Borrelia burgdorferi PCR Detection Kit - Protocol
Contents
1. 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 set 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 set 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 Borrelia burgdorferi PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Borrelia burgdorferi 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 58 C 15 sec Step 3 72 C 30 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C eo D Borrelia burgdorferi 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 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 5 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophore2. a 3430 Schmon Parkway 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 Borrelia burgdorferi PCR Detection Kit Product Insert Product 45200 Pathogen Information Borrelia burgdorferi is a Gram negative bacterium and is the predominant agent of Lyme disease in North America Mice and other small rodents are the primary reservoir for the B burgdorferi bacteria and Ixodes ticks will then attach to these rodents and feed on their blood thereby transmitting the bacteria to the tick larvae Tick larvae become dormant in the winter months however in the spring the young ticks will attach to larger animals including dogs and cats resulting in transmission of the bacteria to the larger hosts and subsequently Lyme disease Typically more dogs contract Lyme disease than cats but both are susceptible Symptoms of Lyme Disease may include loss of neuromuscular function limping in hind leg s fatigue loss of appetite lethargy paralysis muscle and joint pain and fever Serious cases of lyme disease may cause paralysis as well as muscle and heart tissue damage possibly resulting in death For the best possible treatment outcome early diagnosis of Lyme disease is crucial Animals who display any symptoms which could possibly have a diagnosis of Lyme disease should be tested as soon as possible Due to the seriousness of the disease a sensitive and s
3. ater 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert 1 The isolation control is a cloned PCR product The positive control is a cloned PCR product of Borrelia burgdorferi Customer Supplied Reagents and Equipment e Benchtop microcentrifuge e Micropipettors e 96 100 ethanol e 60 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 in performance Norgen s Borrelia burgdorferi PCR Detection Kit contains ready to use Proteinase K and Pronase solutions which are dissolved in a specially prepared storage buffer The Proteinase K and the Pronase are stable for up to 1 year after delivery when stored at room temperature To prolong the lifetime of Proteinase K and Pronase storage at 2 8 C is recommended The 2x BORR Detection PCR Master Mix 2x PCR Control Master Mix BORR Positive Control PosC and the BORR 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 f
4. ch template was added to the reaction 6 How should it be interpreted if only the B burgdorferi target and the B burgdorferi PCR control were amplified in a sample e The sample tested can be considered as B burgdorferi positive 7 How should it be interpreted if only the B burgdorferi target was amplified in a sample e It is recommended that the isolation is repeated 8 How should it be interpreted if only the B burgdorferi PCR control and the B burgdorferi 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 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 B burgdorferi 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 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 DNA isolation or the PCR reactions Related Products Product Dirofilaria immitis PCR Detection Kit 44500 Toxoplasma gondi
5. e at 10 000 RPM Do Not Discard the Flowthrough as it contains the DNA Incubate the column and collection tube at 60 C for 20 minutes After the 20 minute incubation add 450 uL of Binding Buffer Il and 10 uL of BORR Isolation Control IsoC to the lysate in the collection tube mix well by pipeting and then transfer the entire contents of the collection tube back onto the spin column Centrifuge for 1 minute at 10 000 RPM Discard the flow through Apply 450 uL of Wash Solution I to the column and centrifuge for 1 minute at 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Apply 450 uL of Wash Solution II to the column and centrifuge for 1 minute at 14 000 RPM Discard the flow through and reassemble the spin column with its collection tube 9 Apply 450 uL of 96 100 ethanol supplied by the user to the column and centrifuge for 1 minute at 14 000 RPM Discard the flow through and reassemble the spin column with its collection tube 10 Repeat Step 9 a second time 11 Spin the column empty for 1 minute at 14 000 RPM Discard the collection tube 12 Incubate the column horizontally with the lid open for 3 minutes at 60 C 13 Transfer the spin column to a fresh Elution Tube Apply 75 uL of Elution Buffer to the column and centrifuge for 2 minutes at 2 000 RPM followed by 2 minutes at 14 000 RPM 14 The purified DNA sample could be used immediately for PCR as described below It is recommend
6. e of 15mL The label on the bottle has a box that can be checked to indicate that ethanol has been added Preheat an incubator or heating block to 60 C Borrelia burgdorferi solation Control IsoC A BORR Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the BORR Isolation Control IsoC to the lysate during the isolation procedure The BORR Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the BORR Isolation Control IsoC more than 2 times The BORR Isolation Control IsoC must be kept on ice at all times during the isolation procedure The PCR components of the Borrelia burgdorferi PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification It is important to work quickly during this procedure A Isolation of DNA oF NO Add 250 uL of Binding Buffer I for every 1 75 mL urine sample Mix well by inverting a few times Apply up to 650 uL of the urine sample onto a column and centrifuge for 1 minute at 10 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube Note If the entire volume does not pass through into the collection tube the column can be spun for an additional minute Repeat Step 2 until the entire urine sample has passed through the column Add 35 uL of Proteinase K and 35 uL of Pronase to each column and centrifuge for 1 minut
7. ed that samples be placed at 70 C for long term storage B Borrelia burgdorferi 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 BORR 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 PCR reaction using the 2X BORR 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 result For every PCR run one reaction containing BORR 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 e Using a lower volume from the sample than recommended may affect the sensitivity of Borrelia burgdorferi Limit of Detection 1 Prepare the PCR for sample detection Set 1 using 2X BORR Detection PCR Mastermix and control detection Set 2 using 2X PCR Conrtol 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 BORR detection reaction and one control reaction is prepared for each DNA sample Adjust the final volume of the PCR reaction
8. i PCR Detection Kit 44700 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 Norgen s B burgdorferi 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 2011 Norgen Biotek Corp P145200 2
9. ilters 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 Borrelia burgdorferi PCR Detection Kit including the 2x BORR Detection PCR Master Mix 2x PCR Control Master Mix BORR Isolation Control and BORR Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Borrelia burgdorferi 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 Binding Solution I Binding Solution II Wash Solution and Wash Solution II contain guanidine hydrochloride and should be handled with care Guanidine hydrochloride forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these solutions is spilt clean with suitable laborato
10. ive 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 Borrelia burgdorferi PCR Assay Specificity and Sensitivity The specificity of Norgen s Borrelia burgdorferi PCR Detection Kit is first and foremost ensured by the selection of the Borrelia burgdorferi specific primers as well as the selection of stringent reaction conditions The Borrelia burgdorferi specific primers were checked for possible homologies to GenBank published sequences by sequence comparison analysis and published strains F Linear Range The linear range of Norgen s Borrelia burgdorferi PCR Detection Kit was determined by analysing a dilution series of a Borrelia burgdorferi quantification standards ranging from 1pg to 10 ng Each dilution has been tested in replicates n 4 using Norgen s Borrelia burgdorferi PCR Detection Kit on a 1X TAE 1 7 agarose gel The linear range of Norgen s Borrelia burgdorferi PCR Detection Kit has been determined to cover concentrations from 1pg to 10 ng Under the conditions of the Norgen s Borrelia burgdorferi DNA Isolation procedure Norgen s Borrelia burgdorferi 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
11. pecific diagnostic test is also necessary to avoid false negative results Principle of the Test Norgen s Borrelia burgdorferi PCR Detection Kit constituents a ready to use system for the isolation and detection of Borrelia burgdorferi using end point PCR The kit first allows for the isolation of Borrelia burgdorferi DNA from urine samples using spin column chromatography based on Norgen s proprietary resin The Borrelia burgdorferi DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for Borrelia burgdorferi detection using the provided B burgdorferi Master Mix The B burgdorferi Mastermix contains reagents and enzymes for the specific amplification of a 277 bp region of the genome In addition Norgen s Borrelia burgdorferi PCR Detection 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 lsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Binding Solution 8 mL Binding Solution II 4mL Wash Solution 4mL Wash Solution II 12 mL Elution Buffer 3 mL Proteinase K in storage buffer 1 mL Pronase in storage buffer 1 mL Mini Spin Columns 24 Collection Tubes 24 Elution tubes 1 7 mL 24 Nuclease Free W
12. run e Norgen s Borrelia burgdorferi 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 B burgdorferi PCR control nor the B burgdorferi Isolation Control IsoC amplifies e f neither the B burgdorferi PCR control nor the B burgdorferi 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 B burgdorferi PCR control showed amplification but neither the B burgdorferi target nor the B burgdorferi 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 B burgdorferi Isolation Control lsoC was amplified in a sample e The sample tested can be considered as B burgdorferi negative 5 How should it be interpreted if the B burgdorferi PCR control and the B burgdorferi target showed amplification in a sample e The sample tested can be considered positive It could happen when too mu
13. ry 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 are available as convenient PDF files online at www norgenbiotek com Protocol Important Notes Prior to Beginning Protocol A variable 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 First time users should read the entire manual before proceeding with the protocol Do not spin down or filter the urine sample before proceeding with the isolation as this could decrease the DNA yield Ensure that all 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 Always vortex both the Proteinase K and the Pronase before use Prepare a working concentration of Binding Solution Il and Wash Solution I by adding 11 mL of 96 100 ethanol provided by the user respectively to the supplied bottle containing the concentrated Binding Solution Il and Wash Solution I This will give a final volum
14. sis apparatus 3 Sample results are provided below B burgdorferi Nnn M lt NC Pos 2000 1500 1000 750 500 300 150 50 Figure 1 A representative 1 5X TAE 1 7 agarose gel showing the amplification of B burgdorferi under different concentrations using the 2X B burgdorferi Detection PCR Mastermix The size of the B burgdorferi target amplicon corresponds to 277 bp as represented by the provided DNA Marker M NC Negative Control lt Isolation Control lt 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 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 B burgdorferi IsoC Band PCRC Band Target Band 499 bp 150 bp 350 bp aii x x x Valid cay x Valid Sample X X X Posit
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