Urine-Based HSV-1&2 PCR Detection Kit - Protocol
Contents
1. HSV 1 amp 2 PCR control nor the HSV 1 amp 2 Isolation Control amplifies e If neither the HSV 1 amp 2 PCR control nor the HSV 1 amp 2 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 HSV 1 amp 2 PCR conirol showed amplification but neither the HSV 1 or HSV 2 target nor the HSV 1 amp 2 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 HSV 1 amp 2 Isolation Control IsoC was amplified in a sample e The sample tested can be considered as HSV 1 or HSV 2 negative 5 How should it be interpreted if only the HSV 1 or HSV 2 target and the HSV 1 amp 2 PCR control were amplified in a sample e The sample tested can be considered as HSV 1 or HSV 2 positive 6 How should it be interpreted if only the HSV 1 target was amplified in a sample e The sample tested can be considered positive At high HSV 1 viral load the HSV 1 amplicon will be predominant and the HSV 1 amp 2 PCR control as well as the HSV 1 amp 2 Isolation control may not amplify 7 How should it be interpreted if only the HSV 2 target was amplified in a sample e The sample tested can be considered posi2. 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 HSV 1 amp 2 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 HSV 1 amp 2 2x PCR Master Mix the HSV 1 amp 2 Isolation Control IsoC the HSV 1 amp 2 Positive Control PosC and the HSV 1 amp 2 Negative Control NegC 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 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
3. 0 x g 14 000 RPM C HSV 1 amp 2 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 the HSV 1 amp 2 Limit of Detection e HSV 1 amp 2 Negative Control NegC and HSV 1 amp 2 Positive Control PosC must be included during every run e The HSV 1 amp 2 Negative Control NegC and HSV 1 amp 2 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 HSV 1 amp 2 Negative Control NegC and HSV 1 amp 2 Positive Control PosC must be included in every run Pipette 10 uL of HSV 1 amp 2 Negative Control NegC into a PCR tube and add 10 uL of Master Mix Pipette 10 uL of HSV 1 amp 2 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 Prepa
4. 4 Dx 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 Urine Based HSV 1 amp 2 PCR Detection Kit Product Insert Product 31700 Herpes Simplex Virus 1 and 2 HSV 1 amp 2 are both members of the herpes virus family Herpesviridae HSV 1 and HSV 2 have relatively large double stranded DNA genomes HSVs are primarily transmitted by sexual intercourse direct contact with lesions or perinatally Most HSV positive cases are characterised by lesions on the skins and mucous membranes of the mouth and genitals HSV infection can be either primary or a recurrence of a previous infection More than 90 of the primary HSV infections are asymptomatic Primary infection with HSV 1 can lead to gingivostomatitis eczema herpeticum keratoconjunctivitis and encephalitis The primary symptoms of a secondary infection are skin lesions in the nose mouth and genital regions The infection is contagious mainly during an epidemic Principle of the Test Norgen s Urine Based HSV 1 amp 2 PCR Detection Kit constituents a ready to use system for the isolation detection and differentiation of HSV 1 and HSV 2 using end point PCR The 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 inhibi
5. N 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 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 PI31700 5
6. 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 HSV 1 amp 2 PCR Detection Kit the HSV 1 amp 2 2x PCR Master Mix the HSV 1 amp 2 Isolation Control IsoC the HSV 1 amp 2 Negative Control NegC and the HSV 1 amp 2 Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine based HSV 1 amp 2 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 Guanidine hydrochloride forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If li
7. ere with your down stream applications 13 What If I forgot to add the HSV 1 amp 2 Isolation control during the Isolation e The Isolation must be repeated Related Products 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 Urine Based HSV 1 PCR Detection Kit 32600 Urine Based HSV 2 PCR Detection Kit 32400 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 HSV 2 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 NORGE
8. ng with the isolation as this could negatively affect the isolation of viral 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 e An HSV 1 amp 2 Isolation Control IsoC is supplied This allows the user to control the DNA isolation procedure For this assay add the HSV 1 amp 2 Isolation Control IsoC to the lysate during the isolation procedure o The HSV 1 amp 2 Isolation Control lsoC must not be added to the sample material directly o Do not freeze and thaw the HSV 1 amp 2 Isolation Control lsoC more than 2 time
9. quid 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 national instructions for the transport of pathogen material B Isolation of DNA from Urine Notes e Do not spin down or filter the urine sample before proceedi
