Urine-Based HPV 6/16 PCR Detection Kit - Protocol
Contents
1. Spin the column for 2 minutes at 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 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 1 minute at 14 000 RPM C HPV 6 16 PCR Assay Preparation Notes e It is recommended that 10 uL of the DNA elution be used as the PCR sample input volume e Sample volume can be varied between 2 uL 10 uL of the DNA elution PCR grade water should be added to make up the final volume of the PCR reaction to 20 uL e Using a lower volume from the sample than recommended may affect the sensitivity of either HPV 6 and or HPV 16 Limit of Detection e A Negative Control and HPV 6 16 Positive Control PosC must be included during every run e The Nuclease Free Water and HPV 6 16 Positive Control PosC provided are sufficient for eight PCR runs e Before each use all reagents need to be thawed completely mixed by repeated up and down pipetting or quick vortexing and centrifuged briefly 1 Prepare PCR reactions as outlined in Table 2 below For each sample to be run pipette 10 uL of the eluted DNA and 10 uL of the Master Mix into a PCR tube Each PCR reaction will have a final volume of 20 uL 2 A Negative Control and HPV 6 16 Positive Control PosC must be included in every run Pipette 10 uL of Nuclease Free Water as a Negative Control NegC into a PCR tube and add2. 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 Midstream urine samples should be collected as the first flow of urine has been shown to have a higher rate of contamination Morimoto et al 2003 e It is highly recommended that urine samples be collected using Norgen s Urine Collection and Preservation Tubes Cat 18111 The urine samples can be stored for at least one year at room temperature when collected directly using Norgen s Urine Collection and Preservation Tubes e Alternatively urine 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 in 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 natio
3. of stringent reaction conditions The primers were checked for possible homologies in GenBank published sequences by sequence comparison analyses Furthermore the specificity of the HPV 6 16 specific primers were tested against most of the known sexually transmitted pathogens F Linear Range e The linear range analytical measurement of Norgen s Urine Based HPV 6 16 PCR Detection Kit was determined by analyzing a dilution series of HPV quantitative standard ranging from 8 46 x 10 VP ul to 1 x 107 IU ul e Each dilution has been tested in replicates n 4 using Norgen s Urine Based HPV 6 16 PCR Detection Kit on 1X TAE 1 7 Agarose gels e The linear range of Norgen s Urine Based HPV 6 16 PCR Detection Kit has been determined to cover concentrations from 0 2 VP ul to at least 8 x 10 VP ul e Under the conditions of Norgen s Urine DNA Isolation procedure Norgen s Urine Based HPV 6 16 PCR detection Kit covers a linear range from 450 VP mL urine to at least 8 x 10 VP mL urine for the high risk HPV 16 and covers a linear range from 250 VP mL urine to at least 8 x 10 VP mL urine for the Low risk HPV 6 G Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Urine Based HPV 6 16 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 Negat
4. 10 uL of Master Mix Pipette 10 uL of HPV 6 16 Positive Control PosC into a PCR tube and add 10 uL of Master Mix 3 Program the PCR machine according to the program shown in Table 3 below 4 Run PCR Table 2 PCR Assay Preparation Preparation of PCR assay Volume Per PCR Reaction 2X HPV 6 16 PCR Master Mix 10 uL 10 uL 10 uL Sample Eluted DNA 10 uL HPV 6 16 Positive Control PosC 10 uL HPV 6 16 Negative Control NegC 10 uL Total Volume 20 uL 20 uL 20 uL Table 3 HPV 6 16 PCR 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 0 D HPV 6 16 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 e Figure 1 and Table 4 explain how to interpret the PCR assay results 6 NegC PosC A Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of HPV 6 16 at different concentrations Target The size of the HPV 16 target amplicon corresponds to the 391 bp band whereas the size of the HPV 6 target amplicon corr
