qualitative detection and differentiation of types 6 and 11 papiloma

8/10/2019 Qualitative Detection and Differentiation of Types 6 and 11 Papiloma

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For Professional Use Only

GUIDELINES

toAmpliSens HPV6/11-FRTPCR kit

for qualitative detection and differentiation of types 6 and 11

ofhuman papillomavirus (HPV)DNA in clinical material by the

polymerase chain reaction (PCR) with real-time hybridization-

fluorescence detection

Ecoli s.r.o., Studenohorska12841 03 Bratislava 47

Slovak RepublicTel.: +421 2 6478 9336Fax: +421 2 6478 9040

Federal Budget Institute ofScience Central ResearchInstitute for Epidemiology

3A Novogireevskaya StreetMoscow 111123 Russia


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REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 2 of 22

TABLE OF CONTENTSINTENDED USE .................................................................................................................. 3AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (CorbettResearch, Australia) INSTRUMENTS ................................................................................. 3AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)INSTRUMENTS ................................................................................................................. 11

AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)INSTRUMENT ................................................................................................................... 16


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INTENDED USE

Guidelines describe the procedure of the use of AmpliSens

HPV 6/11-FRTPCR kitfor

qualitative detection and differentiation of types 6 and 11 ofhuman papillomavirus (HPV)

DNA by means of real-time hybridization-fluorescence detection with the following real-time PCR instruments:

Rotor-Gene 3000, Rotor-Gene 6000 (Corbett Research, Australia),

iCycler iQ5 (Bio-Rad, USA),

Mx3000P (Stratagene, USA).

AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (Corbett

Research, Australia) INSTRUMENTS

Carry out sample pretreatment and reaction mixture preparation stages according to the

instruction manual. Transparent no domed 0.2-mlPCR tubes (detection through the

bottom of a tube) or 0.1-ml tubes are recommended for use.

Hereinafter, all terms corresponding to different instruments and software are

indicated in the following order: for Rotor-Gene 3000/for Rotor-Gene 6000.

Whenworking with Rotor-Gene 3000 instrument use the program Rotor-Gene version 6.

When working with Rotor-Gene 6000 instrument use the program Rotor-Gene 6000

versions 1.7 (build 67) or higher.

Programming the instrument

1. Turn the instrument on and start Rotor-Gene program.

2. Place the tubes in the rotor so that the first tube is in No. 1 well, insert the rotor in the

reaction module, and secure the cover (the rotor cells are numbered and these

numbers are used for sample order programming).

Balance the rotor if it is not loaded entirely. Fill unused wells with empty tubes. Do

not use tubes from previous runs.No.1 well should be loaded with a test tube from the current experiment (notempty).

3. Program the instrument to run amplification and detection:

In the opened window select Advancedmode. Activate the Newbutton.

Select 36-Well Roto r (or 72-Well Roto r) and No Domed Tubes/Lock ing r ing

attached. Click Next.

In the opened window define the operator and set the reaction volume: React ion

vo lumeis 25 l. Tick off the 15loi l layer vo lum eoption.

set amplification program.


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Table 1

DNAHPVtypes 6 and 11 amplification program for

Rotor-Gene 3000/6000 (Corbett Research, Australia)

Stage emperature, TimeFluorescence

detection

Cycle

repeatsHold 95 15 min 1

Cycling

95 15 s

4560 35 s

FAM/Green,JOE/Yellow,ROX/Orange

This program can be applied for both AmpliSensHPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.

Following amplification programs can be used as well:AmpliSens-1 RG (see

Table 2) and amplification program for HPVHCR DNA types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 3) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 RGamplification program is a universal program and it can be used for runningdifferent tests within one run (for example tests for detection of STIs). Fordetection of DNA ofHCRHPV and HPVDNA types 6, 11 within one runAmpliSens FRT HR HPVScreenRG4x Program can be applied.

Using amplification program for HPVHCR DNA types 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59 (from AmpliSensHPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit.

Table 2

AmpliSens-1 RG amplification program

Step Temperature, TimeFluorescence

detection

Cycle

repeats

Hold 95 15 min 1

Cycling 1

95 5 s

560 20 s

72 15 s

Cycling 2

95 5 s

4060 20 s

FAM/Green,

JOE/Yellow,ROX/Orange,

Cy5/Red

72 15 s

Cy5/Red detection channel should be enabled for running multiplex tests (ifrequired).


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Table 3

Amplification program for HPVHCR DNA types16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59


detectionCycle repeats

Hold 95 15 min 1Hold 2 65 2 min 1

Cycling

95 20 s

564

Touchdown:1 deg. per cycle

25 s

65 55 s

Cycling 2

95 15 s

40

60 25 s

65 25 s

FAM/Green,JOE/Yellow,

ROX/Orange,Cy5/Red

The file AmpliSens FRT HR HPVScreen RG4x Program.proenclosed toAmpliSensHPVHCR screen-titre-FRT PCR kit can be used for programming.

Click the Calibrate/Gain Opt imis at ionbutton in the New Run Wizardwindow.

a) for detection in FAM/Green, JOE/Yellow, ROX/Orange, and Cy5/Redchannels

select Calibrate Acq uir ing /Opt imis e Acq uir ing;

b) click Perform Calibrat ion Before 1st Acquis i t ion/Perform Opt imisat ion

Before 1stAcquis i t ion.

c) click the Edit button in the Auto ga in ca l ib rat ion ch annel set tings window

and set Min Reading as4andMax Reading as8for all channels.

4. Select the Start runbutton for amplification to run. Name the experiment and save it to

the disk (results of the run will be automatically saved in this file).

5. Enter data in the table of samples (opens by default when the run begins). Define

names/numbers of clinical samples in the Namecolumn. Define the Negative Control

of amplification as NCA andthe Positive Control of amplification as C+. Enter the

Unknown type for all clinical and control samples. Indicate the None type for empty

wells.

The sample will not be analyzed if its type is defined as None.

Due to the fact that the PCR kit applies a multiplex control including several DNA matrixes

(HPVtype 6, HPVtype 11 and human DNA) several pages should be created in protocol.

To do this, indicate HPV 6(FAM/Green channel) in the Pagearea of the Name field. Click

New. A new page for information about samples will appear. It contains the same names

of samples as in previous page. Indicate HPV 11(JOE/Yellow channel) in thePagearea


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TROUBLESHOOTING

Review this section before running the experiment

Problem Cause How to identify Ways to eliminateNegative samples areanalyzed as positive

samples by Rotor-Gene program

Incorrectmathematical

processing ofnegative samples inthe presence of dropof fluorescence atthe initial cycles(Fig. 2a)

A normal positivesample has a S-

shaped curve offluorescenceaccumulation (Fig. 1,3-5). The negativesamples processedincorrectly appear asquite straight bottom-up lines (Fig. 7)

Use the Ignore Firstoption: select

5 cycles. If it does nothelp, increase thisvalue by 1-5

The threshold linecrosses descendingfluorescence curvesat the initial cycles

(Fig. 6)

The red threshold linecrosses (or touches)fluorescence curvesat the left (initial

cycles) on the chart ofprocessedfluorescence curves(Fig. 6)

Use the Eliminatecycles beforeoption: select 5(crossing the

threshold withfluorescence curve isignored for the first 5cycles)

Drop of sensitivity dueto contamination ofinstrument lenses

Lensescontamination leadsto reduction ofeffectiveness offluorescenceexcitation anddetection. Primarilythis has an impact

on the samplescontaining smallquantity of specificDNA which showlow fluorescencegrowth

Low backgroundsignal in all fourdetection channels(


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increase can appearafter cleansing theinstrument lensesfrom severcontamination

Desensitization due to

decrease ofpolymerase (TaqF)activity

Incorrect storage or

contamination causeenzymedeterioration

It appears as a fail of

the positive control orif a boundary Ct valueof the positive controlis greater than thethreshold of weaksamples

Use the reagent

stored under requiredcondition (see partStability and Storagein the InstructionManual) or use a newreagent

Note! A report on all parameters specified as well as a report on auto-calibration isavailable if the Sett ings button is activated. In particular, the Messages tab, theAutoca l ib ra tion Log Messages option.

Problem Feature Ways to eliminateContamination of a specificDNA

Detection of a signal of thenegative control in anychannel

Repeat the experiment. Takemeasures to detect andeliminate the source ofcontamination

The volume of a DNA sampleadded to a reaction tube isless than required/DNAsamples is not added

Background signal of asample is much stronger thanthe others (see raw curves,Fig. 2b). The sample isnegative

Repeat PCR for this sample

The volume of the reactionmixture added to a reaction

tube is less thanrequired/reaction mixture isnot added/the volume of aDNA sample added to areaction tube is greater thanrequired

Background signal of asample is much lower than the

others (see raw curves, Fig.2b).

If the sample is negative,repeat the test

The autocalibration parameterf rom 4Fl to 8Flis not set or itis a failure of the first tube inthe rotor (the No.1 well isempty; a volume of DNAsample or reaction mixture

added to the tube is incorrect)

Most of the fluorescencebackground signals are lessthan 1 or greater than 20

Set the parameter in the nextrun. In case of off-scalereading or if the signals aretoo weak (no positive signalsafter processing; abackground is less than 0.5)

the experiment should berepeated beginning with PCR

Polymerase (TaqF) was notadded to a reaction mixture

Positive signals are absent forall samples including apositive control

Repeat the experimentbeginning with PCR. Ensureappropriate preparation ofreaction mixtures


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Unprocessed data (raw channel)

Fig. 1. Raw curves are normalFig. 2. Raw curves have a bend (drop offluorescence at the initial cycles)

Fig. 2b. Level of background signal points on thepossible error:DNA sample was not added to the tube 1;double volume of DNA sample was added to the

tube 2

Processed data (Quantitation analysis)

) processed curves are normal (S-shaped, threshold line crosses curves at the area of

growth of fluorescence)

Fig. 3. FAM channelFig. 4. JOE channel

2

1


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Fig. 5. ROX channelinternal control(-globin gene)

b) curves are processed incorrectly

Fig. 6. Threshold line crosses fluorescencecurves twice

Fig. 7. Incorrect processing of the curves withbend (from Fig. 2)


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AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)

INSTRUMENTS


instruction manual. Transparent domed 0.2-mlPCR tubes (detection through the cap of

the tube) are recommended for use.

1. Switch on the instrument and the power supply of the optical module.

The lamp should be warmed up for at least 15 min before the experiment starts.

2. Open the iQ5 program.

Table 4

DNAHPVtypes 6 and 11 amplification programfor iCycler iQ, iQ5 (Bio-Rad, USA)


detection

Cycle

repeats

1 95 15 min 1

295 20 s

4560 1 min FAM, HEX, ROX

This program can be applied for both AmpliSens

HPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.

Following amplification programs can be used as well:AmpliSens-1 iQ (seeTable 5) and amplification program for HPVHCR DNA types 16, 18, 31,33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 6) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 iQ amplificationprogram is universal program and it can be used for running different testswithin one run (for example tests for detection of STIs). For detection of DNAof HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HRHPV ScreenRG4x Program can be applied.

Using amplification program for HPVHCR DNA types 16, 18, 31, 33, 35,

39, 45, 51, 52, 56, 58, 59 (from AmpliSens

HPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit

Table 5

AmpliSens-1 iQ amplification program


detection

Cycle

repeats

1 95 15 min 1

2

95 5 s

560 20 s

72 15 s

3

95 5 s

4060 30 s FAM, HEX, ROX, Cy5

72 15 s


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Table 6

Amplification program for HPVHCR DNA types

16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59


detection

Cycle

repeats

Cycle 1 95 15 min 1

Cycle 3

95 15 s

665


55 s

65 25 s

Cycle 4

95 15 s

4160 55 s Real-time

65 25 s

The AmpliSens FRT HR HPV Screen iQ5.tmo file enclosed to AmpliSensHPVHCR screen-titre-FRTPCR kitcan be used for programming.

