Urine-Based HSV-1 PCR Detection Kit - Protocol
Contents
1. e Each dilution has been tested in replicates n 4 using Norgen s Urine based HSV 1 PCR Detection Kit on 1X TAE 1 7 Agarose gels e The linear range of Norgen s Urine based HSV 1 PCR Detection Kit has been determined to cover concentrations from 20 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 PCR Detection Kit covers a linear range from 200VP mL urine to at least 8 x 10 VP mL urine H Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Urine Based HSV 1 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 HSV 1 PCR control nor the HSV 1 Isolation Control IsoC amplifies e If neither the HSV 1 PCR control nor the HSV 1 Isolation Control 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 PCR control showed amplification but neither the HSV 1 target nor the HSV 1 Isolatio2. 8 Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube 9 Repeat Step 8 to wash column second time 10 Wash the column a third time by adding another 400 uL of Wash Solution to the column and centrifuge for 1 minute Discard the flow through and reassemble the spin column with its collection tube 11 Spin the column for 2 minutes empty at 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 12 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 HSV 1 PCR Assay Preparation Notes e Before use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly e The amount of HSV 1 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 e For each sample one PCR reaction using the HSV 1 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 e For every PCR run one reaction containing HSV 1 Positive Control and one reaction as no template control must be included for proper interpretation of results e The recomme
3. 1 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 E HSV 1 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 A B C D E F G N amp C 2000 1500 1000 750 500 300 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of HSV 1 at different concentrations The size of the HSV 1 target amplicon corresponds to the 255 bp band represented by the provided DNA Marker M Lanes A G represents samples spiked with different HSV 1 concentrations 6 NTC 4 5 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 suggesti
4. 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 Do not spin down or filter the urine sample before proceeding with the isolation as this could negatively affect the isolation of HSV 1 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 e Prepare a working concentration of Wash Solution by adding 21 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give you a final volume of 30 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Isolation Control IsoC e An Isolation Control IsoC
5. 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 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 2014 Norgen Biotek Corp PI32600 6
6. HSV 1 detection using the provided HSV 1 Master Mix The HSV 1 Master Mix contains reagents and enzymes for the specific amplification of a 255 bp region of the HSV 1 In addition Norgen s Urine Based HSV 1 PCR Detection Kit contains a second Master Mix 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 PCR Control PCRC or the Isolation Control IsoC respectively The amplification and detection of either the HSV 1 Isolation Control IsoC or the PCR control PC does not reduce the detection limit of the analytical HSV 1 PCR The kit is designed to allow for the testing of 24 samples Kit Components Component Contents Solution A 10 mL Solution B 15 mL Wash Solution 9mL 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 IsoC Isolation Control PosC Positive Control The positive control is a cloned HSV 1 PCR 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 RPM Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR tubes 96 100 ethanol 60 C incubator 15 mL tubes Storage Cond
7. g DR NORGEN BIOTEK wie 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 PCR Detection Kit Product 32600 Product Insert Herpes Simplex Virus 1 HSV 1 is a member of the herpes virus family Herpesviridae HSV 1 has a relatively large double stranded DNA genome 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 PCR Detection Kit constituents a ready to use system for the isolation and detection of HSV 1 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 inhibitors and can then be used as the template in a PCR reaction for
8. is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control IsoC as indicated during the isolation procedure e The Isolation Control IsoC must not be added to the sample material directly e Do not freeze and thaw the Isolation Control lsoC more than 2 times e The Isolation Control IsoC must be kept on ice at all times during the isolation procedure e The PCR components of the Urine Based HSV 1 PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification e It is important to work quickly during this procedure Add 300 uL of Solution A to 10 mL urine sample Mix well by vortexing for 10 seconds Note 1 Solution A must be mixed well before every pipetting 2 Centrifuge for 5 minutes at 2 000 RPM then discard the supernatant carefully in order not to dislodge the precipitated slurry pellet 3 Add 500 uL Solution B to the precipitated slurry pellet mix well by vortexing for 10 seconds 4 Add 10 uL Isolation Control IsoC to the mixture from Step 3 5 Add 500 uL of 96 100 Ethanol to the mix from Step 4 mix well by vortexing for 10 seconds 6 Transfer 650 uL from the previous mix into a Mini Filter Spin column and centrifuge for 1 minute at 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 7 Repeat Step 6 until the entire mixture from Step 5 has been transferred to the Mini Filter Spin Column
9. itions 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 The HSV 1 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and HSV 1 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 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 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 HSV 1 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and HSV 1 Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine based HSV 1 PCR Detection Kit is designed for
10. lection Storage and Transport Gen eral Precautions Follow universal precautions All patient specimens should be considered as potentially infectious and handled accordingly Wear personal protective equipment including gloves and lab coats when handling kit reagents Wash hands thoroughly when finished performing the test Do not smoke drink or eat in areas where kit reagents and or human specimens are being used Dispose of unused kit reagents and human specimens according to local provincial or federal regulations 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 Personal protective equipment such as laboratory coats and disposable gloves should be area specific As contamination of patient specimens or reagents can produce erroneous results it is essential to use aseptic techniques Pipette and handle reagents carefully to avoid mixing of the samples Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values Do not substitute or mix reagents from different kit lots or from other manufacturers 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
11. n 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 Isolation Control IsoC was amplified in a sample e The sample tested can be considered as HSV 1 negative 5 How should it be interpreted if only the HSV 1 target and the HSV 1 PCR control were amplified in a sample e The sample tested can be considered as HSV 1 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 PCR control as well as the HSV 1 Isolation control may not amplify 7 How should it be interpreted if only the HSV 1 PCR control and the HSV 1 Isolation 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 varied from the 20 minutes specified in the product manual e Less than 20 minutes will re
12. nded 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 the HSV 1 Limit of Detection 1 Prepare the PCR reaction for sample detection Set 1 using HSV 1 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 HSV 1 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 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 HSV 1 PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 HSV
13. ng 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 HSV 1 IsoC Band PCRC Band Target Band 499bp 150 bp 255 bp Positive xX xX x Valid Control Negative i Control X Valid Sample 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 above please refer to the Troubleshooting Section Ignore any bands that appear between the Isolation Control band and the PCR Control band F Specificity The specificity of Norgen s Urine based HSV 1 PCR Detection Kit is first and foremost ensured by the selection of the HSV 1 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 G Linear Range e The linear range analytical measurement of Norgen s Urine based HSV 1 PCR Detection Kit was determined by analyzing a dilution series of HSV 1 quantitative standard ranging from 8 46 x 10 VP ul to 1 x 10 IU ul
14. research purposes only It is not intended for human or diagnostic use Safety Information This kit is designed for research purposes only It is not intended for human or diagnostic use 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 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 I CAUTION DO NOT add bleach or acidic solutions directly to the sample preparation waste 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 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 parti 1 cular use Protocol A Specimen Collection Storage and Transport Precaution All samples have to be treated as potentially infectious material A Specimen Col
15. sult in lower DNA yields More than 20 minutes may not affect your DNA yields 11 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 12 What If forgot to add the HSV 1 Isolation Control IsoC during the Isolation The isolation must be repeated Related Products Product Urine Based HSV 2 PCR Detection Kit 32400 Urine Based HSV 1 amp 2 PCR Detection Kit 31700 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 1 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
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