCycler II (on request) )
With Quantifast Multiplex + R kit (ref : 204756), Qiagen (Not supplied)
NB:
For the Qiagen Rotor-Gene, we recommend the Rotor-Gene Multiplex PCR Kit, Qiagen (Not
supplied) (Ref: 204774).
For the LightCycler 2.0 (Lc2.0), we specially suggested LightCycler ® TaqMan ® Master, Roche (Not
supplied) (ref: 04535286001).
• PCR 50 μl:
(ABI 7000-7300-7500-7900/ Roche Lc480 / Biorad iCycler-IQ5 / Stratagene MX3000P-
3500P / Qiagen Rotor-Gene)
For the Qiagen Rotor-Gene, we recommend the Rotor-Gene Multiplex PCR Kit, Qiagen
(Not supplied) (Ref: 204774).
25 μl ................................... 2X master mix
5 μl ..................................... Diagenode Mycoplasma pneumoniae and Chlamydophila pneumoniae
double-dye probes & primers
5 μl ..................................... Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
10 μl ................................... Human sample (or Mycoplasma pneumoniae and Chlamydophila
pneumoniae positive control)
5 μl ..................................... Diagenode water
50 μl ................................... Final volume
NB:
With ABI system, you can add 1 μl of the ROX solution (Qiagen)
• PCR 25 μl:
(ABI 7000-7300-7500-7900/ Roche Lc480-Lc2.0 / Biorad iCycler-IQ5-CFX 96 / Stratagene
MX3000P-3005P / Qiagen Rotor-Gene / Cepheid SmartCycler II (on request) )
12.5 μl ................................ 2X master mix
2.5 μl .................................. Diagenode Mycoplasma pneumoniae and Chlamydophila pneumoniae
double-dye probes & primers
2.5 μl .................................. Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
5 μl ..................................... Human sample (or Mycoplasma pneumoniae and Chlamydophila
pneumoniae positive control)
2.5 μl .................................. Diagenode water
25 μl ................................... Final volume
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• PCR 20 μl
Caution
(Roche Lc2.0)
NB:
For LightCycler 2.0 (Lc2.0), we specially suggested LightCycler ® TaqMan ® Master, Roche
(Not supplied) (ref: 04535286001)
4 μl ..................................... 5X master mix
2 μl ..................................... Diagenode Mycoplasma pneumoniae and Chlamydophila pneumoniae
primers & double-dye probe
2 μl ..................................... Diagenode DNA extraction & PCR inhibition control double-dye probe &
primers
10 μl ................................... Human sample (or Mycoplasma pneumoniae and Chlamydophila
pneumoniae positive control)
2 μl ..................................... Diagenode water
20 μl ................................... Final volume
NB:
With Roche Lc 2.0, diagenode DNA extraction & PCR inhibition control double-dye probe
& primers (yellow dye) can not be used in the same capillary. You need to use a second
capillary.
A new mixture must be freshly prepared for each new PCR run . Master mix (not supplied) and
primers/probes supplied in the kit must be placed on ice during the whole PCR preparation .
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
IV . PCR profile
> ABI 7000-7300-7500-7900 / Roche Lc480 / Biorad iCycler-IQ5-CFX96 / Stratagene
MX3000P-3005P / Qiagen Rotor-Gene / Cepheid Smartcycler II (on request)
Stage Description
1 2 minutes at 50°C (1 cycle)
2 10 minutes at 95°C (1 cycle)
3 Cycle program (45 cycles)
Step 1: 15 seconds at 95°C
Step 2: 60 seconds at 60°C
NB:
For the step 2 from the stage 3, it is highly recommended to decrease the ramping rate
(°C/s) by 50%.
> Roche Lc2.0
Stage Description
1 10 minutes at 95°C (1 cycle)
2 Cycle program (45 cycles)
Step 1: 10 seconds at 95°C
Step 2: 40 seconds at 60°C
Step 3: 1 second at 72°C
3 30 seconds at 40°C (1 cycle)
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V . Selection of the appropriate dyes
> ABI 7000-7300-7500-7900
Detector’s name Reporter Quencher
Mycoplasma pneumoniae FAM FAM (none)
Chlamydophila pneumoniae Yellow Dye Vic (none)
DIA-EIC/DNA(DR)-050 Orange Dye NED (none)
DIA-EIC/DNA(Cy5)-050 Cy5 Cy5 (none)
Select Passive Reference: ROX (if using master mix with ROX reference).
