ITD by diagnostic rRTPCR-Part-2

• 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 (Pan EV, Sabin123, Pan PV, Sero1, Sero 2, Sero3).

§  Choose reporter by clicking on the Reporter dropdown box. For:

ü  PAN EV – FAM

ü  SABIN 1 – CY5

ü  SABIN 2 – FAM

ü  SABIN 3 – ROX

ü  PAN PV – FAM

ü  SERO 1 – FAM

ü  SERO 2 – FAM

ü  SERO 3 - FAM

ü  FAM-Pan EV,Pan PV,SeroPV1,PV2,PV3,Sabin2& Sabin 123 VDPV-VP1

ü  Cy5-Sabin 1

ü  ROX-Sabin3,Sabin123 VDPV-3D 

§  Assign colour to the reporter dye from colour drop down box.

a.     FAM – Red

b.     Cy5 – Green

c.      Rox – Blue

·       Define sample

o   Write NC for Negative Control, sample numbers and Positive Control. Save sample name in file and click Add Saved.

In the same window open assign targets and samples

• Click on Assign Targets and samples

o   Select Passive reference as none.

o   First wells are selected and then targets are assigned to the respected well.

o   There are six primers for one samples, so assign sample no to six wells.

o   Now click on open run method.

o   Click on Assign Targets and Samples to well (click well in plate layout and assign Target and Sample name to the 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 for VDPVS123

§  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

o   PAN EV Sabin 123 Pan PV, Sero PV1,PV2,PV3

§  42°C for 45 min - RT reaction

§  95°C for 3 min – For Denaturation

§  95°C for 24 sec – For Denaturation

§  44°C for 30 sec – For Annealing

§  60°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 No of cycles 40.
• Click the start run (green) button to start run and a window will appear on screen to save file. Save the file in the designated place in computer. It will show the time Run started at and Estimated Time remaining..

• 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 the stripes and turn off machine.

• 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 the Y axis,

§  Click on   box, the screen will show a box.

§  Click on Y axis.  A box will appear.

§  Click off Auto adjust range. Set the minimum and maximum value. The graph will change.

·       Manual adjustment of Results:

If the Ct value is less than 15 then an undetermined Ct value is obtained even with positive fluorescence. Then manually adjust the baseline to 3 and to less than the actual positive Ct value.

o   Click on analysis settings.

o   Select Edit default settings and click off Use default settings.

o   Set the baseline.

o   Baseline can be changed by moving the triangle on the bottom of graph to get the baseline.

o   Check threshold box. Threshold box will show on the graph and baseline can be moved to adjust its place.

·       Alternate Methods of analysis of results

• Raw Data Plot
• Multicomponent plot

Take backup on every Friday on hard disk to store data of the samples.

• 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.

• Interpretation:

      The results are interpreted by comparing the amplification curves of PCR products from test samples to those of positive control reactions.

Amplicon size of various products

PRIMER	AMPLICON SIZE  (bp)
Pan EV	145
Pan PV	100
PV Serotype 1 (PV 1)	70
PV Serotype 2 (PV 2)	79
PV Serotype 3 (PV 3)	140
Sabin 1	97
Sabin 2	71
Sabin 3	53

Sr.No.	PCR Result( as evidenced by amplification curve)	Result	Actions to be done
1	All Primers (-Ve)	Non Enterovirus	Perform rRTPCR from other arm if available
2	Pan EV (+ve), PanPV (-ve), all other (-ve)	Non polio Enterovirus	Perform rRTPCR from other arm if available
3	Pan EV (+ve), Pan PV (+ve), Sabins All (-ve),One or more serotypes (+ve)	Wild poliovirus of indicated serotypes	Report as NSL and refer for sequencing
4	Pan EV (+ve),Pan PV (+ve),one or more sabins (+ve) corresponding Serotype(s) (+ve)	Vaccine poliovirus (es) of indicated Serotype(s)	Subject the isolate to corresponding VDPV screen
5	Pan EV (+ve),Pan PV (+ve),One or more sabins positive, more serotypes (+ve) than sabins	Mixture of Wild virus and vaccine poliovirus	Report and refer wild virus to GSL, subject the isolate for VDPV screen for vaccine virus
6	Pan EV (+ve),Pan PV (+ve),One or more sabins positive, All serotypes (-ve)	Discordant	Refer to GSL

1.    Safety conditions

• Virus isolates should be brought in mini cooler to ITD lab.
• Wear powder free gloves to avoid DNAase contamination.
• Alliquoting of primers and enzyme mix addition should be done in biosafety cabinet.
• Take ulmost care to prevent 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.

2.    Documentation

• rRT PCR worksheet file PF-16