5. 6 Isolation Control IsoC must not be added to the sample material directly o Do not freeze and thaw the HPV 6 16 Isolation Control IsoC more than 2 times o The HPV 6 16 Isolation Control IsoC must be kept on ice at all times during the isolation procedure Obtain a 5 mL midstream urine sample Add 700 uL of Binding Solution and mix well by inversion Note Binding Solution contains resin and must be mixed well before every pipeting Centrifuge for 5 minutes at 2 000 RPM Discard the supernatant Add 20 uL of both Proteinase K and Pronase to the precipitated slurry pellet resulting from 5 mL of the urine sample Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes After the 20 minute incubation add 260 uL Binding Solution Il Add 15 uL HPV 6 16 Isolation Control IsoC to the lysate mix well by pipeting and then transfer the entire contents into a Mini Filter Spin column provided Centrifuge for 2 minutes at 10 000 RPM and Do Not discard the flow through Reload the flow through back to the column centrifuge for 2 minutes at 10 000 RPM and 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 Il 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
6. Acta 333 101 102 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 P142200 2
7. Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com a 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 3 Phone 866 667 4362 905 227 8848 Urine Based HPV 6 16 PCR Detection Kit Product Insert Product 42200 More than 70 types of human papillomavirus HPV have been identified and are generally classified as high risk or low risk depending on their relationship or lack of relationship with cancer and high grade cervical intraepithelial neoplasia CIN 2 3 HPV viruses are predominantly sexually transmitted and high risk HPV types are a major risk factor for development of cervical cancer HPV 16 has been considered as a high risk cancer associated HPV type The low risk HPV type 6 has been associated with the presence of genital warts There are many other low risk HPV types that are not associated with genital warts or cervical cancer Until now HPV cannot be cultured in vitro and immunological tests are inadequate to determine the presence of HPV cervical infection On the other hand biopsies can be analyzed by nucleic acid hybridization to directly detect the presence of HPV DNA Principle of the Test Norgen s Urine Based HPV 6 16 PCR Detection Kit constituents a ready to use system for the isolation and detection of high risk HPV 16 and low risk HPV 6 using end point PCR The Urine Based HPV 6 16 PCR Detection Kit can also differentiate between the high risk HPV type 16 and low risk HPV type 6 The
8. Product Urine DNA Isolation Kit 18100 Urine Exfoliated Cell DNA Purification Kit 22300 Urine Exfoliated Cell RNA Purification Kit 22500 Urine Bacteria DNA Purification Kit 22400 Urine Bacteria RNA Purification Kit 23400 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 based HPV 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 References Morimoto M Yanai H Chiba H Matsuno K and Shukuya K 2003 Importance of midstream clean catch technique for urinalysis reconfirmed by urinary flow cytometry Clin Chim
9. esponds to the 280 bp band represented by the provided DNA Marker M The size of the HPV 6 16 Isolation Control IsoC corresponds to the 500 bp band represented by the provided DNA Marker M The 2X HPV 6 16 PCR Master Mix contains a HPV 6 16 PCR Control PCRC The HPV 6 16 PCRC Controls for PCR inhibition The size of the HPV 6 16 PCRC corresponds to the 150 bp band represented by the provided DNA Marker M Lanes 1 6 represents samples spiked with different HPV 16 concentrations isolated from 5 mL urine samples whereas Lanes A F represents samples spiked with different HPV 6 concentrations isolated from 5mL urine samples NegC corresponds to the Negative control whereas PosC corresponds to the positive control The HPV spiked in urine samples is a cloned PCR product Table 4 Interpretation of PCR Assay Results Input Type HPV 6 16 IsoC HPV 16 HPV 6 Target HPV 6 16 Interpretation Band 500 bp Target Band Band 280 PCRC Band 391 bp bp 150 bp Positive Control X X X X Valid Negative Control X Valid Sample X X X X Positive Sample X Negative Sample X Positive Sample X X X Positive Sample X xX Positive For results obtained that are not covered in Table 4 above please refer to the Troubleshooting Section E Specificity The specificity of Norgen s Urine Based HPV 6 16 PCR Detection Kit is first and foremost ensured by the selection of the HPV specific primers as well as the selection
10. eted if only the HPV 6 16 PCR control and the HPV 6 16 solation Control IsoC showed amplification e The sample tested can be considered negative 8 Can I process a different urine volume e The reagents provided with the isolation kit are only sufficient to process 24 urine samples of 5mL 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 both the Proteinase K and the Pronase will be reduced This will result in a reduction in your DNA yields 11 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 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 HPV 6 16 Isolation control during the Isolation e The Isolation must be repeated Related Products
