Mycoplasma pneumoniae Chlamydophila pneumoniae - Mikrogen

mikrogen.de

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).

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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.

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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)

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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

PAGE 21

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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

PAGE 23

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PAGE 24

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

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


PAGE 27

MA-MCpn-V1_24_02_12

www.diagenodediagnostics.com


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

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