Select Passive Reference: none (if using master mix without ROX reference)
DIA-EIC/DNA(DR)-050: ABI 7000 or 7300
DIA-EIC/DNA(Cy5)-050: ABI 7500 or 7900
> Roche Lc480-Lc2 .0
Detector’s name Reporter Quencher
Mycoplasma pneumoniae FAM FAM (none)
Chlamydophila pneumoniae Yellow Dye Vic/Hex (none)
DIA-EIC/DNA(Cy5)-050 Cy5 Cy5 (none)
NB:
Before using our real time PCR kit for the first time on the roche lightcycler system 2.0 or 480, you
must create an application-specific color compensation object.
* For Lc 2 .0, you need to use DIA-EIC/DNA(YD)-050 in a second capillary .
> Biorad iCycler-IQ5-CFX96
Detector’s name Reporter Quencher
Mycoplasma pneumoniae FAM FAM (none)
Chlamydophila pneumoniae Yellow Dye Vic/Hex (none)
DIA-EIC/DNA(Cy5)-050 Cy5 Cy5 (none)
DIA-EIC/DNA(Cy5)-050: ICycler or IQ5 or CFX96
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
> Stratagene MX3000P-3005P
Detector’s name Reporter Quencher
Mycoplasma pneumoniae FAM FAM (none)
Chlamydophila pneumoniae Yellow Dye Vic/Hex (none)
DIA-EIC/DNA(Cy5)-050 Cy5 Cy5 (none)
NB:
In Filter Set Gain Settings:
• FAM: select x8
• Hex/JOE (Yellow Dye): select x2
• Cy5: select x4
> Qiagen Rotor-Gene
Detector’s name Channel
Mycoplasma pneumoniae FAM Green
Chlamydophila pneumoniae Yellow Dye Yellow
DIA-EIC/DNA(Cy5)-050 Cy5 Red
NB:
Perform an optimization at 60 degrees at the beginning of each run for the green and red
channels.
> Cepheid SmartCycler II
Detector’s name Reporter Quencher
Mycoplasma pneumoniae FAM FAM (none)
Chlamydophila pneumoniae Yellow Dye Alexa532 (none)
DIA-DNA/EIC(TR)-050 Texas red Texas red (none)
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

VI . Interpretation of results
Interpretation of results
DNA extraction & PCR
inhibition control
Chlamydophila
pneumoniae
Mycoplasma pneumoniae
Signal detection
Signal detection
Signal detection
FAM
Orange dye
Yellow dye
Cy5
The sample is positive:
Mycoplasma pneumoniae
and negative:
Chlamydophila pneumoniae
Signal is above or below the
threshold
Signal is above the threshold Signal is below the threshold
The sample is negative:
Mycoplasma pneumoniae
and positive:
Chlamydophila pneumoniae
Signal is above or below the
threshold
Signal is below the threshold Signal is above the threshold
The sample is positive:
Mycoplasma pneumoniae
Chlamydophila pneumoniae
Signal is above or below the
threshold
Signal is above the threshold Signal is above the threshold
The sample is negative:
Mycoplasma pneumoniae
Chlamydophila pneumoniae
Signal is below the threshold Signal is below the threshold Signal is above the threshold
The sample is invalid .
See Troubleshooting guide
Signal is below the threshold Signal is below the threshold Signal is below the threshold
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
Troubleshooting guide
> Nucleic Acid isolation
Refer to the manufacturer’s product insert.
NB:
Perform preventive maintenance of workstations for automated extraction (according to the
manufacturer’s recommendations). Check the expiration date and storage conditions for
solutions provided.
> PCR
The signal detected for the positive Mycoplasma pneumoniae and Chlamydophila pneumoniae
control, the DNA extraction & inhibition control (DIA-EIC/DNA-050) are negative or weak.
The PCR conditions do not comply with the protocol:
1. Check extraction procedure.
It’s important to select an appropriate extraction procedure. The extraction procedure
must be validated and limit the presence of inhibitory factors.
2. Check PCR reaction set-up.
To obtain a high sensitivity, you must respect volumes described and the master
mixes suggested.
NB:
Check the calibration of pipettes used (according to the manufacturer’s recommendations).
Check the expiration date and storage conditions of master mixes suggested.
3. Check the PCR profile.
To obtain a high sensitivity, respect the protocol adapted to the real-time PCR used.
4. Check dyes selected.
Check and select dyes adapted for each real-time PCR platform.
5. Interpretation of results.
Check the table and select the appropriate channel.
Important note:
Check storage conditions (during and after experiments) and expiration date . These two
conditions highly influence results obtained .
> Real time PCR system
Refer to the manufacturer’s product insert.
NB:
Perform maintenance of the real-time PCR platform (according to the manufacturer’s
recommendations).
For trouble shootings/questions, please contact us:
Tel .: +32 (0)4 364 20 50 - info@diagenodediagnostics .com
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

Specifications
I. Sensitivity
DNA from “Two Quality Control Exercises Involving Nucleic Acid Amplification Methods for
detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae and Carried Out 2
Years Apart (in 2002 and 2004)” (Loens, Beck et al . 2006) was extracted with the Nucleisens
easyMAG ® system (Biomerieux) (see point II . Sample Extraction/DNA Isolation) and subjected to
PCR amplification. A sample was interpreted as positive if the relative fluorescence crossed the
threshold as determined by the ABI 7000 (Applied Biosystems) detection system software. The
following results were obtained:
II. Specificity
Organism Limit of detection
Mycoplasma pneumoniae 500 CCU/100 μl
Chlamydophila pneumoniae 49 IFU/100 μl
The specific primers and probes were selected from the literature (Templeton, Scheltinga et al .
2003; Welti, Jaton et al . 2003; Templeton, Scheltinga et al . 2005; Loens, Beck et al . 2006). They
were also checked for possible homologies to other known sequences by sequence comparison
analysis. Primers and probes are used routinely in clinical diagnostic laboratories on human
bronchoalveolar liquid (BAL), sputum and swab samples.
Internal control sequence has also been checked by sequence analysis.
Cross-reaction tests have been performed with the following viruses:
Archanobacterium
pyogenes
Candida albicans
Haemophilus influenzae Proteus mirabilis
Haemophilus
parainfluenzae
Proteus vulgaris
Staphylococcus
saprophyticus
Streptococcus
agalactiae
Citrobacter freundii Klebsiella pneumoniae Salmonella typhi Varicella zoster virus
Clostridium sporogenes Legionella pneumophila Serratia marcescens Streptococcus mitis
Enterobacter cloacae Moraxella catarrhalis Shigella sonei
Enterococcus faecalis Neisseria meningitidis
Staphylococcus
epidermidis
Streptococcus
pneumoniae
Adenovirus 3
Cytomegalovirus Epstein barr virus Herpes simplex type 1 Herpes simplex type 2
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
Product Use Limitations
• All reagents must exclusively be used for in vitro diagnostics.
• A strict compliance with the user manual is required for optimal results.
• Reliable results are dependent on adequate specimen collection, transport, storage and
processing procedures.
• This test has been validated for use with with bronchoalveolar lavage, vesicular fluid,
ocular fluid, swabs corneal scraping, cerebrospinal fluid, amniotic fluid, plasma, serum,
tissue/biopsy.
• This test has been validated for use with the ABI 7000-7300-7500-7900/ Roche Lc480-
Lc2.0 / Biorad iCycler-IQ5-CFX96 / Stratagene MX3000P-3500P / Qiagen Rotor-Gene /
Cepheid SmartCycler II real Time PCR systems.
Quality Control
Diagenode has obtained ISO 9001 and ISO 13485 certifications for the design, the production and
the sale of clinical diagnostic kits based on the real time PCR technology.
