Plum Pox Virus RT-PCR Detection Kit - Protocol
Contents
1. Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Disclaimers The Lysis Solution contains guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Safety Information Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Working with RNA RNases are very stable and robust enzymes that degrade RNA Autoclaving solutions and glassware is not always sufficient to actively remove these enzymes The first step when preparing to work with RNA is to create an RNase free environment The following precautions are recommended as your best defense against these enzymes The RNA area should be located away from microbiological work stations Clean disposable gloves should be worn at all times when handling reagents samples pipettes disposable tubes etc It is recommended that gloves are changed frequently to avoid contamination There should be designated solutions tips tubes lab coats pipette2. C for long term Make appropriate aliquots and store at 20 C if needed e All other kit components may be stored at room temperature e The PPV 2X Detection RT PCR Mastermix and the Control 2X RT PCR Mastermix Positive Control and Isolation Control should not undergo repeated freeze thaw a maximum freeze thaw of three times e For RT PCR e Allow reagents to thaw at room temperature prior to use e When thawed mix the components and centrifuge briefly e Work quickly on ice e After addition of RT PCR Mastermix use within one hour General Precautions The user should exercise the following precautions when using the kit e Use sterile pipette tips with filters e Store and extract positive material specimens controls and amplicons separately from all other reagents and add it to the reaction mix in a spatially separated facility e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Work quickly on ice Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s PPV RT PCR Detection Kit including the PPV 2x RT PCR Master Mix Control 2X RT PCR Mastermix Isolation Control and PPV Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s PPV RT PCR Detection Kit is designed for research purposes only
3. PCR control gt C E E P Figure 2 A representative 1X TAE 1 5 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X RT PCR Mastermix The size of the Isolation Control amplicon and PCR Control amplicon correspond to 499 bp and 150 bp respectively as represented by the provided DNA Marker M Lanes 1 to 6 showed detection of both Isolation Control and PCR Control suggesting that the RNA isolation as well as the RT PCR reaction was successful Lane 7 and 8 showed only the detection of PCR Control suggesting that while the RT PCR was successful the isolation failed to recover even the spiked in Isolation control Table 5 Interpretation of One Step RT PCR Assay Results Input Type Target Control Reaction Interpretation reaction PPV Target IsoC Band PCRC Band Band 295 bp 499 bp 150 bp Positive xX x x Valid Control Po PE x Valid Sample X X X Positive Sample X X Negative Sample X Re test Sample Re test Sample X Negative Sample X X Positive Sample X X Positive Sample X Re test For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section E PPV RT PCR Assay Specificity and Sensitivity The specificity of Norgen s PPV RT PCR Detection Kit is first and foremost ensured by the selection of the PPV specific primers as well as the selection of st
4. be applied to this procedure b Using a pipette transfer the lysate into an RNAase free microcentrifuge tube not provided c Spin the lysate for 2 minutes to pellet any cell debris Transfer the supernatant to another RNase free microcentrifuge tube Note the volume of the supernatant lysate Note Ensure that only the clear supernatant is transferred avoiding any of the debris If necessary repeat Step 1c if visible precipitates are still present after the first spin d Add an equal volume of 70 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 A RNA to Column Assemble a column with one of the provided collection tubes b Add 10 uL of Isolation Control soC to the lysate mixture c Apply up to 600 uL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 14000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute d Depending on your lysate volume repeat step 2c if necessary Column Wash a Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the colum
5. the PPV 2X Detection RT PCR Mastermix and one RT PCR reaction using Control 2X RT PCR Mastermix should be set up in order to have a proper interpretation of the results For every RT PCR run one reaction containing PPV Positive Control PPV PosC and one reaction as no template control Nuclease Free Water must be included for proper interpretation of results e The recommended minimum number of RNA samples tested per RT PCR run is 6 Using a lower volume from the sample than recommended may affect the sensitivity of PPV Limit of Detection 1 Prepare the RT PCR reaction for sample detection Set 1 using PPV 2X Detection RT PCR Mastermix and the RT PCR reaction for control detection Set 2 using Control 2X RT PCR Mastermix as shown in Table 1 below The recommended amount of sample RNA to be used is 1 2 uL Ensure that one PPV detection reaction and one control reaction is prepared for each RNA sample Adjust the final volume of the RT PCR reaction to 20 uL using the Nuclease Free Water provided Table 1 RT PCR Assay Preparation RT PCR Components Volume Per RT PCR Reaction Sample RNA 2 uL Nuclease Free Water 8 uL Total Volume 20 uL 2 For every RT PCR run prepare one positive control RT PCR as shown in Table 2 below Table 2 RT PCR Positive Control Preparation RT PCR Components Volume Per RT PCR Reaction Total Volume 20 uL 3 For every RT PCR run prepare one no template control RT
6. 3430 Schmon Parkway mel lt Thorold ON Canada L2V 4Y6 3 Phone 866 667 4362 905 227 8848 Fax 905 227 1061 BIOTEK s CORPORATION Email techsupport norgenbiotek com Plum Pox Virus RT PCR Detection Kit Product Insert Product 33400 Pathogen Information Plum pox virus PPV is a member of the genus Potyvirus and the virus is considered the most serious pathogen affecting stone fruits including apricots cherry nectarine peaches and plums The symptoms of plum pox disease or Sharka include fruit abnormality loss of taste and fruits that drop prematurely These symptoms may cause losses as high as 80 100 of a crop and consequently it is a disease of economic significance in every country where stone fruits are grown To achieve successful eradication or effective management of the associated disease sensitive detection of the virus in infected plants is required Principle of the Test Norgen s Plum Pox Viurs PPV RT PCR Detection Kit constituents a ready to use system for the isolation and detection of PPV using end point one step RT PCR The kit first allows for the isolation of PPV RNA from plant tissues using spin column chromatography based on Norgen s proprietary resin The RNA is isolated free from inhibitors and can then be used as the template in a one step RT PCR reaction for PPV detection using the provided PPV Master Mix The PPV Mastermix contains reagents and enzymes for the specific amplification of a 295 b
7. PCR as shown in Table 3 below Table 3 RT PCR Negative Control Preparation RT PCR Components Volume Per RT PCR Reaction Nuclease Free Water 10 uL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate RT PCR reactions C One Step RT PPV PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step RT PCR Table 4 PPV Assay Program One Step RT PCR Cycle Step Temperature Duration Cycle 1 Step 1 50 C 30 min Cycle 2 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 3 40x Step 2 55 C 30 sec Step 3 72 C 45 sec Cycle 4 Step 1 72 C 5 min Cycle 5 Step 1 4 C oo D PPV One Step RT PCR Assay Results Interpretation 1 For the analysis of the RT PCR data the entire 20 uL RT PCR Reaction should be loaded on a 1X TAE 1 5 Agarose RNA gel along with 10 uL of Norgen s DNA Marker provided 2 The RT PCR products should be resolved on the 1X TAE 1 5 Agarose gel at 150V for 20 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below 23 45 67M PPV gt Figure 1 A representative 1X TAE 1 5 agarose gel showing the amplification of PPV negative Lanes 6 and 7 positive Lanes 1 to 5 controls The size of the PPV target amplicon corresponds to 295 bp as represented by the provided DNA Marker M Isolation control
8. at the integrity of the RNA is not compromised While the provided procedure does not rely on the use of liquid nitrogen to homogenize the sample both fresh and frozen tissues can optionally be processed using other homogenization methods including grinding with liquid nitrogen Isolation Control soC An Isolation Control soC is supplied This allows the user to control the RNA isolation procedure For this assay add the Isolation Control IsoC to the lysate during the isolation procedure The Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the Isolation Control IsoC more than 2 times The PPV Isolation Control soC must be kept on ice at all times during the isolation procedure The RT PCR components of the PPV RT PCR Detection Kit should remain at 20 C until RNA is extracted and ready for RT PCR amplification It is important to work quickly during this procedure 1 Lysate Preparation 2 3 a Transfer lt 100 mg of plant tissue into a mortar that contains 800 uL of Lysis Solution The volume of plant tissue and Lysis Solution can be increased proportionally For instance 0 5g of plant tissue requires 4 mL of Lysis Solution Extra lysis solution can be purchased separately See Related Products table Grind the sample using a pestle until the tissue is completely macerated Note Other homogenization devices such as Bioreba extraction bag and a homogenizer can also
9. n If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps 3a and 3b to wash column a second time d Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute e Discard the flowthrough and reassemble the spin column with its collection tube Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 9 4 RNA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 50 uL of Elution Buffer to the column c Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a 1 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volume has not been eluted spin the column at 14 000 x g 14 000 RPM for 1 additional minute d The purified RNA sample could be used immediately for RT PCR as described below It is recommended that samples be placed at 70 C for long term storage B PPV RT PCR Assay Preparation Notes e Before use suitable amounts of all RT PCR components should be completely thawed at room temperature gently vortexed and centrifuged briefly The amount of PPV 2X RT PCR Master Mix provided is enough for up to 32 RT PCR reactions 24 sample RT PCR 4 positive control RT PCR and 4 no template control RT PCR For each sample one RT PCR reaction using
10. not amplify therefore the problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the PPV RT PCR control showed amplification but neither the PPV target nor the PPV Isolation control amplified for a sample e This indicates a poor isolation The isolation procedure must be repeated 4 How should it be interpreted if only the Isolation Control soC was amplified in a sample e The sample tested can be considered as PPV negative 5 How should it be interpreted if the PPV RT PCR control and the PPV target showed amplification in a sample e The sample tested can be considered positive It could happen when too much template was added to the reaction 6 How should it be interpreted if only the PPV target and the PPV RT PCR control were amplified in a sample e The sample tested can be considered as PPV positive 7 How should it be interpreted if only the PPV target was amplified in a sample e The sample tested should be considered as PPV positive At high PPV input the PPV amplicon will be predominant and thus the PPV PCR control as well as the PPV Isolation control may not amplify as they compete for PCR resources 8 How should it be interpreted if only the PPV RT PCR control and the Isolation control showed amplification in a sample e The sample tested can be considered negative 9 What if I forgot to do a dry spin after my third wash e Your first RNA elution will be contaminated wi
11. one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2010 Norgen Biotek Corp PI33400 10
12. p region of the virus genome In addition Norgen s PPV RT PCR Detection Kit contains a second Mastermix the Control 2X RT PCR Master Mix which can be used to identify possible PCR inhibition and or inadequate isolation via a separate RT PCR reaction with the use of the provided PCR control PCRC or Isolation Control IsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Lysis Solution 25 mL Wash Solution 11 mL Elution Buffer 2mL Mini Spin Columns 24 Collection Tubes 24 Elution tubes 1 7 mL 24 Nuclease Free Water 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert 1 IsoC Isolation Control PosC Positive Control The isolation control is a RNA transcript product The positive control is PPV RNA transcript Customer Supplied Reagents and Equipment e Benchtop microcentrifuge 1 5 mL microcentrifuge tubes 96 100 ethanol 70 ethanol Mortar and pestle or other homogenization device Storage Conditions and Product Stability e The Positive Control PPV PosC red cap and Isolation Control IsoC orange cap should be stored at 70 C If needed make aliquots of the controls according to the volume used in the protocol 10 uL of PPV PosC or 10 uL of IsoC prior to freezing e The PPV 2X Detection RT PCR Mastermix green cap and the Control 2X RT PCR Mastermix yellow cap should be stored at 20 C upon receipt 70
13. ringent reaction conditions The PPV universal primers were checked for possible homologies to all plant viruses in GenBank published sequences by sequence comparison analysis and published PPV strains F Linear Range The linear range of Norgen s PPV RT PCR Detection Kit was determined by analysing a dilution series of an PPV quantification standards ranging from 100 ag to 1 pg Each dilution has been tested in replicates n 4 using Norgen s PPV RT PCR Detection Kit ona 1X TAE 1 5 agarose gel The linear range of Norgen s PPV RT PCR Detection Kit has been determined to cover concentrations from 100 ag to 1 ng Under the conditions of the Norgen s PPV RNA Isolation procedure Norgen s PPV RT PCR Detection Kit covers a linear range from 100 copies to 1 x 10 copies Frequently Asked Questions 1 How many samples should be included per RT PCR run e Norgen s PPV RT PCR Detection Kit is designed to test 24 samples For every 6 samples a non template control Nuclease Free Water and a Positive Control must be included It is preferable to pool and test 6 samples at a time 2 How can interpret my results if neither the PPV RT PCR conirol nor the Isolation Control IlsoC amplifies e f neither the PPV RT PCR control nor the PPV Isolation Control soC amplifies the sample must be re tested If the positive control showed amplification then the problem occurred during the isolation where as if the Positive control did
14. s etc for RNA only All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water Clean all surfaces with commercially available RNase decontamination solutions When working with purified RNA samples ensure that they remain on ice during downstream applications Protocol A PPV Total RNA Isolation Important Notes Prior to Beginning Protocol All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields may be observed Ensure that all solutions are at room temperature prior to use Prepare a working concentration of the Wash Solution by adding 25 mL of 95 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 36 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Both fresh or frozen samples may be used for this procedure Samples should be flash frozen in liquid nitrogen and transferred immediately to a 70 C freezer for long term storage Do not allow frozen samples to thaw prior to grinding with the mortar and pestle in order to ensure th
15. th the Wash Solution This may dilute the RNA yield in your first elution and it may interfere with the PCR detection as ethanol is known to be a PCR inhibitor 10 What if forgot to add the Isolation Control soC during the isolation e It is recommended that the isolation is repeated Related Products Product Lysis Solution 100 mL 25806 Plant RNA DNA Purification Kit 24400 Plant Fungi RNA Purification Kit 25800 Viroid RNA Purification Kit 32800 Bacterial Genomic RNA Isolation Kit 17900 Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Plum Pox Viurs PPV RT PCR Detection Kit or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 or call
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