Welcome to the
presentation
onthe
the
Welcome to
presentation on the
qPCR set/kit for the detection of
potato leafroll virus and potato
virus Y
Dr. Marcel Lüscher
Number of Slides: 12
Duration: 4 min
by BIOREBA AG 2017
Introduction
The qPCR set enables the simultaneous detection (multiplex) of
potato leafroll virus (PLRV) and potato virus Y (PVY) with real
time PCR.
Viral RNAs extracted from potato samples (with our “potato
DNA/RNA rapid extraction set”) are amplified in a one-step
RT-PCR reaction.
The increase of the amplified cDNAs can be monitored in real
time since the specific probes are labeled with fluorophores.
Suitable tissues for the source of viral RNA are
Leaves
From tubers:
Stolon, peel
and sprouts
From dormant
tubers use:
Heel
For the procedure of nucleic acid extraction please
refer to the Potato DNA/RNA rapid extraction set
The qPCR set:
The qPCR sets are available for 96 or 192
reactions:
Each set contains:
Taq Master Mix (2x)
Primers/Probes/IC Mix_PLRV/PVY (10x)
RT Master Mix (50x)
Nuclease free water
AND
There are 4 qPCR kits available.
Each kit contains
a “rapid extraction set” and a “qPCR set”:
839610 qPCR PLRV/PVY kit 96/10:
For 96 extractions
and a pool size of up to 10 tuber
samples
839625 qPCR PLRV/PVY kit 192/10:
For 192 extractions
and a pool size of up to 10 tuber
samples
839210 qPCR PLRV/PVY kit 96/25:
For 96 extractions
and a pool size of up to 25 tuber
samples
839225 qPCR PLRV/PVY kit 192/25:
For 192 extractions
and a pool size of up to 25 tuber
samples
Material and Equipment required
Rnase-free filter tips and micropipets
Optical Rnase-free tubes
Disposable latex or venyl gloves
Thermal cycler for real time PCR
Please note!
Always wear
gloves
Use tips with
filter
…and for your
safety
Preparation of the qPCR samples
Gently thaw the kit components on ice or at
4°C.
Mix the tubes briefly and spin the liquid
down.
Determine the number of reactions to
prepare, then increase the number by 1-2 to
compensate for pipetting losses.
Preparation of the qPCR samples II
Prepare the master mix reaction by
combining the kit components according to
the table below:
Preparation of the qPCR samples III
Add the reaction mix to each PCR tube or
well of an optical grade plate.
Add 2-5 μl RNA template to the samples
reactions.
Seal the tubes or plates, centrifuge briefly
and protect from light before thermal cycling.
Thermal cycling
Thermal cycle the samples using the following
settings:
Monitoring the PCR amplification
Monitor the simultaneous PCR amplification
with a thermal cycler which is able to
measure the fluorescence of the following
fluorohores: