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For Professional Use Only

GUIDELINES
to

AmpliSens HPV 6/11-FRT PCR kit

for qualitative detection and differentiation of types 6 and 11


of human papillomavirus (HPV) DNA in clinical material by the
polymerase chain reaction (PCR) with real-time hybridizationfluorescence detection

Ecoli s.r.o., Studenohorska


12
841 03 Bratislava 47
Slovak Republic
Tel.: +421 2 6478 9336
Fax: +421 2 6478 9040

Federal Budget Institute of


Science Central Research
Institute for Epidemiology
3A Novogireevskaya Street
Moscow 111123 Russia

TABLE OF CONTENTS
INTENDED USE .................................................................................................................. 3
AMPLIFICATION AND DATA ANALYSIS WITH Rotor-Gene 3000/6000 (Corbett
Research, Australia) INSTRUMENTS ................................................................................. 3
AMPLIFICATION AND DATA ANALYSIS WITH iCycler iQ5 (Bio-Rad, USA)
INSTRUMENTS................................................................................................................. 11
AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)
INSTRUMENT ................................................................................................................... 16

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

INTENDED USE
Guidelines describe the procedure of the use of AmpliSens HPV 6/11-FRT PCR kit for
qualitative detection and differentiation of types 6 and 11 of human papillomavirus (HPV)
DNA by means of real-time hybridization-fluorescence detection with the following realtime 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-ml PCR 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.
When working 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 (not
empty).
3. Program the instrument to run amplification and detection:
In the opened window select Advanced mode. Activate the New button.
Select 36-Well Rotor (or 72-Well Rotor) and No Domed Tubes/Locking ring
attached. Click Next.
In the opened window define the operator and set the reaction volume: Reaction
volume is 25 l. Tick off the 15 l oil layer volume option.
set amplification program.
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 3 of 22

Table 1
DNA HPV types 6 and 11 amplification program for
Rotor-Gene 3000/6000 (Corbett Research, Australia)
Stage

emperature,

Time

Hold

95
95

15 min
15 s

60

35 s

Cycling

Fluorescence
detection

FAM/Green,
JOE/Yellow,
ROX/Orange

Cycle
repeats
1
45

This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 RG (see
Table 2) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 3) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 RG
amplification program is a universal program and it can be used for running
different tests within one run (for example tests for detection of STIs). For
detection of DNA of HCR HPV and HPV DNA types 6, 11 within one run
AmpliSens FRT HR HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit.
Table 2
AmpliSens-1 RG amplification program
Step

Temperature,

Time

Hold
Cycling 1

95
95
60
72
95

15 min
5s
20 s
15 s
5s

Cycling 2

60

20 s

72

15 s

Fluorescence
detection

FAM/Green,
JOE/Yellow,
ROX/Orange,
Cy5/Red

Cycle
repeats
1
5

40

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

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 4 of 22

Table 3
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
Step

Temperature,

Time

Hold
Hold 2

95
65
95
64
Touchdown:
1 deg. per cycle
65
95
60

15 min
2 min
20 s

Fluorescence
detection

25 s

55 s
15 s
25 s

65

25 s

FAM/Green,
JOE/Yellow,
ROX/Orange,
Cy5/Red

Cycling

Cycling 2

Cycle repeats
1
1

40

The file AmpliSens FRT HR HPV Screen RG4x Program.pro enclosed to


AmpliSens HPV HCR screen-titre-FRT PCR kit can be used for programming.
Click the Calibrate/Gain Optimisation button in the New Run Wizard window.
a) for detection in FAM/Green, JOE/Yellow, ROX/Orange, and Cy5/Red channels
select Calibrate Acquiring/Optimise Acquiring;
b) click Perform Calibration Before 1st Acquisition/Perform Optimisation
Before 1st Acquisition.
c) click the Edit button in the Auto gain calibration channel settings window
and set Min Reading as 4 and Max Reading as 8 for all channels.
4. Select the Start run button 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 Name column. Define the Negative Control
of amplification as NCA and the 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
(HPV type 6, HPV type 11 and human DNA) several pages should be created in protocol.
To do this, indicate HPV 6 (FAM/Green channel) in the Page area 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 the Page area
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 5 of 22

of the Name field. Click New. Third page for information about samples will appear.
Indicate Human (ROX/Orange channel) in the Page area of the Name field.
Data analysis
Review row data in all three channels (FAM/Green, JOE/Yellow and ROX/Orange) by
selecting Cycling A buttons on the Channels bar. Pay attention to the followings:

among all signals detected in the channel at least one should be positive (a positive
signal has typical S-shape, see Fig.1). If the experiment is correct, the signal of
positive control C+ must be detected.
If positive signals were not detected in a channel the result of data analysis can
be incorrect.