Create a new plate setup in the Edit Plate Setup tab. Indicate samples as Unknown.

Proceed to the Select fluoro forestab and set fluorescence detection for all tubes in the

FAM, HEX and ROX channels. Go back to the Samp les: Whole plate loadingand click

the Per Dye Layerbutton.

Click Run. Select Well factor (both well factor by experiment tubes and fixed well factor

are acceptable for use but the latter is recommended). Run the experiment.

Data analysis

1. Proceed to thePCR Quant i f icat iontab in theData Analysismode.

2. Review data in one channel at a time.

3. Ensure that the threshold line was set correctly for each channel. If it is required,

activate User Def ined, 2 through 10 cycles parameter. Normally threshold line

should cross s-shaped curves of signal accumulation of positive samples and controls

and not cross the base line. Otherwise, raise the threshold line up to the level where it

crosses positive samples only.

4. Activate the Resultsbutton (below the fluorofore buttons).

5. Proceed to the PCR Standard Curv etab, review parameters of the reaction, copy the

results.

6. Click on the results grid using the mouse right button. Select Export to Excelfrom the

drop-down menu. Agree to save the file. If Microsoft Excel program is loaded on the

computer it will open automatically (Microsoft Excel program is required for further


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data analysis). Results in the FAM, HEX, ROX, and then Cy5 channels follow

consistently.

TROUBLESHOOTING

Problem Cause How to identify Ways to eliminate

Drop of sensitivity dueto deterioration ofprobes

Incorrect storage orutilizing kitcomponents (hightemperature,multiple openingreagent tubes, foulworking conditions)can cause destroy ofoligonucleotides

Probe destruction canbe find out as a resultof comparison ofexperimental dataobtained at the firstuse of the reagentsand after a period oftime or in comparisonwith the reagents ofthe same lot storedproperly. It isappeared as an

increase ofbackgroundfluorescence(fluorescence isassessed in theBackground

subtractedmode atthe beginning of theexperiment) twice asmuch in differentexperiments (in casethe same instrument

is used). Note!Effectof f luorescenceincrease can appearin case the lamp waschanged, opticalsystem was cleaned,or calibration wasdone)

Use mixtures storedunder statedconditions untillabeled expire date(see part Stabilityand Storage in theInstruction Manual)

Drop of sensitivity dueto decrease of activityof polymerase (TaqF)

Incorrect storage orcontamination causeenzyme

deterioration

It appears as a fail ofthe positive control orif a boundary Ct value

of the positive controlis much greater thanthe normal one

Use the reagentstored under requiredcondition (see part

Stability and Storagein the InstructionManual) or use a newreagent

Problem Feature Ways to eliminate

Contamination of a specificDNA

Detection of a signal for anegative control in anychannel


The volume of a DNA sampleadded to a reaction tube is

less than required/DNAsamples is not added

Background signal of thesample is much stronger than

the others (see raw curvesBackground subtractedmode). The sample isnegative



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The volume of a reactionmixture added to a reactiontube is less thanrequired/reaction mixture isnot addedorthe volume of aDNA sample added to a

reaction tube is greater thanrequired

Background signal of thesample is much lower than theothers (see raw curves-Background subtractedmode)


Incorrect adjustment of thethreshold level

Threshold line is located alongwith negative samples orabove some or all positivecurves (S-shaped)

Set the threshold line so that itcrosses only sigmoid curvesof f luorescence accumulationor at the level of betweenfinal values of negative andpositive samples

Drops were not removed fromthe tube walls beforeexperiment started

Curves of f luorescenceaccumulation have negativeor positive steps (Fig. 2)

Select the BaseLineThresholdwindow (the rightmouse button on the graph offluorescence) and indicate

that base line is to becalculated beginning with thefirst cycle after the step




Unprocessed (raw) data (Background su bt racted mode).

