1. Welcome to the presentation on the Welcome to the 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

2. 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.

3. 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

4. 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)
AND
Nuclease free water

5. There are 4 qPCR kits available. Each kit contains
a “rapid extraction set” and a “qPCR set”:
qPCR PLRV/PVY kit 96/10:
For 96 extractions
and a pool size of up to 10 tuber samples
qPCR PLRV/PVY kit 192/10:
For 192 extractions
and a pool size of up to 10 tuber samples
qPCR PLRV/PVY kit 96/25:
For 96 extractions
and a pool size of up to 25 tuber samples
qPCR PLRV/PVY kit 192/25:
For 192 extractions
and a pool size of up to 25 tuber samples

6. 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

7. Please note!
Always wear gloves
Use tips with filter
…and for your safety

8. 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.

9. Preparation of the qPCR samples II
Prepare the master mix reaction by combining the kit components according to the table below:

10. 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.

11. Thermal cycling
Thermal cycle the samples using the following settings:

12. Monitoring the PCR amplification
Monitor the simultaneous PCR amplification with a thermal cycler which is able to measure the fluorescence of the following fluorohores: