Dirofilaria immitis PCR Detection Kit - Protocol
Contents
1. PCR reaction using the 2X DIR Detection PCR Mastermix and one PCR reaction using 2X PCR Control Mastermix should be set up in order to have a proper interpretation of the results For every PCR run one reaction containing DIR Positive Control 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 DIR Limit of Detection 1 Prepare the PCR reaction for sample detection Set 1 using 2X DIR Detection PCR Mastermix and the PCR reaction for control detection Set 2 using 2X PCR Control Mastermix as shown in Table 1 below The recommended amount of sample DNA to be used is 2 5 uL However a volume between 1 and 5 uL of sample DNA may be used as template Ensure that one DIR detection reaction and one control reaction is prepared for each DNA sample Adjust 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 2 5 uL Nuclease Free Water 7 5 pL 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 P2. 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 En gt lt Dirofilaria immitis PCR Detection Kit Product Insert Product 44500 Pathogen Information Heartworm Dirofilaria immitis is a parasitic roundworm that causes a serious and potentially fatal parasitic disease which primarily infects dogs cats and a number of wild animals including wolves coyotes foxes lions raccoons The parasite is transmitted from host to host by mosquito bites whereby tiny heartworm larvae are injected into the animals bloodstream Heartworms damage the blood vessels and reduce the heart s pumping ability resulting in severe lung and heart disease The signs of heartworm disease are usually detectable only after the disease has progressed and much damage has already been done to the internal organs The animal may also tire easily during exercise and collapse due to heart failure Principle of the Test Norgen s Dirofilaria immitis PCR Detection Kit constituents a ready to use system for the isolation and detection of Dirofilaria immitis using end point PCR The kit first allows for the isolation of Dirofilaria immitis DNA from the blood samples using spin column chromatography The Dirofilaria immitis DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for Dirofilaria immitis detecti
3. CR 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 D Dirofilaria immitis PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Dirofilaria immitis Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 35x Step 2 60 C 15 sec Step 3 72 C 30 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C oo E Dirofilaria immitis PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 15 20 uL PCR Reaction should be loaded on a 1X TAE 1 5 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 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 Dirofilaria 276 bp Figure 1 A representative 1X TAE 1 5 agarose gel showing the amplification of Dirofilaria at different concentrations Dirofilaria Target The size of the Dirofilaria target amplicon corresponds to 276 bp as represented by the provided DNA Marker M NTC Negative Control Isolation Control PCR Control Figure 2 A representative 1X TAE 1 5 agarose gel showing the amplificatio
4. he 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 I forgot to add the DIR Isolation Control soC during the isolation e It is recommended that the isolation is repeated 11 What if forgot to run the Control PCR for the sample and only ran the Detection PCR and l obtained a positive result e The result can be considered positive However any negative result must be verified by running the associated control PCR to ensure that it is a true negative and not a false negative due to problems with the RNA isolation or the PCR reactions Related Products Product Blood Genomic DNA Isolation Kit 46300 Toxoplasma gondii PCR Detection Kit 44700 Leptospira interrogans PCR Detection Kit 44600 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 Dirofilaria immitis 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 sugges
5. ific primers were checked for possible homologies to GenBank published sequences by sequence comparison analysis and published Dirofilaria strains F Linear Range The linear range of Norgen s Dirofilaria immitis PCR Detection Kit was determined by analysing a dilution series of a Dirofilaria immitis quantification standards ranging from 100 ag to 1 pg Each dilution has been tested in replicates n 4 using Norgen s Dirofilaria immitis PCR Detection Kit on a 1X TAE 1 7 agarose gel The linear range of Norgen s Dirofilaria immitis PCR Detection Kit has been determined to cover concentrations from 100 ag to 1 ng Under the conditions of the Norgen s Dirofilaria immitis DNA Isolation procedure Norgen s Dirofilaria immitis 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 PCR run e Norgen s Dirofilaria immitis 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 DIR PCR control nor the DIR Isolation Control IlsoC amplifies e If neither the DIR PCR control nor the DIR Isolation Control soC amplifies the sample must be re tested If the positive con
6. n of Isolation Control and PCR Control under different conditions using the 2X PCR Control 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 5 showed detection of both Isolation Control and PCR Control suggesting that the DNA isolation as well as the PCR reaction was successful Lane 6 showed only the detection of PCR Control suggesting that while the PCR was successful the isolation failed to recover even the spiked in Isolation control NTC Negative Control Table 5 Interpretation of PCR Assay Results Input Type Target Control Reaction Interpretation Reaction Dirofilaria IsoC Band PCRC Band Target Band 499 bp 150 bp 276 bp Positive x X x Valid Control Negative j Control fs 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 Dirofilaria immitis PCR Assay Specificity and Sensitivity The specificity of Norgen s Dirofilaria immitis PCR Detection Kit is first and foremost ensured by the selection of the Dirofilaria immitis specific primers as well as the selection of stringent reaction conditions The Dirofilaria immitis spec
7. n performance Norgen s Dirofilaria immitis PCR Detection Kit contains a ready to use Proteinase K solution which is dissolved in a specially prepared storage buffer The Proteinase K is stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of the Proteinase K storage at 2 8 C is recommended The 2x DIR Detection PCR Master Mix 2x PCR Control Master Mix DIR Positive Control PosC and the Isolation Control soC 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 Dirofilaria immitis PCR Detection Kit including the 2x DIR Detection PCR Master Mix 2x PCR Control Master Mi
8. on using the provided Dirofilaria immitis Detection Master Mix The Dirofilaria immitis Detection Mastermix contains reagents and enzymes for the specific amplification of a 276 bp region of the genome In addition Norgen s Dirofilaria immitis PCR Detection Kit contains a second Mastermix the PCR Control Master Mix which can be used to identify possible PCR inhibition and or inadequate isolation via a separate 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 18 mL Wash Solution 12 mL Elution Buffer 6 mL Proteinase K 0 6 mL Spin Columns inserted 25 Into Collection Tubes Collection Tubes 25 Elution tubes 1 7 mL 25 Nuclease Free Water 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert IsoC Isolation Control PosC Positive Control The isolation control is a cloned PCR product The positive control is a fragment of Dirofilaria cloned in a plasmid Customer Supplied Reagents and Equipment e Benchtop microcentrifuge e 1 5 mL microcentrifuge tubes e 96 100 ethanol e lsopropanol e 55 C water bath or incubator 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 year without showing any reduction i
9. r PCR amplification e It is important to work quickly during this procedure Lysate Preparation Add 12 uL of Proteinase K to a microcentrifuge tube Transfer 500 uL of blood sample to the tube containing Proteinase K Add 600 uL of Lysis Solution to the blood and mix well by gentle vortexing for 10 seconds Briefly spin the tube to collect any drops of liquid from the inside of the lid Incubate at 55 C for 10 minutes If any debris is present in the sample centrifuge for 2 minutes at 14 000 x g 14 000 RPM to precipitate Transfer the clean supernatant to a microcentrifuge tube prior to Step 7 Briefly spin the tube to collect any drops of liquid from the inside of the lid Add 240 uL of Isopropanol to the sample and mix well by gentle vortexing for 10 seconds Briefly spin the tube to collect any drops of liquid from the inside of the lid Specimen DNA Purification Following the lysate preparation DNA can be extracted from the patient specimens using the supplied buffers and solutions according to the following protocol 1 2 3 DnR Add 10 uL of Isolation Control IsoC to the lysate mixture Obtain a spin column assembled with its collection tube Apply up to 650 uL of the lysate to the column and centrifuge for 1 minute at 6 000 x g 8 000 RPM Discard the flowthrough Reassemble the column and the collection tube Note Ensure that all of the lysate has passed through into the collection tube If the entire l
10. solutions become clear again e 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 e For best results the use of whole blood collected into tubes containing an anticoagulant is highly recommended e Both fresh and frozen anticoagulated blood may be used with this procedure Ensure that frozen blood is thawed at room temperature prior to starting the protocol e Prepare a working concentration of Wash Solution by adding 28 mL of 96 100 ethanol provided by the user to the supplied bottle containing concentrated Wash Solution This will give a final volume of 40 mL The label on the bottle has a box that can be checked to indicate that ethanol has been added e Always vortex the Proteinase K before use e isolation Control soC An Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control soC 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 Isolation Control IsoC must be kept on ice at all times during the isolation procedure e The PCR components of the Dirofilaria immitis PCR Detection Kit should remain at 20 C until DNA is extracted and ready fo
11. tions 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 2011 Norgen Biotek Corp P144500 5
12. trol 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 DIR PCR control showed amplification but neither the DIR target nor the DIR 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 DIR Isolation Control soC was amplified in a sample e The sample tested can be considered as Dirofilaria immitis negative 5 How should it be interpreted if the DIR PCR control and the DIR 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 DIR target and the DIR PCR control were amplified in a sample e The sample tested can be considered as Dirofilaria immitis positive 7 How should it be interpreted if only the DIR target was amplified in a sample e It is recommended that the isolation is repeated 8 How should it be interpreted if only the DIR PCR control and the DIR Isolation control showed amplification in a sample e The sample tested can be considered negative 9 What if forgot to do a dry spin after my third wash e Your first DNA elution will be contaminated with the Wash Solution This may dilute t
13. x Isolation Control and DIR Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Dirofilaria immitis PCR Detection Kit is designed for research purposes only 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 Blood of all human and animal subjects is considered potentially infectious All necessary precautions recommended by the appropriate authorities in the country of use should be taken when working with whole blood 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 Important Notes Prior to Beginning Protocol e 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
14. ysate volume has not passed centrifuge for an additional 2 minutes Repeat step B2 and B3 with remaining lysate Discard the collection tube containing flow through Assemble a spin column with a new collection tube 7 Apply 500 uL of Wash Solution ensure ethanol was added to the column and centrifuge for 1 minute at 6 000 x g 8 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 8 Wash column a second time by adding 500 uL of Wash Solution and centrifuging for 1 minute at 6 000 x g 8 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 9 Spin the column for 2 minutes in order to thoroughly dry the resin at 14 000 x g 14 000 RPM Discard the collection tube 10 Place the column into a provided 1 7 mL elution tube 11 Add 200 uL of Elution Buffer to the column 12 Centrifuge for 1 minute at 6 000 x g 8 000 RPM 13 The purified DNA sample may be stored at 4 C for a few days It is recommended that samples be placed at 20 C for long term storage C Dirofilaria immitis 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 2X DIR Detection PCR Master Mix and 2X PCR Control 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 each sample one
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