11. 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 Urine Based HPV 6 16 PCR Detection Kit the HPV 6 16 2x PCR Master Mix the HPV 6 16 Isolation Control lsoC and the HPV 6 16 Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Based HPV 6 16 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 information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com The Binding Solution Binding Solution Il Wash Solution and Wash Solution Il contain guanidine hydrochloride and should be handled with care
12. ive 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 HPV 6 16 PCR control nor the HPV 6 16 Isolation Control IsoC amplifies e f neither the HPV 6 16 PCR control nor the HPV 6 16 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 HPV 6 16 PCR control showed amplification but neither the HPV 6 16 target nor the HPV 6 16 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 HPV 6 16 Isolation Control IsoC was amplified in a sample e The sample tested can be considered as HPV 6 16 negative 5 How should it be interpreted if only the HPV 6 16 target and the HPV 6 16 PCR control were amplified in a sample e The sample tested can be considered as HPV 6 16 positive 6 How should it be interpreted if only the HPV 6 16 target was amplified in a sample e The sample tested can be considered positive At high HPV 6 16 viral load the HPV 6 16 amplicon will be predominant and the HPV 6 16 PCR control as well as the HPV 6 16 Isolation control may not amplify 7 How should it be interpr
13. kit first allows for the isolation of total DNA including viral DNA from the urine samples using spin column chromatography based on Norgen s proprietary resin The viral DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for the detection and the differentiation between HPV 6 and HPV 16 using the provided HPV 6 16 Master Mix The HPV 6 16 Master Mix contains reagents and enzymes for the specific amplification of either a 391 bp region of high risk HPV 16 and or a 280 bp region of low risk HPV 6 In addition Norgen s Urine Based HPV 6 16 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 HPV 6 16 Isolation Control IsoC or the PCR control PCRC does not reduce the detection limit of the analytical HPV 6 16 PCR The kit is designed to allow for the testing of 24 samples Kit Components Component Contents Binding Solution 20 mL Proteinase K 0 6 mL Pronase 0 6 mL Binding Solution II 3 mL Wash Solution 4mL Wash Solution II 12 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 1 mL Product Insert 1 The positive control is a cloned HPV6 and HPV16 products The isolation control is a cloned PCR product C
14. nal instructions for the transport of pathogen material B Isolation of DNA from Urine Notes Do not spin down or filter the urine sample before proceeding with the isolation as this could negatively affect the isolation of HPV 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 both the Proteinase K and the Pronase before use Preheat an incubator or heating block to 60 C Prepare a working concentration of Binding Solution Il and Wash Solution by adding the proper volume of 96 100 ethanol provided by the user indicated in Table 1 below to the supplied bottle containing the concentrated Binding Solution Il and Wash Solution I The label on the bottle has a box that may be checked to indicate that the ethanol has been added Elevated levels of bilirubin 215 mg dl and lipids 2800 mg dl and haemolytic samples do not influence the system Table 1 Volume of Ethanol to be added to Binding Buffer Il and Wash Buffer I Binding Solution I 10 mL Volume Provided Ethanol 96 100 Volume to Add Final Volume Wash Solution 15 mL 10 11 12 e An HPV 6 16 Isolation Control IsoC is supplied This allows the user to control the DNA isolation procedure For this assay add the HPV 6 16 Isolation Control IsoC to the lysate during the isolation procedure o The HPV 6 1
15. ustomer Supplied Reagents and Equipment e Disposable powder free gloves Centrifuge with a swinging bucket rotor capable of 2000 RPM Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR tubes 96 100 ethanol 60 C incubator 15 mL 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 Urine Based HPV 6 16 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 HPV 6 16 2x PCR Master Mix the HPV 6 16 Isolation Control IsoC the HPV 6 16 Positive Control PosC and the Nuclease Free Water 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 while using the kit e Use sterile pipette tips with filters e Store and extract positive material specimens controls and amplicons separately
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