Fig. 1 Raw curves are normal (FAM) Fig. 2. Baseline has a step:drops werenot removed from the tube walls before theexperiment in the sample indicated as Err

Err


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Processed data

Processed curves are normal; S-shaped; thresholds are located correctly (PCR Base Line

Subtracted Curv e Fit mode)

Fig. 3. FAM channel Fig.4. HEX channel

Fig 5. ROX channelinternal control(-globin gene)


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AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)

INSTRUMENT


instruction manual. Transparent domed 0.2-mlPCR tubes (detection through the cap of

the tube) are recommended for use.

1. Switch on the instrument and open the Mx3000P program.

2. In the New Experiment Option swindow select Quantitative PCR (Multiple Standards )

and selectTurn lamp on for warm-up.

The lamp should be warmed up for at least 15 min before the experiment starts.

3. Place the tubes in the instrument, secure the locking tool and close the lid.4. Select Opt ics Conf igura tion in the Opt ions menu. In the Dye Assignm ent tab

indicate JOE next to the HEX/JOE fi lter s et option; indicate FAM next to the FAM

filter setoption.

5. In theInst rumentmenu of the Mx3000P program select the Filter Set Gain Sett ings

option. In the opened window set the following parameters of multiplication factors:

Cy5 x4

ROX x1

HEX/JOE x4

FAM x4

Prior to analysis, remove drops from tube walls, because drops falling duringamplification cause signal errors and complicate data analysis. Do not turnstrip/plate over when loading into the PCR instrument.

6. Define parameters of fluorescence acquiring in the Plate Setupmenu. To do this:

a) hold Ctrland use the mouse to select all cells in which test tubes are inserted.

b) define all selected cells as Unknown in the Well type window. In the Collect

f luorescenc e data option select FAM, JOE, ROX, and Cy5. Double click each

cell and enter a sample name (Well Inform at ionwindow). Indicate the positive

control as +, the negative control as . Sample names can be entered during or

after the amplification run in the Plate Setupmenu.

7. Proceed to the Thermal Prof i le Setup tab and set the amplification program (see

Table 7):


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Table 7

DNA HPVtypes 6 and 11 amplification program

for Mx3000P, Mx3005P (Stratagene, USA)

Step Temperature, Time Fluorescencedetection Cyclerepeats

1 95 15 min 1

295 20 s

4560 1 min FAM, HEX/JOE, ROX

This program can be applied for both AmpliSensHPV6/11-FRTandAmpliSensHPV16/18-FRTPCR kits.

Following amplification programs can be used as well:AmpliSens-1 Mx (seeTable 2) and amplification program for HPV HCRDNA types 16, 18, 31,

33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 9) (this program is applied forAmpliSensHPVHCR screen-titre-FRT PCR kit). AmpliSens-1 Mxamplification program is universal program and it can be used for runningdifferent tests within one run (for example tests for detection of STIs). Fordetection of DNA of HPVHCR and HPV DNA types 6, 11 within one runAmpliSens FRT HR HPVScreenRG4x Program can be applied.

Using amplification program for HPV HCR DNA types 16, 18, 31, 33, 35,39, 45, 51, 52, 56, 58, 59 (from AmpliSensHPVHCR screen-titre-FRT PCRkit) does not affect analytical performances of this PCR kit

Table 8

AmpliSens-1 Mx amplification program


detection

Cycle

repeats

Segment 1 95 15 min 1

Segment 2(Cycling)

95 5 s

560 20 s

72 15 s

Segment 3

(Cycling)

95 5 s

4060 30 sFAM, HEX/JOE, ROX,

Cy572 15 s



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Table 9Amplification program for HPV HCRDNA types

16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59


detection

Cycle

repeats

Segment 1 95 15 min 1Segment 2 65 2 min 1

Segment 3(Cycling)

95 20 s

564


25 s

65 55 s

Segment 4(Cycling)

95 20 s

4060 25 s

65 55 sCy5, FAM,

HEX/JOE, ROX

The AmpliSens FRT HR HPV Screen Mx.mxp file enclosed to AmpliSensHPVHCR screen-titre-FRTPCR kitcan be used for programming.

8. In the Plate Setup tab of the Setupmode indicate names of the samples. Define all

samples as Unknown. Select FAM, JOE/HEX, ROX dyes for all samples.

