Phomopsis viticola PCR Detection Kit - Protocol
Contents
1. PI34900 22. year without showing any reduction in performance The P viticola 2x PCR Master Mix P viticola Positive Control PosC and the P viticola Isolation Control IsoC should be kept tightly sealed and stored at 20 C for up to 1 year without showing any reduction in performance Repeated thawing and freezing gt 2 x should be avoided as this may reduce the sensitivity If the reagents are to be used only intermittently they should be frozen in aliquots 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 Phomopsis viticola PCR Detection Kit including the Phomopsis viticola 2x PCR Master Mix Phomopsis viticola Isolation Control IsoC and Phomopsis viticola Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Phomopsis viticola PCR Detection Kit is designed for research purposes only It is not intended for human o
3. 1 2 3 4 5 6 7 M Iso control Target PCR control Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of Phomopsis viticola at different concentrations Target The size of the P viticola target amplicon corresponds to 300 bp as represented by the provided DNA Marker M The size of the P viticola Isolation Control so C corresponds to 529 bp as represented by the provided DNA Marker M The P viticola 2X PCR Master Mix contains a P viticola PCR Control PCRC The P viticola PCRC controls for PCR inhibition The size of the P viticola PCRC corresponds to 171bp as represented by the provided DNA Marker M The amplification from each lane is interpreted as shown below Lane 1 to 4 P viticola detected Lanes showed the detection of all three PCR amplicons Lane 5 No IsoC and Template Only P viticola PCRC was detected Lane 6 P viticola not detected Detection of P viticola IsoC and P viticola PCRC suggesting that the DNA isolation was successful but no P viticola DNA was present in the sample Lane 7 Positive All three PCR amplicons were detected Table 5 Interpretation of PCR Assay Results Input Type P viticola Iso P viticola Target P viticola PCR Interpretation C Band 529 Band 300 bp C Band 150 bp bp Positive Control X X X Valid Negative Control X X Valid Sample X X X Positive Sample X X Negative Sample X Negative Sample X X Positiv
4. 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com NORGEN BIOTEK wi CORPORATION E gt gt lt Phomopsis viticola PCR Detection Kit Product Insert Product 34900 Pathogen Information Phomopsis cane and leaf spot caused by Phomopsis viticola occurs in most grape growing regions and is an important disease of grapes worldwide The fungus attacks all green parts of the vine including canes leaves flowers rachises and berries However most economic damage is caused by rachis and berry infection Infected leaves have small yellowish spots with dark brown centers and may be puckered With successive cool wet springs the inoculum will build up and infections will become more severe over time Since symptoms appear 21 to 30 days after infection early detection of the pathogen in the vineyard is therefore crucial for its control Principle of the Test Norgen s Phomopsis viticola PCR Detection Kit constituents a ready to use system for the isolation and detection of P viticola using end point PCR The kit first allows for the isolation of fungal DNA using spin column chromatography based on Norgen s proprietary resin Fungal DNA can be isolated from fungi growing on culture plates or from plant tissue or fruit using this kit The DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for P viticola
5. C during the isolation e It is recommended that the isolation is repeated Related Products Product Bacterial Genomic DNA Isolation Kit 17900 Fungi yeast Genomic DNA isolation kit 27300 Plant Fungi DNA Isolation Kit 26200 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 Urine DNA Isolation Mini Kit Slurry Format 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 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
6. Fruit Wash the tissue or fruit with an appropriate amount of DNAse free water with vortexing Transfer up to 1 mL of washed spores and wet mycelium to a microcentrifuge tube provided by user Centrifuge at 14 000 x g 14 000 RPM for 2 minutes to pellet the cells Pour off the supernatant carefully so as not to disturb or dislodge the cell pellet Add 500 uL of Lysis Solution to the cell pellet Resuspend the cells by gentle vortexing Transfer the mixture to a provided Bead Tube and secure the tube horizontally on a flat bed vortex pad with tape or in any commercially available bead beater equipment e g Scientific Industries Disruptor Genie Vortex for 5 minutes at maximum speed or optimize the condition for any commercially available bead beater equipment Note Foaming during the homogenization is common This foaming is due to detergents present in the Lysis Buffer and will not affect the protocol Incubate the Bead Tube with lysate at 65 C for 10 minutes Occasionally mix the lysate 2 or 3 times during incubation by inverting the tube Briefly spin the tube to remove liquid from the cap and transfer all of the lysate including cell debris to a DNase free microcentrifuge tube provided by the user by pipetting Ensure that the beads are not transferred during the pipetting Centrifuge the tube for 2 minutes at 14000 x g 14 000 RPM Carefully transfer clean supernatant to a new DNase free microcentrifuge tube provided by