Mycoplasma/Chlamydophila pneumoniae Real-Time PCR is tested against predetermined
specifications to ensure the product quality.
References
Loens, K., T. Beck, et al. (2006). "Two quality control exercises involving nucleic acid amplification
methods for detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae and carried
out 2 years apart (in 2002 and 2004) ." J Clin Microbiol 44(3): 899-908.
Templeton, K. E., S. A. Scheltinga, et al. (2003). "Comparison and evaluation of real-time PCR,
real-time nucleic acid sequence-based amplification, conventional PCR, and serology for
diagnosis of Mycoplasma pneumoniae ." J Clin Microbiol 41(9): 4366-71.
Templeton, K. E., S. A. Scheltinga, et al. (2005). "Improved diagnosis of the etiology of communityacquired
pneumonia with real-time polymerase chain reaction ." Clin Infect Dis 41(3): 345-51.
Welti, M., K. Jaton, et al. (2003). "Development of a multiplex real-time quantitative PCR assay
to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in
respiratory tract secretions ." Diagn Microbiol Infect Dis 45(2): 85-95.
Europe Diagenode sa / CHU - Tour GIGA - B34 - 3 e étage // Avenue de l’Hôpital, 1 // 4000 Liège (Sart Tilman) // Belgium // Phone: (+32) 4 364 20 50 // Mail: info@diagenode.com

Explanation of Symbols
yyyy-mm
Catalogue number
Batch code
Number of experiments
Temperature limitation
Expiry date
In vitro diagnostic medical device
Refer to user manual
Biological risk
Control
Negative control
Positive control
Protected from light
Manufacturer
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DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
Notice to purchaser
This product is optimized for use in the polymerase chain reaction (PCR) covered by patents owned
by Roche Molecular Systems, Inc., and F. Hoffmann-La Roche ltd. (Roche). No license under these
patents to use the PCR process is conveyed expressly or by implication to the purchaser by the
purchase of this product. A license to use the PCR process for certain research and development
activities accompanies the purchase of certain reagents from licensed suppliers, when used in
conjunction with an Authorized Thermal Cycler, or is available from Applied Biosystems. Further
information on purchasing licenses to practice the PCR process may be obtained by contacting
the Director of Licensing at Applied Biosystems, 850 Lincoln Center Drive, Foster City, California
94404 or at Roche Molecular Systems, Inc, 1145 Atlantic Avenue, Alameda, California 94501.
• ABI (Applied Biosystems) Prism 7000 or 7300 or 7500 or 7900 are a trademark of Applera
Corporation
• Roche Lightcycler 480 and 2.0 are a trademark of Roche
• Biorad iCycler or IQ5 or CFX96 are a trademark of Biorad
• Stratagene MX3000P or 3500P are a trademark of Stratagene
• Qiagen Rotor-Gene is a trademark of Qiagen
• Cepheid SmartCycler II is a trademark of Cepheid
• Nuclisens easyMAG ® System is a trademark of Biomerieux
• MagNa pure LC system is a trademark of Roche
• QIAamp UltraSens Virus Kit (50) is a trademark of Qiagen
• Quantifast Multiplex+R kit is a trademark of Qiagen
* Class Ea1: virus which can cause diseases to animals, and present the following
characteristics (with variable degrees): limited geographic importance, weak
interspecies transmissibility, vector or bearer non-existent . It does not normally
require particular measures of seclusion . There is usually disease prevention and/
or an effective treatment .
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PAGE 26 26 DIAGENODE MYCOPLASMA PNEUMONIAE / CHLAMYDOPHILA PNEUMONIAE REAL-TIME PCR USER MANUAL
Diagenode s.a.
Avenue de l’hôpital,1 • Tour GIGA, 3rd Floor • 4000 Liège • Belgium
Tel: +32 4 364 20 50 • Fax: +32 4 364 20 51 • info@diagenodediagnostics.com
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PAGE 27
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Diagenode sa
CHU, Tour GIGA, 3e étage
Avenue de l’hôpital, 1
4000 Liège - BELGIUM
Tel. +32 4 364 20 50
info@diagenodediagnostics.com
www.diagenodediagnostics.com