Rabies RT-PCR Detection Kit - Protocol
Contents
1. For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section E Rabies RT PCR Assay Specificity and Sensitivity The specificity of Norgen s Rabies RT PCR Detection Kit is first and foremost ensured by the selection of the Rabies specific primers as well as the selection of stringent reaction conditions The Rabies specific primers were checked for possible homologies to all GenBank published sequences by sequence comparison analysis and published strains F Linear Range The linear range of Norgen s Rabies RT PCR Detection Kit was determined by analysing a dilution series of a Rabies quantification standards ranging from 100 ag to 1 pg Each dilution has been tested in replicates n 4 using Norgen s Rabies RT PCR Detection Kit on a 1X TAE 1 7 agarose gel The linear range of Norgen s Rabies RT PCR Detection Kit has been determined to cover concentrations from 100 ag to 1 ng Under the conditions of the Norgen s Rabies RNA Isolation procedure Norgen s Rabies 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 Rabies 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 If not the2. 5X TAE 1 7 agarose gel showing the amplification of Rabies under different concentration Target using the Rabies 2X RT PCR Mastermix The size of the Rabies target amplicon corresponds to 280 bp as represented by the provided DNA Marker M NC Negative Control 1 2 3 4 5 6 NC 2000 1500 1000 750 500 300 Isolation Control 150 PCR Control Figure 2 A representative 1X TAE 1 7 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 5 showed detection of both Isolation Control and PCR Control suggesting that the RNA isolation as well as the RT PCR reaction was successful Lane 6 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 NC Negative Control Table 5 Interpretation of One Step RT PCR Assay Results Input Type Target Control Reaction Interpretation reaction Rabies Target IsoC Band Rabies PCRC Band 280 bp 499 bp Band 171 bp foil x x x Valid K 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
3. provided Positive Control is enough to run 3 samples at a time 2 How can interpret my results if neither the Rabies RT PCR control nor the Isolation Control IsoC amplifies e f neither the Rabies PCR control nor the Rabies 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 not amplify therefore the problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the Rabies PCR control showed amplification but neither the Rabies target nor the 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 Rabies negative 5 How should it be interpreted if the Rabies PCR control and the Rabies 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 Rabies target and the Rabies PCR control were amplified in a sample e The sample tested can be considered as Rabies positive 7 How should it be interpreted if only the Rabies target was amplified in a sample e It is recommended that the isolation is repeated 8 How shou
4. 20 uL 3 For each RT PCR run prepare one no template control RT 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 PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step RT PCR Table 4 Rabies Assay Program One Step RT PCR Cycle Step Temperature Duration Cycle 1 Step 1 50 C 25 min Cycle 2 Step 1 95 C 5 min Step 1 94 C 15 sec Cycle 3 35x Step 2 60 C 30 sec Step 3 72 C 45 sec Cycle 4 Step 1 72 C 5 min Cycle 5 Step 1 4 C eo D Rabies One Step RT PCR Assay Results Interpretation 1 For the analysis of the RT PCR data the entire 15 20 uL RT PCR Reaction should be loaded on a 1X TAE 1 7 Agarose RNA gel along with 10 uL of Norgen s RNA Marker provided Prepare enough agarose gel for running one set of RT PCR of Rabies detection and one set of RT PCR for controls detection 2 The RT PCR products should be resolved on the 1X TAE 1 7 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below 2000 1500 1000 750 500 300 lt Target 150 50 Figure 1 A representative 1
5. Fax 905 227 1061 BIOTEK ad CORPORATION Email techsupport norgenbiotek com cok 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 z Phone 866 667 4362 905 227 8848 Rabies RT PCR Detection Kit Product Insert Product 45300 Pathogen Information Rabies is a viral disease that results in acute encephalitis inflammation of the brain in mammals Rabies is caused by the rabies virus which is a non enveloped virus with a single stranded RNA genome The virus is transmitted via saliva and transmission of the disease almost always occurs as a result of an infected animal biting a non infected animal The virus does not survive long outside the host and will remain viable in the carcass of an infected animal for less than 24 hours After coming into contact with the virus rabies will spread through the nerves towards the brain The virus is slow moving and the average time of incubation from exposure to brain involvement is between 3 to 8 weeks in a dog and 2 to 6 weeks in a cat However incubation periods as long as 6 months have been reported After the virus reaches the brain it will then spread to the salivary glands where it can spread through bites The virus is shed at high levels in saliva There is no treatment for the disease once rabies develops it is fatal Currently the only way to diagnose rabies in animals is to submit the brain for post mortem examinations Since it is possible to be exposed to rabies without becoming in
6. e RT PCR reaction using the Rabies 2X 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 result For every RT PCR run one reaction containing Rabies Positive Control Rabies PosC and one reaction as no template control must be included for proper interpretation of results e The recommended minimum number of RNA samples tested per RT PCR run is 6 e Using a lower volume from the sample than recommended may affect the sensitivity of Rabies Limit of Detection 1 Prepare the RT PCR reaction for sample detection Set 1 using Rabies 2X 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 2 5 uL However a volume between 1 and 5 uL of sample RNA may be used as template Ensure that one Rabies 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 5 uL Nuclease Free Water 7 5 pL Total Volume 20 uL 2 For each 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
7. e available as convenient PDF files online at www norgenbiotek com General Precautions e Follow universal precautions All specimens should be considered as potentially infectious and handled accordingly e Wear personal protective equipment including gloves and lab coats when handling kit reagents Wash hands thoroughly when finished performing the test e Dispose of unused kit reagents and specimens according to local provincial or federal regulations e Workflow in the laboratory should proceed in a uni directional manner beginning in the pre amplification area s i e specimen collection and RNA extraction and moving to the amplification detection area s RT PCR and gel electrophoresis e Do not use supplies and equipment across the dedicated areas of specimen extraction and sample preparation No cross movement should be allowed between the different areas e Personal protective equipment such as laboratory coats and disposable gloves should be area specific e Only use the protocol provided in this insert Alterations to the protocol and deviations from the times and temperatures specified may lead to erroneous results 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 b
8. est defense against these enzymes e The RNA area should be located away from microbiological work stations e 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 e There should be designated solutions tips tubes lab coats pipettes etc for RNA only e All RNA solutions should be prepared using at least 0 05 DEPC treated autoclaved water or molecular biology grade nuclease free water e Clean all surfaces with commercially available RNase decontamination solutions e When working with purified RNA samples ensure that they remain on ice during downstream applications INSTRUCTIONS FOR USE Important Notes Prior to Beginning Protocol e 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 e 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 e Avariable speed microcentrifuge 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 Pre
9. fected methods for the diagnosis of disease by other methods is urgently required This will not only help in the prevention of the spread of disease but may also spare animals that would otherwise be euthanized Principle of the Test Norgen s Rabies RT PCR Detection Kit constituents a ready to use system for the isolation and detection of rabies virus using end point one step RT PCR The kit first allows for the isolation of rabies RNA from the blood samples 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 rabies detection using the provided Rabies Detection Mastermix The Rabies Detection Mastermix contains reagents and enzymes for the specific amplification of a 280 bp region of the viral genome In addition Norgen s Rabies RT PCR Detection Kit contains a second Mastermix the RT PCR Control 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 10 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 Ma
10. ld it be interpreted if only the Rabies PCR control and the Rabies 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 with 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 11 What if forgot to run the Control RT PCR for the sample and I only ran the Detection RT PCR and I obtained a positive result e The result can be considered positive However any negative result must be verified by running the associated control RT PCR to ensure that it is a true negative and not a false negative due to problems with the RNA isolation or the RT PCR reactions Related Products Product Total RNA Purification Kit 17200 Feline Leukemia Virus RT PCR Detection Kit 44000 Feline Calicivirus RT PCR Detection Kit 43900 Feline Herpes Virus PCR Detection Kit 44300 Feline infectious peritonitis RT PCR Detection Kit 44400 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 q
11. nding on lysate volume repeat steps B2 and B3 Apply 400 uL of Wash solution and centrifuge for one minute at 14 000 rpm 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 through spin for an additional minute Discard the flowthrough and reassemble the spin column with its collection tube Repeat steps B5 and B6 two more times for a total of 3 washes Spin the column for 2 minutes to thoroughly dry the resin at 14 000 rpm Discard the collection tube 9 Place the column into a new 1 7 mL Elution tube 10 Add 50 uL of Elution Solution to the column 11 Centrifuge for 2 minutes at 2 000 rpm followed by a 2 minute spin at 14 000 rpm Note the volume eluted from the column If the entire 50 uL has not been eluted spin the column for an additional minute at 14 000 rpm 12 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 CoO C Rabies RT PCR Assay Preparation Notes Before use suitable amounts of all RT PCR components should be completely thawed at room temperature vortexed and centrifuged briefly The amount of Rabies 2X RT PCR Mastermix and Control 2X RT PCR Mastermix 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 each For each sample on
12. o the specimen as soon as possible within 6 hours It is important to work quickly during this procedure A SPECIMEN LYSATE PREPARATION Blood Lysate Preparation 1 2 Poo Add 350 uL of the Lysis Solution to an RNase free microcentrifuge tube Add up to 100 uL of blood Vortex for 10 seconds to mix Note Rabies has a poor survival rate outside the infected body It is important to add the Lysis Solution to the specimen as soon as possible within 6 hours In the presence of the Lysis Solution components the virus could be stable for hours if stored at room temperature and gt 1 month if stored at 70 C Add 10 uL of the Isolation Control IsoC to the lysate Vortex for 10 seconds to mix Add 250 uL of 95 ethanol to the lysate Vortex for 10 seconds to mix Proceed to RNA Isolation Step B B SPECIMEN RNA PURIFICATION Following the lysate preparation viral RNA can be extracted from the specimens using the supplied buffers and solutions according to the following protocol 1 2 oR Assemble a column with one of the provided collection tubes Apply the lysate with ethanol up to 650 uL to the column and centrifuge for 1 minute at 14 000 rpm 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 through spin for an additional minute Discard the flowthrough and reassemble the spin column with its collection tube Depe
13. pare 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 Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood It is important to work quickly during this procedure Isolation Control soC An Isolation Control IsoC 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 Isolation Control fsoC must be kept on ice at all times during the isolation procedure The RT PCR components of the Rabies RT PCR Detection Kit should remain at 20 C until RNA is extracted and ready for RT PCR amplification It is recommended that no more than 100 uL of blood be used in order to prevent clogging of the column We recommend the use of this kit to isolate RNA from non coagulating fresh blood using EDTA or heparin as the anti coagulant Rabies has a poor survival rate outside the infected body It is important to add the Lysis Solution t
14. rker 0 1 mL Product Insert 1 IsoC Isolation Control PosC Positive Control The isolation control is an RNA transcript The positive control is Rabies RNA transcript Customer Supplied Reagents and Equipment e Benchtop microcentrifuge 95 100 ethanol Thermocycler and or Real Time PCR System Micropipettes with an accuracy range between 1 10 uL 10 100 uL and 100 1000 uL Laminar flow hood for extractions Vortex Sterile nuclease free aerosol barrier micropipettor tips Microcentrifuge tube rack Disposable latex gloves B mercaptoethanol Storage Conditions and Product Stability e The Rabies Positive Control and Rabies Isolation Control should be stored at 70 C If needed make aliquots of the controls according to the volume used in the protocol 10 uL of Rabies PosC or 20 uL of IsoC prior to freezing e The Rabies 2X RT PCR Mastermix and the Control 2X RT PCR Mastermix should be stored at 20 C upon receipt 70 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 Rabies 2X 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 When thawed mix the components and centrifuge briefly Work quickly on ice After addition of RT PCR Mastermix u
15. se within one hour Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Rabies RT PCR Detection Kit including the Rabies 2x RT PCR Mastermix Control 2X RT PCR Mastermix Isolation Control and Rabies Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Rabies RT 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 If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite 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 ar
16. uestions or experience any difficulties regarding Norgen s Rabies 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 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 2012 Norgen Biotek Corp P145300 4
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