7. NA Elution a Place the column into a fresh 1 7 mL Elution tube provided with the kit b Add 75 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 5 Storage of DNA The purified DNA may be stored at 20 C for a few days It is recommended that samples be placed at 70 C for long term storage B Phomopsis viticola PCR Assay Preparation Notes Before use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly The amount of P viticola 2X PCR Master Mix provided is enough for up to 32 PCR reactions 24 sample PCR 4 positive control PCR and 4 no template control PCR For every PCR run one reaction containing P viticola Positive Control PosC and one reaction as no template control must be included for proper interpretation of results e The recommended minimum number of DNA samples tested per PCR run is 6 Using a lower volume from the sample than recommended may affect the sensitivity of P viticola Limit of Detection 1 Prepare the PCR for sample detection as shown in Table 1 below The recommended amount of sample DNA to be used is 5 uL However a volume between 1 and 10 uL of sample DNA may be used as template A
8. detection using the provided P viticola Master Mix The P viticola Mastermix contains reagents and enzymes for the specific amplification of a 300 bp region of the fungal genome In addition Norgen s Phomopsis viticola PCR Detection Kit contains a second heterologous amplification system to identify possible PCR inhibition and or inadequate isolation The amplification and detection of either the Isolation Control IsoC or the PCR control PC does not reduce the detection limit of the analytical P viticola PCR This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Lysis Solution 15 mL Wash Solution 9 mL Elution Buffer 3 mL Bead Tubes 24 Mini Filter 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 IsoC Isolation Control PosC Positive Control The isolation and PCR control are a cloned PCR product The positive control is P viticola genomic DNA Customer Supplied Reagents and Equipment e Disposable powder free gloves Benchtop microcentrifuge 1 5 mL microcentrifuge tubes 65 C water bath or heating block 96 100 ethanol 70 ethanol RNase A optional Lyticase optional Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C Buffers can be stored for up to 1
9. djust the final volume of the PCR reaction to 20 uL using the Nuclease Free Water provided Table 1 PCR Assay Preparation PCR Components Volume Per PCR Reaction Sample DNA 2to5 ul Nuclease Free Water Up to 10 uL Total Volume 20 uL 2 For every PCR run prepare one positive control PCR as shown in Table 2 below Table 2 PCR Positive Control Preparation PCR Components Volume Per PCR Reaction Total Volume 20 uL 3 For every PCR run prepare one no template control PCR as shown in Table 3 below Table 3 PCR Negative Control Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 10 uL Total Volume 20 uL C Phomopsis viticola PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run PCR Table 4 P viticola Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 40x Step 2 60 C 30 sec Step 3 72 C 45 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C D Phomopsis viticola PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 20 uL PCR Reaction should be loaded on a 1X TAE 1 7 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided 2 The PCR products should be resolved on the 1X TAE 1 7 Agarose gel at 150V for 30 minutes 3 Sample results are provided below M
10. e Sample X X Positive Sample X Positive For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section E Fungi Phomopsis viticola PCR Assay Specificity and Sensitivity The specificity of Norgen s Phomopsis viticola PCR Detection Kit is first and foremost ensured by the selection of the P viticola specific primers as well as the selection of stringent reaction conditions The primers were checked for possible homologies to all in GenBank published sequences by sequence comparison analysis The specific detectability of all relevant strains has thus been ensured by a database alignment and by PCR amplification with the following fungi commonly found in plant samples Aspergillus niger Cladosporium sp Phomopsis viticola Mucor racemosus Alterneria tenuissima Rhizopus oryzae Penicillum sp Fusarium oxysporum 0000000 0 F Linear Range e The linear range analytical measurement of Norgen s Phomopsis viticola PCR Detection Kit was determined by analysing a dilution series of a P viticola quantification standards ranging from 1 x 10 cfu ul to 1 x 10 cfu ul e Each dilution has been tested in replicates n 4 using Norgen s Phomopsis viticola PCR Detection Kit on a 1X TAE 1 7 agarose gel e The linear range of Norgen s Phomopsis viticola PCR Detection Kit has been determined to cover concentrations of genomic DNA from 10 pg to 100 ng e Under the conditions
