- A window of Experiment profile appears. In this window do the following:
o
Experiment Name-Write date, name of Primers and
experiment number e.g. 030212vdpvs123Exp-5.
o
Click on 7500 (96 well)
o
Click Quantitation –Standard curve
o
Click TaqMan Reagent
o
Click Standards (~ 2 hours Compete a run)
Click on the Set up plate in the experiment
menu.
- A setup window is opened. In this
window do the following to define Targets and samples:
o
Define Target
§
Write target in the target name box ( VDPV123)
§
Choose reporter by clicking on the Reporter
dropdown box. For:
ü S1
VDPV – FAM
ü S2
VDPV – FAM
ü S3
VDPV – FAM
FAM- Sabin 123 VDPV-VP1
ü
Sabin123 VDPV-3D
- Assign colour to the reporter dye from colour drop down box.
ü
FAM – Red
ü Rox
– Blue
o Define
sample
§
Write NC, sample numbers and PC in the same
window.
§
Write Sabin 123 VDPV-VP1and sample number, and
also save sample name in file and click Add Saved.
o
In the same window open assign targets and
samples
o
Click on Assign Targets and samples
o
Select Passive reference as none.
o
Click on Assign Targets and Samples to well
o
Select wells in plate layout and assign Target by
clicking on respective target. Then assign sample no to respective well.
Click on the Run Methods in experiment menu.
- A
Default Run Method window is opened. In this window do the
following to set up the thermal programme to get the right temperature (following
stages and temperatures are to be set that can be done by clicking on add
stage or add step) :
o
Non-Degenerate conditions Sabin 123 VDPV
§
42°C for 45 min - RT reaction
§
95°C for 3 min – For Denaturation
§
95°C for 24 sec – For Denaturation
§
50°C for 30 sec – For Annealing
§
65°C for 24 sec – For Extension
§
Ramp rate 25% between anneal and extension
§
Cycles – 40.
- Click on the window at stage 3, step 2 (anneal step) forData collection.
- Set the sample volume at 25µl and number of cycles 40.
- Click the start button (green) to start run, it will show the time Run started at and Estimated Time remaining. And a window will appear on screen to save file. Save the file in the designated place in the computer.
- When the cycle is over, a window will appear as Finish run. Click the red x button at the upper right. Boxes will popup on the screen to save the changes before closing. Click yes to save the file.
- Take out stripes and turn off machine.
o
Analysis
of data
§
Open the 7500 software v2.0.1
§
Click on analysis in advanced setup. Find the
saved file and open the run file.
§
A window appears.
§
Select Linear (not Log) in the drop down Graph
Type button.
§
Click on Amplification Plot in experiment menu
to view the results.
§
Select sample well. Select well in plot color
for all the primers except SM. For SM select target from dropdown of plot
color.
§
Look for the amplification by looking at the
different color amplification curves. Color of amplification curve will tell
about the dye and the target.
§
If amplification is to be seen only for single
target then select target from target dropdown.
§
Select positive and negative control to validate
experiment first. Then Select each well and click it and see the graph.
§
If amplification curve is too low then adjust
manually as described in section 8.3.
§
Backup is taken at every 3 Month on hard disk to
store data of the samples.
o Report
Generation:
- After completion of the cycle, generate report of the experiment for documentation and future referral.
- Click on export in tool bar.
- Click export to power point.
- A box will appear on the screen.
- Choose the file that we want to export.
- Click on create slides.
- Select plate lay out option and amplification plot.
- Click on create slides. A power point file will open in which experiment summary, plate layout and amplification plot will be generated.
o
Interpretation:
§
The results are interpreted by comparing the
amplification curves for VP1 dye only of PCR products from test samples to
those of positive control reactions. First results are to be validated by
negative control (No curve) and positive control (amplification curve).
§
If amplification occurs then a logarithmic curve
appears and if no amplification then no curve appears.
|
Sr.No.
|
VP1
|
3D
|
Result
|
Action to be done
|
|
1
|
+
|
+
|
SL
|
Report as SL and refer to GSL
for banking
|
|
2
|
-
|
-
|
NSL
|
Report as Discordant and refer to GSL for Sequencing
|
|
3
|
+
|
-
|
SL
|
Report as SL and refer to GSL
for banking
|
1.
Safety conditions
- Use a separate room for Pre PCR and PCR procedures.
- Use separate sets of reagents for pre and post PCR procedures.
- Virus isolates should be brought in mini cooler to ITD lab.
- Wear powder free gloves to avoid DNAase contamination.
- Alliquoting of primers, isolates, and enzyme mix addition should be done in biosafety cabinet.
- Take utmost care to prevent cross contamination of primers.
- Change gloves frequently to avoid cross contamination.
- No infectious material should be taken in Pre PCR room.
- Attach the thermocycler with UPS so that in electricity failure, cycler does not stop.
- Thermal cycling conditions for degenerate and Non degenerate primers should be appropriate.
- Add non sample component (master mix) to the reaction tube before adding the samples and Positive controls.
1.
Documentation
·
Log Book
of 7500 rRT PCR PR-51.
·
PCR
worksheets file PF 15.
·
Log Book
of Biosafety Cabinet No.1(Dengue) PR-47
·
Log Book of Biosafety Cabinet No.7 PR-48
1.
References used to draw SOP
- Manual of ABI 7500 thermocycler
- Manual On Intratypic differentiation of Polioviruses by Real time RT-PCR of CDC
- Kit Literature.
Example of summary sheet for final results of ITD
|
Sample No
|
Primers(ITD)
|
ITD
results
|
Comments
|
Primers (VDPV)
|
VDPV result
|
Final Result
|
|||||||||
|
|
Pan
EV
|
Pan
PV
|
Sero
1
|
Sero
2
|
Sero
3
|
Sabin Mutiplex
|
|
|
VP1
|
|
|
||||
|
|
S1
|
S2
|
S3
|
|
|
PV1
|
PV2
|
PV3
|
|
|
|||||
|
NC
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
Valid
|
|
|
|
|
|
|
|
S1
|
+
|
+
|
+
|
-
|
-
|
+
|
-
|
-
|
P1SL
|
P1VDPV screen
|
SL
|
NR
|
NR
|
P1 SL
|
Report as P1SL
|
|
S2
|
+
|
+
|
-
|
+
|
-
|
-
|
+
|
-
|
P2 SL
|
P2 VDPV screen
|
NR
|
SL
|
NR
|
P2 SL
|
Report as P2SL
|
|
S3
|
+
|
+
|
-
|
-
|
+
|
-
|
-
|
+
|
P3 SL
|
P3 VDPV screen
|
NR
|
NR
|
SL
|
P3 SL
|
Report as P3SL
|
|
S4
|
+
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
NPEV
|
Perform ITD
PCR on other
arm
|
|
|
|
|
|
|
S5
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
-
|
|
Perform ITD PCR on other arm
|
|
|
|
|
|
|
S5
|
+
|
+
|
+
|
-
|
-
|
-
|
-
|
-
|
P1 NSL
|
Report and refer for sequencing
|
|
|
|
|
|
|
S6
|
+
|
+
|
+
|
-
|
+
|
+
|
-
|
+
|
P1, P3 SL
|
P1,P3V screen
|
SL
|
NR
|
SL
|
P1,P3
SL
|
Report as P1,P3
SL
|
|
S7
|
+
|
+
|
+
|
-
|
-
|
+
|
-
|
-
|
P1 SL
|
P1VDPV screen
|
NSL
|
NR
|
NR
|
P1
NSL
|
Report as P1 discordant
and refer for sequencing
|
|
S8
|
+
|
+
|
+
|
+
|
-
|
-
|
+
|
-
|
P1 NSL, P2SL
|
Report and refer PI NSL for sequencing, P2 VDPV
screen
|
NR
|
SL
|
NR
|
P2 SL
|
Report as P1SL,
P2 SL
|
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