10. ration Preparation of PCR assay Volume Per PCR Reaction HSV 1 amp 2 2X PCR Master Mix 10 uL 10 uL 10 uL Sample Eluted DNA 10 uL HSV 18 amp 2 Positive Control PosC 10 uL HSV 1 amp 2 Negative Control NegC 10 uL Total Volume 20 uL 20 uL 20 uL Table 3 HSV 1 amp 2 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 HSV 1 amp 2 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 M A B C D E F GWNegCM 1 2 3 4 5 6 7 NegCM 2000 1500 1000 750 Isolation Control IsoC 500 HSV2 Target 300 HASV1Target PCR Control 150 PCRC 50 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of HSV 1 and HSV 2 at different concentrations Target The size of the HSV 1 target amplicon corresponds to the 275bp band represented by the provided DNA Marker M The size of the HSV 2 target amplicon corresponds to the 350bp band represen
11. rimers were checked for possible homologies in GenBank published sequences by sequence comparison analyses Furthermore the specificity of the HSV 1 and HSV 2 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 HSV 1 amp 2 PCR Detection Kit was determined by analyzing a dilution series of HSV 1 and HSV 2 quantitative standard ranging from 8 46 x 10 VP ul to 1 x 10 IU ul e Each dilution has been tested in replicates n 4 using Norgen s Urine Based HSV 1 amp 2 PCR Detection Kit on 1X TAE 1 7 Agarose gels e The linear range of Norgen s Urine Based HSV 1 amp 2 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 HSV 1 amp 2 PCR detection Kit covers a linear range from 200VP mL urine to at least 8 x 10 VP mL urine G Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Urine Based HSV 1 amp 2 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
12. s o The HSV 1 amp 2 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 I to the urine sample 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 x g 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 260uL Binding Solution Il Add 15 uL HSV 1 amp 2 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 6 700 x g and discard the flow through Apply 450 uL of Wash Solution I to the column and centrifuge for 1 minute 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 Discard the flow through and reassemble the spin column with its collection tube Spin the column for 1 minute 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 200 x g 2 000 RPM followed by 1 minute at 14 00
13. ted by the provided DNA Marker M The size of the HSV 1 amp 2 Isolation Control IsoC corresponds to the 500bp band represented by the provided DNA Marker M The HSV 1 amp 2 2X PCR Master Mix contains a HSV 1 amp 2 PCR Control PCRC The HSV 1 amp 2 PCRC Controls for PCR inhibition The size of the HSV 1 amp 2 PCRC corresponds to the 150bp band represented by the provided DNA Marker M Lanes A G represents samples spiked with different HSV 1 concentrations isolated from 5mL urine samples interpreted as positive results Lanes 1 7 represents samples spiked with different HSV 2 concentrations isolated from 5mL urine samples interpreted as positive results The HSV 1 and HSV 2 spiked in urine samples is a cloned PCR product Table 4 Interpretation of PCR Assay Results Input Type HSV 1 amp 2 IsoC HSV 1 275 HSV 1 amp 2 PCRC Interpretation Band 500 bp bp or HSV 2 Band 150 bp 350 bp Target Band Positive Control X X X Valid Negative Control xX Valid Sample X X X Positive Sample X Negative Sample xX Positive Sample X X Positive Sample X 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 HSV 1 amp 2 PCR Detection Kit is first and foremost ensured by the selection of the HSV 1 and HSV 2 specific primers as well as the selection of stringent reaction conditions The p
14. tive At high HSV 2 viral load the HSV 2 amplicon will be predominant and the HSV 1 amp 2 PCR control as well as the HSV 1 amp 2 Isolation control may not amplify 8 How should it be interpreted if only the HSV 1 amp 2 PCR control and the HSV 1 amp 2 Isolation Control IsoC showed amplification e The sample tested can be considered negative 9 Can 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 interf
15. tors and can then be used as the template in a PCR reaction for HSV 1 and HSV 2 detection using the provided HSV 1 amp 2 Master Mix The HSV 1 amp 2 Master Mix contain reagents and enzymes for the specific amplification of a 275bp region of HSV 1 and 350 bp region of HSV 2 In addition Norgen s Urine Based HSV 1 amp 2 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 HSV 1 amp 2 Isolation Control lsoC or the PCR control PCRC does not reduce the detection limit of the analytical HSV 1 or HSV 2 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 HSV 1 amp 2 Negative Control NegC 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert The positive control is a cloned HSV 1 and HSV 2 product The isolation control is a cloned PCR product Customer Supplied Reagents and Equipment e Disposable powder free gloves Centrifuge with a swinging bucket rotor capable of 2000 x g Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR tubes 96 100 ethanol 60 C
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