If background signal of some samples greatly differs from all other samples it indicates
incorrect dispensing reaction mixture or DNA sample during tube preparation (see
Fig. 2a).

1. Make sure that all analyzed samples are activated on the legend at the right.
2. Activate the Analysis button then select the Quantitation mode. Activate the Cycling
A. FAM/Cycling A. Green button; click Show.
3. Set Threshold = 0.03.
4. Select Linear scale. Activate the Dynamic tube and Slope Correct buttons in the
main window menu (Quantitation analysis).
5. Select the More Settings/Outlier Removal parameter and set the NTC threshold
value as 10 %.
6. Carry out data analysis for JOE/Yellow and ROX/Orange channels similarly to that for
FAM/Green channel.
In some rare cases due to descending character of the curves at the beginning
of the experiment, fluorescent curves can cross the threshold line at first cycles
and Rotor-Gene program can accept these results as positive (Fig. 7). Use
Eliminate cycles before option and enter 5.
7. For data analysis select Analysis then Quantitation.

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 6 of 22

TROUBLESHOOTING
Review this section before running the experiment
Problem
Cause
How to identify
Negative samples are
analyzed as positive
samples by RotorGene program

Incorrect
mathematical
processing of
negative samples in
the presence of drop
of fluorescence at
the initial cycles
(Fig. 2a)

The threshold line


crosses descending
fluorescence curves
at the initial cycles
(Fig. 6)

Drop of sensitivity due


to contamination of
instrument lenses

Drop of sensitivity due


to deterioration of
probes

Lenses
contamination leads
to reduction of
effectiveness of
fluorescence
excitation and
detection. Primarily
this has an impact
on the samples
containing small
quantity of specific
DNA which show
low fluorescence
growth
Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause
oligonucleotides
destruction

Ways to eliminate

A normal positive
sample has a Sshaped curve of
fluorescence
accumulation (Fig. 1,
3-5). The negative
samples processed
incorrectly appear as
quite straight bottomup lines (Fig. 7)
The red threshold line
crosses (or touches)
fluorescence curves
at the left (initial
cycles) on the chart of
processed
fluorescence curves
(Fig. 6)
Low background
signal in all four
detection channels
(<1) along with
maximum value of the
gain parameter (10)

Use the Ignore First


option: select
5 cycles. If it does not
help, increase this
value by 1-5

Probe destruction can


be find out as a result
of comparison of
experimental data
obtained at the first
use of the reagents
and after a period of
time or in comparison
with the reagents of
the same lot stored
properly. It can be
revealed as a
reduction of value of
the gain multiplying
coefficient (selected
automatically) by
more than 2 units in
different experiments
(in case the same
instrument is used).
Note! Effect of the
gain parameter

Use mixtures stored


under stated
conditions until
labeled expire date
(see part Stability
and Storage in the
Instruction Manual)

Use the Eliminate


cycles before
option: select 5
(crossing the
threshold with
fluorescence curve is
ignored for the first 5
cycles)
Clean horizontal and
vertical lenses of the
instrument with a dry
cotton pad at least
once a month

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 7 of 22

Desensitization due to
decrease of
polymerase (TaqF)
activity

Incorrect storage or
contamination cause
enzyme
deterioration

increase can appear


after cleansing the
instrument lenses
from sever
contamination
It appears as a fail of
the positive control or
if a boundary Ct value
of the positive control
is greater than the
threshold of weak
samples

Use the reagent


stored under required
condition (see part
Stability and Storage
in the Instruction
Manual) or use a new
reagent

Note! A report on all parameters specified as well as a report on auto-calibration is


available if the Settings button is activated. In particular, the Messages tab, the
Autocalibration Log Messages option.
Problem
Contamination of a specific
DNA

Feature
Detection of a signal of the
negative control in any
channel

The volume of a DNA sample


added to a reaction tube is
less than required/DNA
samples is not added

Background signal of a
sample is much stronger than
the others (see raw curves,
Fig. 2b). The sample is
negative
Background signal of a
If the sample is negative,
sample is much lower than the repeat the test
others (see raw curves, Fig.
2b).