11. of the Norgen s Phomopsis viticola DNA Isolation procedure Norgen s Phomopsis viticola PCR Detection Kit covers a linear range from 500 copies to 5 x 10 copies Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Phomopsis viticola 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 If not the provided Positive Control is enough to run 3 samples at a time 2 How can interpret my results if neither the P viticola PCR control nor the P viticola Isolation Control IsoC amplifies e If neither the P viticola PCR control nor the P viticola Isolation Control lsoC 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 not amplify therefore the Problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the P viticola PCR control showed amplification but neither the P viticola target nor the P viticola Isolation Control IsoC 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 P viticola Isolation Control IsoC was amplified in a sample e The sample tested can be considered as P
12. r diagnostic use 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 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 Protocol A Phomopsis viticola Genomic DNA Isolation Important Notes Prior to Beginning Protocol 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 isolation solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again Prepare a working concentration of the Wash Solution by adding 21 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 30 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added Lysate can be prepared from either fungi growing on plates plant tissue or fruit Please en
13. sure that you follow the proper procedure for lysate preparation in Step 1a For the isolation of genomic DNA from fungi growing on plates Collection Solution must be prepared Collection Solution consists of 0 9 w v NaCl prepared with distilled water Preheat a water bath or heating block to 65 C P viticola Isolation Control BC IsoC A P viticola Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the P viticola Isolation Control soC to the lysate during the isolation procedure The P viticola Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the P viticola Isolation Control soC more than 2 times The P viticola Isolation Control soC must be kept on ice at all times during the isolation procedure The PCR components of the Phomopsis viticola PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification 1 Lysate Preparation a Fungi Growing on Plates Add approximately 5 mL volume can be adjusted based on density of fungal growth of Collection Solution see notes prior to Beginning Protocol to the plate and gently collect fungal spores and mycelium with an inoculation loop or autoclaved pipette tip ensuring not to collect any agar debris Transfer up to 1 mL of washed spores and wet mycelium to a microcentrifuge tube provided by user Fungi from Plant Tissue or
14. the user without disturbing the pellet Note the volume 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 Binding to Column 2 Binding DNA to Column oD Assemble a spin column with one of the provided collection tubes Add 10 uL of P viticola Isolation Control soC to the lysate mixture Apply up to 600 uL of the lysate with ethanol onto the column and centrifuge for 1 minute at 14 000 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 Depending on your lysate volume repeat step 2c if necessary 3 Column Wash a Apply 500 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 column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the column with its collection tube c Repeat step 3a to wash column a second time d Discard the flowthrough and reassemble the spin column with its collection tube e Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 D
15. viticola negative 5 How should it be interpreted if the P viticola PCR control and the P viticola target showed amplification in a sample e The sample tested can be considered positive It could happen when Isolation Control failed to be amplified due to the incorrect spiking volume of Isolation Control or low Isolation Control recovery in the elution with high P viticola input 6 How should it be interpreted if only the P viticola target and the P viticola PCR control were amplified in a sample e The sample tested can be considered as P viticola positive 7 How should it be interpreted if only the P viticola target was amplified in a sample e The sample tested should be considered as P viticola positive At high P viticola cell input the P viticola amplicon will be predominant and thus the P viticola PCR control as well as the P viticola solation control may not amplify as they compete for PCR resources 8 How should it be interpreted if only the P viticola PCR control and the P viticola Isolation Control IsoC 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 second wash e Your first DNA elution will be contaminated with the Wash Solution This may dilute the DNA 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 P viticola Isolation Control Iso
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