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Presentation transcript:

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

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) AND Nuclease free water

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: