1.
Scope
·
This procedure explains the method of performing
VDPV rRT PCR on vaccine virus isolates as identified by rRTPCR –ITD.
2.
Definition
- VDPV -
Vaccine Derived Polio Virus.
3.
Materials
- Materials required:
- Powder free gloves
- Micro Amp optical 8 tube strip (0.2 ml)
- Micro Amp optical 8 cap strip
- Micropipettes (10µl to 200µl variable).
- Aerosol resistant tips (20µl, 30µl, 200µl)
- Discarding beaker
- Permant marker
- Rack to hold Micro centrifuge tubes
- Tissue paper
- Micro centrifuge tube (0.2 ml, 0.5
ml)DNase, RNase free.
- Reagent Required:
- Vaccine Virus isolates
- Positive controls
- Non degenerate primers in Buffer A – (S1 , S2, S3 VDPV primers)
- Enzyme mix
·
Equipments required:
- Biosafety cabinet Class II.
- Vortex mixture
- Minicentrifuge.
- Deep freeze (-15°c to -25°c).
1. Procedure
PCR Room:
·
Prepare rRT VDPV PCR protocol for isolates to be
tested along with negative control, isolates and positive control for the
primer.
·
Each vaccine virus isolate is to be tested
against its corresponding VDPV primers only (Sab1 VDPV type, Sab2 VDPV type, Sab3 VDPV type).
·
For example
o
Sample1
– P1SL – Sab1 VDPV primer.
o
Sample2
– P1+P2SL – Sab1 VDPV primer and Sab2 VDPV primer.
o
Sample3
– P2SL – Sab2 VDPV primer.
o
Sample4
– P3SL – Sab1 VDPV primer.
o
Sample5
– P1+P2+P3SL – Sab1 VDPV primer, Sab2 VDPV primer and Sab3 VDPV primer.
·
If the isolate is mixture of vaccine and wild by
ITD of rRTPCR then choose only VDPV of vaccine serotype. Do not put the VDPV test
wild serotype.
·
Negative control – Master mix of each primer is
taken as negative control. 1st well is taken as negative control.
Preparation of Master Mix:
·
All the
steps are to be performed in Biosafety cabinet II in pre PCR room.
·
Calculation for the preparation of master mix:
o
Master Mix- 19µlof each Primer in Buffer A+ 5 µl of +E (Enzyme Mix)
·
Take three 0.5ml microcentrifuge tubes and label
each with one primer (S1 VDPV, S2 VDPV, and S3 VDPV.
·
Thaw PCR reagents (Primers and Enzymes mix).
·
Mix each vial of primer by vortex for 30 seconds
and spin in the microcentrifuge for 1 minute.
·
Calculate the quantity of master mix required
according to the no of isolates one negative control and one positive control.
·
For example if there are 8 isolates then total
no of reactions for each primer willl be 10 that includes controls also. So the
calculation for Buffer A and +E (master Mix)
will be as follows:
o
10
samples × 19µl Buffer A=190µl
o
10samples×
5µl +E=50µl
o Total
volume (240µl) for 10 samples = Buffer
A (190 µl) + Enzyme Mix (50µl) Mix each vial on vortex for 30 seconds and spin
in microcentrifuge for 1 minute.
·
Transport these microcentrifuge tubes in
minicooler (at 40 C) to PCR Laboratory.
|
No of Samples/
reactions
|
Quantitiy of Buffer A (ul)
|
Quantitiy of Enzyme Mix
( ul)
|
Total Volume
(ul)
|
|
1
|
19
|
5
|
24
|
|
2
|
38
|
10
|
48
|
|
3
|
57
|
15
|
72
|
|
4
|
76
|
20
|
96
|
|
5
|
95
|
25
|
120
|
|
6
|
114
|
30
|
144
|
·
Transfer microcentrifuge tubes to another
minicooler in PCR Laboratory and send it back to Pre PCR Laboratory.
Distribution of Mastermix:
·
Positive controls are used in the following
manner:
o
For P1 Sl isolates - Control RNAsPV1/PV2/PV3)
·
Arrange Microcentrifuge strips according to the
protocol in Microcentrifuge box.
·
Virus isolate:
o
Positive vaccine virus isolates (LR/LLR or RLR
/RRLR) as identified by rRTPCR-ITD PCR are brought to PCR room in biosafety
cabinet in vaccine carrier.
·
Take one positive and one negative control for
each primer set.
·
Mix each vial mastermix on vortex for 30 seconds
and spin in microcentrifuge for 1 minute.
Distribution of Mastermix
·
Arrange the required no of Micro Amp TM 8-tube strip
wells in the PCR work station.
·
Dispense 24µl of master mix (reaction volume) of
respective master mix in to each of its respective well with sterile RNAase
free microtip. Use separate tip for different primers.
·
Discard the microtips in discarding container.
Addition of virus isolate:
o
Each primer is to be tested with one positive
control and one negative control.
o
Arrange the samples as per the protocol.
o
0.5µl of virus isolate is taken from the
supernatant with RNAase free microtip. Do not go deep in the bottom.
o
Add 0.5 µl of virus isolate in its respective master
mix reaction well as per protocol.
o
Add 0.5 ul of virus isolate in each master mix
reaction well (all 6 primers) as per protocol.
o
Use separate micro tip for different isolates
and primers. Discard the microtips in discarding container.
o
Add 1µl of respective control RNA for positive
control and add into last reaction well for each primer. Add 24µl of respective
master mix in the 1st well as negative control for each primer.
o
Close each tube with Micro Amp TM 8-cap strip.
Fix the caps completely by pressing with fingers.
o
Label the edge of the first well with strip
number and opposite edge with primer name.
o
Spin all strips in minicentrifuge for 1 minute.
·
Place strips in the PCR work station properly,
check that caps are closed completely and keep in Real Time Thermocycler (ABI
7500) and set the thermal cycle as follows:
o
RT reaction 42°C, 45 min.
o
In activate RT, 95°C, 3 min.
o
PCR
cycles for:
- Non-degenerate primers (Sab1VDPV, Sab2VDPV, Sab3VDPV primers):
o
95°c for 24 sec, 50°c for 30 sec to 65°c for 24
sec, and 25% ramp speed for 40 cycles.
- Operation of ABI 7500 for real time RT-PCR
for VDPV screen (rRT PCR):
o
Start the Main power switch and then switch on
the UPS. Start the computer.
o
Put the power switch on of the cycler. Open the
cycler by pushing the button in front of cycler.
o
Keep the strip with holder in proper position
inside the cycler and close it.
o
In computer monitor, click on 7500 soft ware
icon.
A
window appears. Double click on Advanced Set up (Software Version 2.0) icon.
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