The volume of the reaction


mixture added to a reaction
tube is less than
required/reaction mixture is
not added/the volume of a
DNA sample added to a
reaction tube is greater than
required
The autocalibration parameter
from 4Fl to 8Fl is not set or it
is a failure of the first tube in
the rotor (the No.1 well is
empty; a volume of DNA
sample or reaction mixture
added to the tube is incorrect)

Polymerase (TaqF) was not


added to a reaction mixture

Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample

Most of the fluorescence


background signals are less
than 1 or greater than 20

Set the parameter in the next


run. In case of off-scale
reading or if the signals are
too weak (no positive signals
after processing; a
background is less than 0.5)
the experiment should be
repeated beginning with PCR

Positive signals are absent for


all samples including a
positive control

Repeat the experiment


beginning with PCR. Ensure
appropriate preparation of
reaction mixtures

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 8 of 22

Unprocessed data (raw channel)

Fig. 2. Raw curves have a bend (drop of


fluorescence at the initial cycles)

Fig. 1. Raw curves are normal

Fig. 2b. Level of background signal points on the


possible 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 channel

Fig. 4. JOE channel

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 9 of 22

Fig. 5. ROX channel internal control


(-globin gene)
b) curves are processed incorrectly

Fig. 6. Threshold line crosses fluorescence


curves twice

Fig. 7. Incorrect processing of the curves with


bend (from Fig. 2)

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 10 of 22

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


INSTRUMENTS
Carry out sample pretreatment and reaction mixture preparation stages according to the
instruction manual. Transparent domed 0.2-ml PCR 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
DNA HPV types 6 and 11 amplification program
for iCycler iQ, iQ5 (Bio-Rad, USA)
Step

Temperature,

Time

95
95
60

15 min
20 s
1 min

Fluorescence
detection

FAM, HEX, ROX

Cycle
repeats
1
45

This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 iQ (see
Table 5) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 6) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 iQ amplification
program is universal program and it can be used for running different tests
within one run (for example tests for detection of STIs). For detection of DNA
of HCR HPV and HPV DNA types 6, 11 within one run AmpliSens FRT HR
HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit
Table 5
AmpliSens-1 iQ amplification program
Step

Temperature,

Time

95
95
60
72
95
60
72

15 min
5s
20 s
15 s
5s
30 s
15 s

Fluorescence
detection

FAM, HEX, ROX, Cy5

Cycle
repeats
1
5

40

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 11 of 22

Cy5/Red detection channel should be enabled for running multiplex tests (if
required).
Table 6
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
15 min
15 s

Fluorescence
detection

Cycle
repeats
1

55 s

25 s
15 s
55 s
25 s

Real-time

Step

Temperature,

Time

Cycle 1

95
95
65
Touchdown:
1 deg. per cycle
65
95
60
65

Cycle 3

Cycle 4

41

The AmpliSens FRT HR HPV Screen iQ5.tmo file enclosed to AmpliSens


HPV HCR screen-titre-FRT PCR kit can be used for programming.

Create a new plate setup in the Edit Plate Setup tab. Indicate samples as Unknown.
Proceed to the Select fluorofores tab and set fluorescence detection for all tubes in the
FAM, HEX and ROX channels. Go back to the Samples: Whole plate loading and click
the Per Dye Layer button.

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 the PCR Quantification tab in the Data Analysis mode.
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 Defined, 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 Results button (below the fluorofore buttons).
5. Proceed to the PCR Standard Curve tab, review parameters of the reaction, copy the
results.
6. Click on the results grid using the mouse right button. Select Export to Excel from 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
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 12 of 22

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

How to identify

Drop of sensitivity due


to deterioration of
probes

Problem

Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause destroy of
oligonucleotides

Drop of sensitivity due


to decrease of activity
of polymerase (TaqF)

Incorrect storage or
contamination cause
enzyme
deterioration

Probe destruction can


be find out as a result
of comparison of
experimental data
obtained at the first
use of the reagents
and after a period of
time or in comparison
with the reagents of
the same lot stored
properly. It is
appeared as an
increase of
background
fluorescence
(fluorescence is
assessed in the
Background
subtracted mode at
the beginning of the
experiment) twice as
much in different
experiments (in case
the same instrument
is used). Note! Effect
of fluorescence
increase can appear
in case the lamp was
changed, optical
system was cleaned,
or calibration was
done)
It appears as a fail of
the positive control or
if a boundary Ct value
of the positive control
is much greater than
the normal one

Problem
Contamination of a specific
DNA

Feature
Detection of a signal for a
negative control in any
channel

The volume of a DNA sample


added to a reaction tube is
less than required/DNA
samples is not added

Background signal of the


sample is much stronger than
the others (see raw curves
Background subtracted
mode). The sample is
negative

Ways to eliminate
Use mixtures stored
under stated
conditions until
labeled expire date
(see part Stability
and Storage in the
Instruction Manual)

Use the reagent


stored under required
condition (see part
Stability and Storage
in the Instruction
Manual) or use a new
reagent

Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 13 of 22

The volume of a reaction


mixture added to a reaction
tube is less than
required/reaction mixture is
not added or the volume of a
DNA sample added to a
reaction tube is greater than
required
Incorrect adjustment of the
threshold level

Background signal of the


If the sample is negative,
sample is much lower than the repeat the test
others (see raw curves Background subtracted
mode)

Drops were not removed from


the tube walls before
experiment started

Curves of fluorescence
accumulation have negative
or positive steps (Fig. 2)

Polymerase (TaqF) was not


added to a reaction mixture

Positive signals are absent for


all samples including a
positive control

Threshold line is located along


with negative samples or
above some or all positive
curves (S-shaped)

Set the threshold line so that it


crosses only sigmoid curves
of fluorescence accumulation
or at the level of between
final values of negative and
positive samples
Select the BaseLine
Threshold window (the right
mouse button on the graph of
fluorescence) and indicate
that base line is to be
calculated beginning with the
first cycle after the step
Repeat the experiment
beginning with PCR. Ensure
appropriate preparation of
reaction mixtures

Unprocessed (raw) data (Background subtracted mode).

Err

Fig. 1 Raw curves are normal (FAM)

Fig. 2. Baseline has a step: drops were


not removed from the tube walls before the
experiment in the sample indicated as Err

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 14 of 22

Processed data
Processed curves are normal; S-shaped; thresholds are located correctly (PCR Base Line
Subtracted Curve Fit mode)

Fig.4. HEX channel

Fig. 3. FAM channel

Fig 5. ROX channel internal control


(-globin gene)

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 15 of 22

AMPLIFICATION AND DATA ANALYSIS WITH Mx3000P (Stratagene, USA)


INSTRUMENT
Carry out sample pretreatment and reaction mixture preparation stages according to the
instruction manual. Transparent domed 0.2-ml PCR 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 Options window select Quantitative PCR (Multiple Standards)
and select Turn 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 Optics Configuration in the Options menu. In the Dye Assignment tab
indicate JOE next to the HEX/JOE filter set option; indicate FAM next to the FAM
filter set option.
5. In the Instrument menu of the Mx3000P program select the Filter Set Gain Settings
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 during
amplification cause signal errors and complicate data analysis. Do not turn
strip/plate over when loading into the PCR instrument.
6. Define parameters of fluorescence acquiring in the Plate Setup menu. To do this:
a) hold Ctrl and 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
fluorescence data option select FAM, JOE, ROX, and Cy5. Double click each
cell and enter a sample name (Well Information window). Indicate the positive
control as +, the negative control as . Sample names can be entered during or
after the amplification run in the Plate Setup menu.
7. Proceed to the Thermal Profile Setup tab and set the amplification program (see
Table 7):

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 16 of 22

Table 7
DNA HPV types 6 and 11 amplification program
for Mx3000P, Mx3005P (Stratagene, USA)
Step

Temperature,

Time

95
95
60

15 min
20 s
1 min

Fluorescence
detection

FAM, HEX/JOE, ROX

Cycle
repeats
1
45

This program can be applied for both AmpliSens HPV 6/11-FRT and
AmpliSens HPV 16/18-FRT PCR kits.
Following amplification programs can be used as well: AmpliSens-1 Mx (see
Table 2) and amplification program for HPV HCR DNA types 16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 (see Table 9) (this program is applied for
AmpliSens HPV HCR screen-titre-FRT PCR kit). AmpliSens-1 Mx
amplification program is universal program and it can be used for running
different tests within one run (for example tests for detection of STIs). For
detection of DNA of HPV HCR and HPV DNA types 6, 11 within one run
AmpliSens FRT HR HPV ScreenRG4x 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 AmpliSens HPV HCR screen-titre-FRT PCR
kit) does not affect analytical performances of this PCR kit
Table 8
AmpliSens-1 Mx amplification program
Step

Temperature,

Time

Segment 1
Segment 2
(Cycling)

95
95
60
72
95

15 min
5s
20 s
15 s
5s

Segment 3
(Cycling)

