- Scope
·
This procedure explains the method of real time
RTPCR using kit provided by CDC.
Principle:
The Poliovirus diagnostic real time RT PCR is performed on poliovirus isolates.
The viral RNA is converted to complementary DNA (cDNA) using reverse
transcriptase. The cDNA is amplified in a PCR reaction using Taq Polymerase.
The PCR products are detected and identified by hybridization with specific
Taqman probes. Both the cDNA synthesis and the PCR reaction use multiple sets
of oligonucleotide primers which are tagged with probes with different
specifities. This combination of primers will result in the serotype
identification and intratypic
differentiation of poliovirus isolates.
- Definition
·
rRTPCR – Real time reverse transcriptase Polymerase
Chain Reaction
3.
Materials
·
Equipments required:
o
Pre PCR cabinet room
o
BSL2 cabinet
o
Vortex
o
Minicentrifuge
o
Pipette sets
o
Deep freeze (-15°c to -25°c)
·
Materials required:
o
Powder free gloves
o
Micro Amp optical 8 tube strip (0.2 ml)
o
Micro Amp optical 8 cap strip
o
Aerosol
resistant tips (10µl, 20µl, 200µl, 1000µl)
o
Discarding beaker
o
Permant marker
o
Micro centrifuge tube box
o
Tissue
paper
o
Micro
centrifuge tube (0.2 ml, 0.5 ml) DNase, RNase free.
·
Reagents required:
o
Virus isolates to be tested
o
Positive
control RNA(PV1,PV2,PV3)
o
Primers in Buffer A –
§ All
primers (Pan EV, Sabin multiplex primers i.e. sabin 1,2 and 3, Pan PV, Sero 1,
Sero 2, Sero 3)
o
Enzyme Mix
1. Procedure
·
Preparation of Master Mix:
o
All the
steps are to be performed in Biosafety cabinet II in pre PCR room.
o
Calculation of master mix:
§
19ul of each Primer in Buffer A for each sample
§
5 ul of +E (Enzyme Mix) for each sample
o
Take six 0.5ml microcentrifuge tubes and label
each with one primer (Pan EV, sabin Multiplex, Pan PV, Sero type 1, Sero type
2, Sero type 3).
o
Calculate the quantity of master mix required
according to the no of isolates and controls. Take one positive and one
negative control for each primer set.
o Total
volume (24µl) per sample= Buffer A
(19 µl) + Enzyme Mix (5µl)
(for one sample, 6 vials of master mix
each having its own primer)
o
For example if there are 8 isolates then total
no of reactions for each primer will be 10 that includes controls also. So the
the calculation for Buffer A and +E ( master Mix) for 10 samples will be as
follows:
§ 10 samples × 19µl Buffer A=190µl
§ 10samples× 5µl +E=50µl
§
Total volume (240µl) for 10 samples = Buffer A (190 µl) + Enzyme Mix (50µl) .( 6 vials of
master mix , each having its own primer)
o
Mix each vial on vortex for 30 seconds and spin
in microcentrifuge for 1 minute.
o
Transport these microcentrifuge tubes in
minicooler (at 40 C) to PCR Laboratory.
o
Transfer microcentrifuge tubes to another minicooler in PCR Laboratory
and send it back to Pre PCR Laboratory.
Distribution
of mastermix:
o Prepare
rRT PCR protocol for isolates to be tested along with negative control and
positive control for each primer.
o Mix
each vial of primer by vortex for 30 seconds and spin in the microcentrifuge
for 1 minute.
o
All the
steps are to be performed in Biosafety cabinet II in PCR room.
o
Arrange the required no of Micro Amp TM optical
8-tube strips according to the protocol in the PCR work station.
o
Dispense 24µl (reaction volume) of respective
master mix in to each of its respective well with sterile 30 µl RNAase free
microtip. Discard the microtips in discarding container.
o
Use separate tip for different primers.
o
Each isolate is to be tested against six primers
(Pan EV, Sabin Multiplex, Pan PV, Sero type 1, Sero type 2, and Sero type 3)
o
Negative control – Master mix of each primer is
taken as negative control.
- Addition of virus isolate:
o Prepare the protocol
o
Each primer is to be tested with one positive
control and negative control.
o
0.5 µl 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 each master mix
reaction well (all 6 primers) as per protocol with 30 µl RNAase free microtip. Discard
the microtips in discarding container.
o
Use separate microtip for different isolates and
primers.
o
After completion of virus isolate take out
positive control from the deep freeze. Thaw them, vortex it and spin for 30
sec. in minicentrifuge.
o
Positive controls are used in the following
manner:
§ For
Pan EV and PAN PV - any of the control RNAs( PV1/PV2/PV3)
§ For
Sabin multiplex- Sabin multiplex
§ For
Serotype P1 – PV1
§ For
Serotype P2 – PV2
§ For
Serotype P3 – PV3
o
Add 1µl of control RNA for positive control and
add into last reaction well for each primer as mentioned in the protocol.
o
Add 24µl of respective master mix in the 1st
well as negative control for each primer.
o
Close each tube with Micro Amp TM optical cap
strip. Fix the caps completely by pressing with fingers.
o
Label the edge near the first well with strip
number and other edge with primer name. Check that caps are closed completely.
o
Spin all strips in minicentrifuge for 1 minute.
o
Place strip again in the PCR work station properly
and keep in Real Time Thermocycler (ABI 7500) and set the thermal cycle as follows;
§
RT reaction 42°C, 45 min.
§
In activate RT, 95°C, 3 min.
§ PCR
cycles for:
All Primers
(Pan EV and Sabin Pan PV, PV1, PV2, and PV3 primers)
95°c for 24 sec, 44°c for 30 sec, and then a
25% ramp speed to 60°c for24 sec, for 40 cycles.
- Interpretation:
o
The results are interpreted by looking at the
amplification curve. First results are to be validated by negative control (No
curve) and positive control (amplification curve).
o
If amplification occurs then a logarithmic curve
appears and if no amplification then no curve appears.
o
Positive amplification is recorded as Pos for
the respective primer and No amplification is recorded as Neg.
·
Operation
of ABI 7500 for real time RT-PCR (rRT PCR);
- Start the Main power switch and then switch on the UPS. Start the computer.
- Put the power switch ON of the cycler.
- Open the cycler by pushing the button in front of cycler.
- Keep the strip with holder in proper position inside the cycler and close it.
- In computer monitor, click on 7500 soft ware icon (Software Version 2.0.1).
A
window appears. Double click on Advanced Set up icon.
- A window of Experiment profile appears. In this window do the following:
o
Experiment Name-Write date, Primer names, Experiment
Number e.g. 030112evsmpvsero123Exp-1
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.
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