9. Run the program.

10. Proceed to the analysis of results after the end of the run.

Data analysis

1. Proceed to the Analys is item by clicking the button on the toolbar in the Mx300P

program.

2. Ensure that all test samples are enabled (the cells are tinted) in the opened Analys is

Selection/Setuptab. Otherwise, hold Ctrland select all test samples with the mouse.

3. Proceed to the Results tab.

4. On the Dyes shown toolbar activate displaying FAM channel and deactivate

displaying all others channels. Set 150in the Thresho ld f luorescencefield for FAM

channel. One at a time activate next channel and deactivate the previous one and

enter threshold values according to the table:

Cy5 150

ROX 150

HEX/JOE 150

FAM 150

5. Activate displaying all channels (FAM, HEX and ROX buttons should be selected in

the Dyes Show n field at the bottom of the program window).

6. Ensure that the JOE, FAM and ROX fluorescence channels are ticked off in the

Thresho ld f luorescencefield.


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7. Ensure that the threshold line is set correctly. Normally, a threshold line should cross

S-shaped curves of signal accumulation of positive samples and controls and should

not cross the base line. Otherwise, the threshold should be raised.

8. Select Text Report option in the Area to analyzefield. Review the results and export

data for further analysis (for example export to Excel:Fi le>Export Instrument

Data>Export Instrum ent Data to Excel).

TROUBLESHOOTING

Problem Cause How to identify Ways to eliminateDrop of sensitivity dueto deterioration ofprobes

Incorrect storage orutilizing kitcomponents (hightemperature,multiple opening

reagent tubes, foulworking conditions)can cause destroy ofoligonucleotides

Probe destruction canbe find out as a resultof comparison ofexperimental dataobtained at the first

use of the reagentsand after a period oftime or in comparisonwith the reagents ofthe same lot storedproperly. It isappeared as anincrease ofbackgroundfluorescence(fluorescence isassessed in the Rmode at thebeginning of theexperiment) twice asmuch in differentexperiments (in casethe same instrumentis used). Note!Effectof f luorescenceincrease can appearafter cleansing theoptical system

Use mixtures storedunder statedconditions untillabeled expire date(see part Stability

and Storage in theInstruction Manual)

Drop of sensitivity dueto decrease ofpolymerase (TaqF)activity

Incorrect storage orcontamination causeenzymedeterioration

It appears as a fail ofthe positive control orif a boundary Ct valueof the positive controlis greater than thethreshold of weaksamples

Use the reagentstored under requiredcondition (see partStability and Storagein the InstructionManual) or use a newreagent


20/22


Problem Feature Ways to eliminate

Contamination of a specificDNA

Detection of a signal for anegative control in anychannel


The volume of a DNA sampleadded to a reaction tube isless than required/DNAsamples is not added

Background signal of asample is much stronger thanthe others (see raw curvesR-mult icompon ent viewmode) The sample is negative


The volume of a reactionmixture added to a reactiontube is less thanrequired/reaction mixture isnot addedorthe volume of aDNA sample added to areaction tube is greater than

required

Background signal of asample is much lower than theothers (see raw curves-R-mul t icompo nent v iewmode)


Incorrect adjustment of thethreshold level

Threshold line is located alongwith negative samples orabove some or all positivecurves (S-shaped)

Set the threshold line so that itcrosses only sigmoid curvesof f luorescence accumulationor at the level of betweenfinal values of negative andpositive samples





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Unprocessed data(R mode)

Fig. 1. Raw curves are normal Fig. 2. Level of background signal pointon a possible problem: DNA samplewas not added to the Err tube

Processed data

Normal curves that were processed, dRmode (S-shaped)

Fig. 3. FAM channel

Fig. 4. HEX channel

Fig. 5. ROX channelinternal control

(-globin gene)

Err


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List of Changes Made in the Instruction Manual

VERLocation of

changesEssence of changes

02.04.14

SA

Cover page

Footer

Address of European representative was added

REF R-V11(RG,iQ,Mx)--B was deleted

qualitative detection and differentiation of types 6 and 11 papiloma

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