60

30 s

72

15 s

Fluorescence
detection

FAM, HEX/JOE, ROX,


Cy5

Cycle
repeats
1
5

40

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

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 17 of 22

Table 9
Amplification program for HPV HCR DNA types
16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59
15 min
2 min
20 s

Fluorescence
detection

Cycle
repeats
1
1

25 s

55 s
20 s
25 s

Cy5, FAM,
HEX/JOE, ROX

Step

Temperature,

Time

Segment 1
Segment 2

95
65
95
64
Touchdown:
1 deg. per cycle
65
95
60
65

55 s

Segment 3
(Cycling)

Segment 4
(Cycling)

40

The AmpliSens FRT HR HPV Screen Mx.mxp file enclosed to AmpliSens


HPV HCR screen-titre-FRT PCR kit can be used for programming.
8. In the Plate Setup tab of the Setup mode 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 Analysis 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 Analysis
Selection/Setup tab. Otherwise, hold Ctrl and 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 150 in the Threshold fluorescence field 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 Shown field at the bottom of the program window).
6. Ensure that the JOE, FAM and ROX fluorescence channels are ticked off in the
Threshold fluorescence field.
REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 18 of 22

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 analyze field. Review the results and export
data for further analysis (for example export to Excel: File>Export Instrument
Data>Export Instrument Data to Excel).
TROUBLESHOOTING
Cause

How to identify

Drop of sensitivity due


to deterioration of
probes

Problem

Incorrect storage or
utilizing kit
components (high
temperature,
multiple opening
reagent tubes, foul
working conditions)
can cause destroy of
oligonucleotides

Drop of sensitivity due


to decrease of
polymerase (TaqF)
activity

Incorrect storage or
contamination cause
enzyme
deterioration

Probe destruction can


be find out as a result
of comparison of
experimental data
obtained at the first
use of the reagents
and after a period of
time or in comparison
with the reagents of
the same lot stored
properly. It is
appeared as an
increase of
background
fluorescence
(fluorescence is
assessed in the R
mode at the
beginning of the
experiment) twice as
much in different
experiments (in case
the same instrument
is used). Note! Effect
of fluorescence
increase can appear
after cleansing the
optical system
It appears as a fail of
the positive control or
if a boundary Ct value
of the positive control
is greater than the
threshold of weak
samples

Ways to eliminate
Use mixtures stored
under stated
conditions until
labeled expire date
(see part Stability
and Storage in the
Instruction Manual)

Use the reagent


stored under required
condition (see part
Stability and Storage
in the Instruction
Manual) or use a new
reagent

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 19 of 22

Problem
Contamination of a specific
DNA

Feature
Detection of a signal for a
negative control in any
channel

The volume of a DNA sample


added to a reaction tube is
less than required/DNA
samples is not added

Background signal of a
sample is much stronger than
the others (see raw curves
R-multicomponent view
mode) The sample is negative
Background signal of a
If the sample is negative,
sample is much lower than the repeat the test
others (see raw curves - Rmulticomponent view mode)

The volume of a reaction


mixture added to a reaction
tube is less than
required/reaction mixture is
not added or the volume of a
DNA sample added to a
reaction tube is greater than
required
Incorrect adjustment of the
threshold level

Polymerase (TaqF) was not


added to a reaction mixture

Threshold line is located along


with negative samples or
above some or all positive
curves (S-shaped)

Positive signals are absent for


all samples including a
positive control

Ways to eliminate
Repeat the experiment. Take
measures to detect and
eliminate the source of
contamination
Repeat PCR for this sample

Set the threshold line so that it


crosses only sigmoid curves
of fluorescence accumulation
or at the level of between
final values of negative and
positive samples
Repeat the experiment
beginning with PCR. Ensure
appropriate preparation of
reaction mixtures

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 20 of 22

Unprocessed data (R mode)

Err

Fig. 1. Raw curves are normal

Fig. 2. Level of background signal point


on a possible problem: DNA sample
was not added to the Err tube

Processed data
Normal curves that were processed, dR mode (S-shaped)

Fig. 4. HEX channel


Fig. 3. FAM channel

Fig. 5. ROX channel internal control


(-globin gene)

REF R-V11(RG,iQ,Mx)-CE / VER 17.02.12-02.04.14 / Page 21 of 22

List of Changes Made in the Instruction Manual


VER
02.04.14
SA

Location of
changes
Cover page
Footer

Essence of changes
Address of European representative was added
REF R-V11(RG,iQ,Mx)--B was deleted

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