Sputum-Based Mycobacterium tuberculosis PCR Detection Kit
Contents
1. Total Volume 20 uL 3 For each 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 D Mycobacterium tuberculosis PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Mycobacterium tuberculosis 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 eo D Mycobacterium tuberculosis PCR Assay Interpretation e For the analysis of the PCR data the entire 20 uL PCR reaction should be loaded on a 1X TAE 2 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided e The PCR products should be resolved on the 1X TAE 2 Agarose gel at 150V for 30 minutes Gel running time will vary depending on an electrophoresis apparatus M 1 2 3 4 5 6 7 8 Neg Pos M 2000 1500 1000 750 500 300 150 50 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of Mycobacterium tuberculosis at different concentrations The size of the Mycobacterium tuberculosis target amplicon corresponds to the 319 bp band represented by the provided DNA Marker M Lanes 1 8 represents samples2. s Sputum Based Mycobacterium tuberculosis PCR Detection Kit is designed to test 24 samples For every 6 samples a Negative Control and a Positive Control must be included It is preferable to pool and test 6 samples at a time If not the provided Negative Control and Positive Control are enough to run 3 samples at a time 2 How can interpret my results for a sample if neither the Mycobacterium tuberculosis PCR control nor the Mycobacterium tuberculosis Isolation Control IsoC amplifies e If neither the Mycobacterium tuberculosis PCR control nor the Mycobacterium tuberculosis Isolation Control IsoC 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 the problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the Mycobacterium tuberculosis PCR control showed amplification but neither the Mycobacterium tuberculosis target nor the Mycobacterium tuberculosis 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 Mycobacterium tuberculosis Isolation Control IsoC was amplified in a sample e The sample tested can be considered as Mycobacterium tuberculosis negative 5 How should it be interpreted if only the Mycobacterium tuberculosis target and the M
3. 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 2013 Norgen Biotek Corp P142100 4
4. 4 gt lt NORGEN BIOTEK wi CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Sputum Based Mycobacterium tuberculosis PCR Detection Kit Product Insert Product 42100 Mycobacterium tuberculosis is a pathogenic bacterial species belonging to the genus Mycobacterium and is the causative agent of tuberculosis Tuberculosis TB is a multifaceted disease and challenging public health problem in both industrialized and developing countries According to the WHO 8 8 million active cases of TB are diagnosed each year and of these almost 2 million die Once thought to be under control or even close to extinction TB infection levels are rising and the threat is compounded by new virulent and drug resistant strains Although most cases 80 occur in the developing world increasing population mobility combined with the ease of transmission means that no country is immune from the resurgence of TB TB control programs are currently facing a number of constraints Worldwide fewer than 25 of all tuberculosis cases are detected Of utmost concern is the absence of a timely and accurate test for the diagnosis of mycobacterial disease Early diagnosis is crucial for the prevention of further spread of the disease Principle of the Test Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit is a ready to use system for the iso
5. What If my incubation varied from the 20 minutes specified in the product manual e Less than 20 minutes will result in lower DNA yields More than 20 minutes may not affect your DNA yields 12 What If I forgot to do a dry spin after my second wash e Your DNA elution will be contaminated with the Wash Solution This may dilute the DNA yield in your elution and it may interfere with your down stream applications 13 What If I forgot to add the Mycobacterium tuberculosis solation control during the Isolation e The Isolation must be repeated Related Products Product Urine Based Mycobacterium Tuberculosis PCR Detection Kit 41200 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 Sputum based Mycobacterium tuberculosis 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
6. above please refer to the Troubleshooting Section Ignore any bands that appear between the Isolation Control band and the PCR Control band E Specificity The specificity of Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit is first and foremost ensured by the selection of the Mycobacterium tuberculosis specific primers as well as the selection of stringent reaction conditions The primers were checked for possible homologies in GenBank published sequences by sequence comparison analyses F Linear Range The linear range analytical measurement of Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit was determined by analyzing a dilution series of Mycobacterium tuberculosis quantitative standard ranging from 8 46 x 10 copies ul to 1 x 10 copies ul Each dilution has been tested in replicates n 4 using Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit on 1X TAE 1 7 Agarose gels The linear range of Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit has been determined to cover concentrations from 2 copies l to at least 8 x 10 copies ul Under the conditions of Norgen s Sputum DNA Isolation procedure Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit covers a linear range from 1000 copies mL sputum to at least 8 x 10 copies mL sputum G Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen
7. erculosis cloned in a plasmid Customer Supplied Reagents and Equipment e Disposable powder free gloves Centrifuge with a swinging bucket rotor capable of 2 000 RPM Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters Lysozyme solution 20 mg mL Dithiothreitol 100 g mL or other solution for upstream sputum homogenization PCR tubes 96 100 ethanol 60 C incubator 15 mL conical tubes Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C for up to 1 year without showing any reduction in performance Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit contains ready to use Proteinase K solutions which are 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 Proteinase K storage at 2 8 C is recommended The Mycobacterium tuberculosis 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and Mycobacterium tuberculosis Positive Control PosC 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 precau
8. g the Mycobacterium tuberculosis 2X Detection PCR Master Mix and one PCR reaction using Control 2X PCR Master Mix should be set up in order to have a proper interpretation of the results For every PCR run one reaction containing Mycobacterium tuberculosis Positive Control and one reaction as no template control must be included for proper interpretation of results 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 the Mycobacterium tuberculosis Limit of Detection 1 Prepare the PCR reaction for sample detection Set 1 using Mycobacterium tuberculosis 2X Detection PCR Mastermix and the PCR reaction for control detection Set 2 using Control 2X PCR 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 Malaria 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 uL Total Volume 20 uL 2 For each 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
9. lation and detection of Mycobacterium tuberculosis using end point PCR The kit first allows for the isolation of mycobacterial DNA from sputum samples using spin column chromatography based on Norgen s proprietary resin The mycobacterial DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for detection using the provided Mycobacterium tuberculosis Master Mix This Master Mix contains reagents and enzymes for the specific amplification of a 319 bp region of the Mycobacterium tuberculosis genome In addition Norgen s Sputum Based Mycobacterium tuberculosis PCR Detection Kit contains a second Master Mix the Control 2x PCR 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 The kit is designed to allow for the testing of 24 samples Kit Components Component Contents Binding Solution 50 mL Proteinase K 0 6 mL Binding Solution II 3 25 mL Wash Solution 6 6 mL Elution Buffer 3 mL 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 5 mL Product Insert IsoC Isolation Control PosC Positive Control The isolation control is a cloned PCR product P The positive control is a fragment of Mycobacterium tub
10. n a shatterproof leak proof transport container as a matter of principle Thus a potential danger of infection due to a leakage of sample can be avoided e The samples should be transported following the local and national instructions for the transport of pathogenic material B Isolation of DNA from Sputum Notes e Do not spin down or filter the sputum sample before proceeding with the isolation as this could negatively affect the isolation of DNA 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 the Proteinase K solution before use Preheat an incubator or heating block to 60 C Prepare a working concentration of Binding Solution I Binding Solution II and Wash Solution by adding the appropriate volume of 96 100 ethanol to the supplied bottles containing the concentrated solutions see Table 1 below The labels on the bottles have a box that may be checked to indicate that the ethanol has been added Prior to the DNA isolation it is recommended that the viscous sputum sample be liquefied This can be accomplished by adding a reducing agent such as dithiothreitol to the sample and heating at 37 C for 20 minutes to completely homogenize the sample We recommend preparing a solution of DTT at a concentration of 100 g mL and then adding an equal volume to the sputum sample to give a final conce
11. ntire contents into a Mini Filter Spin column provided Centrifuge for 1 minutes at 10 000 RPM and discard the flow through Apply 500 uL of Wash Solution 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 500 uL of Wash Solution 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 Apply 500 uL of 95 ethanol and centrifuge for 1 minute at 14 000 RPM Discard the flow through and reassemble the spin column with its collection tube 11 Spin the column for 2 minutes at 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 12 Incubate the column horizontally with the lid open at 60 C for 10 minutes 13 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 100 uL of Elution Buffer to the column and centrifuge for 2 minutes at 2 000 RPM followed by a 1 minute spin at 14 000 RPM C Mycobacterium tuberculosis 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 Mycobacterium tuberculosis 2X Detection PCR Master Mix and Control 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 each sample one PCR reaction usin
12. ntration of 50 ug mL Table 1 Volume of Ethanol to be added to Binding Buffer Binding Buffer Il and Wash Buffer Ethanol 96 100 Solution Volume Provided Volume to be Added Final Volume by User Binding Solution 50 mL 50 mL 50 mL Binding Solution II 3 25 mL 3 25 mL 6 5 mL Wash Solution 6 6 mL 18 4 mL 25 mL a 5 gt oo N e Isolation Control soC A Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control IsoC to the lysate during the isolation procedure The Isolation Control soC 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 Add 4 mL of Binding Solution for 1 mL of sputum sample Mix well by pipetting up and down several times Note Binding Solution contains resin and must be mixed well before every pipeting Centrifuge for 5 minutes at 2 000 RPM and discard the supernatant Add 20 uL of both Proteinase K and Lysozyme user supplied to the precipitated slurry pellet resulting from the sputum sample Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes After the 20 minute incubation add 260 uL of Binding Solution Il Add 10 uL Isolation Control IsoC to the lysate mix well by pipeting and then transfer the e
13. ormation please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Binding Solution I Binding Solution II and the Wash Solution 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 buffers is spilled clean with suitable laboratory detergent and water If the spilled liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite 1 Protocol A Specimen Collection Storage and Transport Precaution All samples have to be treated as potentially infectious material 1 Specimen Collection and Sample Storage e Expectorated or induced sputum samples may be collected e It is highly recommended that sputum samples be collected using Norgen s Sputum DNA Collection Preservation and Isolation Kit Cat 46100 The sputum samples can be stored for at least one year at room temperature when collected directly using Norgen s Sputum DNA Collection Preservation and Isolation Kit e Alternatively sputum samples collected using any other collection and preservation systems or reagents are also compatible with this kit 2 Sample Transport e Sample material should be transported i
14. spiked with different Mycobacterium tuberculosis concentrations isolated from 1 mL sputum samples interpreted as positive results The Mycobacterium tuberculosis spiked in sputum samples is purified plasmid DNA M 1 2 3 4 5 6 NTC lt Isolation Control 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 PCR Master Mix 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 Reaction Control Reaction Interpretation Mycobacterium IsoC Band PCRC Band tuberculosis 499bp 150 bp Target Band 319 bp Positive f Control X X X Valid Negative Control X Valid Sample x x x Positive Sample X Negative Sample X Negative Sample X Re Test Sample Re Test Sample X X Positive Sample X X Positive Sample X Re Test For results obtained that are not covered in Table 5
15. tions while using the kit e All biological samples should be considered as potentially infectious Proper biosafety measures should therefore be carried out when using this 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 Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Mycobacterium tuberculosis 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and Mycobacterium tuberculosis Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Sputum based Mycobacterium tuberculosis PCR Detection Kit is designed for research purposes only It is not intended for human or 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 inf
16. ycobacterium tuberculosis PCR control were amplified in a sample e The sample tested can be considered as Mycobacterium tuberculosis positive 6 How should it be interpreted if only the Mycobacterium tuberculosis target was amplified in a sample e The sample tested can be considered positive At high Mycobacterium tuberculosis concentration the Mycobacterium tuberculosis amplicon will be predominant and the Mycobacterium tuberculosis PCR control as well as the Mycobacterium tuberculosis Isolation control may not amplify 7 How should it be interpreted if only the Mycobacterium tuberculosis PCR control and the Mycobacterium tuberculosis Isolation Control IsoC showed amplification e The sample tested can be considered negative 8 Can process a different sputum volume e The reagents provided with the isolation kit are only sufficient to process 24 sputum samples of 1 mL each 9 What If added more or less of the specified reagents volume during DNA isolation e Adding less volume may reduce your DNA yields Adding more may not affect the DNA yields EXCEPT if more Elution Buffer was added Eluting DNA in higher volumes of Elution Buffer will result in diluting your DNA 10 What If my incubation temperature varied from the specified 60 C e The incubation temperature can be in the range of 55 C 65 C At other temperatures the activity of the Proteinase K will be reduced This will result in a reduction in your DNA yields 11
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