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User Guide PCR Cherry Leaf Roll Virus Set Version 03 – 13/04/2021 Powered by Qualiplante 1 / 3 User Guide PCR Cherry leaf roll virus set Version 03 – 13/04/2021 1 / 3 powered by Qualiplante Information pest: Cherry leaf roll virus Set format and content Cherry leaf roll virus (CLRV) is common in many wild Two sets are available for 24 and 96 tests. and cultivated woody plant species. This virus is Article N° Product name known to infect at least 36 plant families and natural PCR Cherry leaf roll virus 7CLRV-P2 hosts including olive, elm, ash, elderberry, beech, set 24 rhubarb, dogwood and lilac. It was first described in PCR Cherry leaf roll virus 7CLRV-P9 1955 by Posnette and Cropley as causing a disease set 96 of sweet cherry (Prunus avium L.) in England. CLRV belongs to the subgroup-C of the genus Content set 24 set 96 Nepovirus (family Comoviridae). 24 tests 2x48 tests Direct Master Mix 7CLRV-P2-DM- 7CLRV-P9-DM- Symptoms include leaf roll, leaf yellowing, early 24 tests 96 tests RT-Enzyme dropping of leaves, stunted growth, and plant dieback. 7CLRV-P2-RT- 7CLRV-P9-RT- Plants can also be infected without exhibiting 3 tests 8 tests Positive Control symptoms. 7CLRV-P2-PC- 7CLRV-P9-PC- 3 tests 8 tests Negative Control CLRV is readily transmitted by grafting and by seed 7CLRV-P2-NC- 7CLRV-P9-NC- and pollen in several host species. Introduction Storage conditions The PCR Cherry leaf roll virus set has been This set can be shipped at room temperature but upon developed by Qualiplante based on Werner et al. receipt it should be stored immediately at the (1997). A verification was performed by Qualiplante recommended storage temperature: from -30 ° C to - (data not published) and the performance 10 ° C. characteristics of the set are the same as the original Avoid prolonged exposure to light and repeated publication. The primer pair amplifies a 416 bp freeze and thaw cycles. product of the CLRV coat protein gene. This product should be used only for research purposes. Shelf life Intended use If the set is correctly stored, at constant-temperature freezer, its performance is guaranteed until the The PCR set is validated for the detection of Cherry expiration date indicated on the tubes label. leaf roll virus (CLRV) in One-Step End-Point RT-PCR. Materials and equipment (not provided) The distribution of the virus in a tree can be irregular; however, the detection from flowers, young leaves, . RNA extraction tools and reagents old leaves, fruit, dormant wood, and roots is possible. Nuclease-free filter tips and micropipettes The time of year when the sample is taken is also . Optical grade nuclease-free tubes/plate critical and can greatly affect the test results. Optimal . Disposable latex or vinyl gloves sampling is during spring, or early summer, but can . DNA ladder and loading-dye buffer vary from season to season depending on the . PCR thermal cycling weather conditions. CLRV has been reported to be . Agarose gel reagents and apparatus easily detectable throughout the year in tissue of male inflorescences, leaf buds, leaves, single seeds and Nucleic acids extraction cortical tissues of young twigs. The virus can also be detected in roots, meristems and within tubules in Extract RNA from samples according to your usual pollen, ovules and mature seeds. protocol. Upon request, Qualiplante can recommend you an extraction method. Version 03 – 13/04/2021. Adaptations from last version: complete redesign of the protocol Qualiplante SAS www.qualiplante.eu Cap Alpha – Avenue de l’Europe [email protected] FR-34830 Clapiers User Guide PCR Cherry leaf roll virus set Version 03 – 13/04/2021 2 / 3 powered by Qualiplante Preparation of the CLRV 1-Step master mix Agarose gel electrophoresis a) Slowly thaw Direct Master Mix and RT-Enzyme by Prepare an agarose gel at 1,5% w/v in 1X-TAE placing it on ice or at 4°C. buffer. b) Shake briefly Direct Master Mix and RT-Enzyme Gel loading: and spin down the liquid. - load the DNA ladder (for example 100-1.000 bp In a new tube called CLRV 1-Step master mix, c) DNA step ladder) mix 17,5 µl of Direct Master Mix and 0,5 µl of RT- - load 10 µl of PCR products from the previous Enzyme per reaction. Do not forget to count the step adding the loading-dye buffer (not provided Positive Control and the Negative Control in the in the set). number of reactions to prepare. Run: run the gel electrophoresis for 50-60 minutes at Example: 1 rxn 10 rxns 80V. Direct Master Mix 17,5 µl 175,0 µl RT-enzyme 0,5 µl 5,0 µl Results analysis d) Store the CLRV 1-Step master mix by placing it on ice or at 4°C. ANALYSIS VALIDATION Cherry leaf roll virus is detected when a 416 bp DNA Reaction set-up fragment is observed. The analysis is validated when: 1 DNA fragment of 416 bp is visible in the a) Shake briefly CLRV 1-Step master mix and spin positive control lane. down the liquid. No DNA fragment is visible in the negative b) Add 18 µl of CLRV 1-Step master mix (without control lane. RNA template) to each PCR tubes or wells of an optical-grade PCR plate. The picture below represents a 1X-TAE 1,5% agarose c) Add 2 µl of RNA template to the CLRV 1-Step gel showing the DNA amplification in a sample master mix. Do not forget to prepare a PCR tube infected by CLRV: or well of an optical-grade PCR plate for the Positive Control and the Negative Control. Volume/PCR tube Components or well RNA template or Positive control or 2 μl Negative control CLRV 1-Step master mix 18 μl Total Volume / PCR tube or well 20 μl In order to confirm the absence of any reagent’s contamination, we strongly recommend including a no-template control (e.g. DEPC water) in the assay. MK: DNA ladder – 1 to 5: CLRV positive sample at different Run and thermal cycling concentration or Positive Control - 1: 20 ng of total RNA - 2: 2 ng of total RNA - 3: 0,2 ng of total RNA - 4: 0,1 ng of total RNA - 5: a) Seal carefully the PCR tubes or PCR plate. 0,02 ng of total RNA - 6: healthy sample or Negative Control - 7: no Centrifuge briefly to collect components at the template control. bottom of the PCR tubes or wells of the plate. RESULTS INTERPRETATION Protect from light before thermocycling. b) Load the PCR tubes or plate into the thermal- The specific product of CLRV is a 416 bp DNA fragment. cycler and follow the thermal cycling below: A sample is positive when a 416 bp specific Temp Steps Time Cycle(s) DNA fragment is present in the PCR reaction. (°C) A sample is negative when no fragment is Reverse transcription 50°C 15 min 1 present in the PCR reaction. Enzyme activation 95°C 10 min 1 The table below summarizes the results Denaturation 95°C 30 sec interpretation: 45 Annealing and Fragment size 60°C 60 sec Interpretation elongation 416 bp Storage 4°C ∞ - - Negative POSITIVE Cherry leaf roll virus Version 03 – 13/04/2021. Adaptations from last version: complete redesign of the protocol Qualiplante SAS www.qualiplante.eu Cap Alpha – Avenue de l’Europe [email protected] FR-34830 Clapiers User Guide PCR Cherry leaf roll virus set Version 03 – 13/04/2021 powered by Qualiplante 3 / 3 Special handling instructions Troubleshooting This set was designed to be used by laboratory staff Post-PCR data analysis shows no amplification, or trained to follow the usual molecular biology amplification plots look grossly abnormal: precautions. Always perform the tests in a nuclease- Possible causes Corrective actions free work environment. Always wear gloves when Evaporation of the Repeat the test using the handling samples containing DNA/RNA and the sample due to appropriate tools to seal components of the set. Do not touch any set inadequate sealing of correctly the plate components with an ungloved hand. Use appropriate the plate laboratory disposable parts. Use nuclease-free tubes Repeat the test using Consumables are not and filter tips to avoid degradation and cross- consumables appropriate for the recommended by the contamination. Do not use components from sets with method different batch numbers in the same test procedure. thermal cycler supplier Do not interchange reagents with other sets. To avoid Repeat the extraction step. Ensure that the method of cross-contamination, use separate rooms for (a) The quality of nucleic extraction has been nucleic acids extraction, (b) preparation of the Master acid extracted is low Mix and (c) amplification. To avoid cross- performed correctly. In case contamination and obtain reliable results, it is of doubt, contact us essential to strictly follow the protocol in this manual. Centrifuge the plate briefly to spin down the contents Avoid unnecessary freeze-thaw cycles of the set Abnormal amplification components. Do not use reagents after their and eliminate any air bubbles expiration date. No amplification reaction is observed in the positive control well, while other samples are positive: Possible causes Corrective actions The positive control provided with the set Repeat the test. If the was not added into the problem persists, contact us reaction well An amplification plot is observed in the negative control well: Possible causes Corrective actions Repeat the test by Contamination of the applying appropriate negative control or the quality procedures to Master Mix with target- prevent contamination. positive nucleic acid Seal the plate correctly Warranty and Responsibilities Qualiplante SAS guarantees the buyer exclusively concerning the quality of reagents and of the components used to produce the Sets.
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