Urine-Based HIV RT-PCR Detection Kit - Protocol
Contents
1. T cells macrophages and dendritic cells HIV progresses to AIDS at a variable rate affected by viral host and environmental factors HIV specific treatment delays this process Most will progress to AIDS within 10 years of HIV infection some will have progressed much sooner and some will take much longer Treatment with anti retrovirals increases the life expectancy of people infected with HIV Even after HIV has progressed to diagnosable AIDS the average survival time with antiretroviral therapy was estimated to be more than 5 years as of 2005 Without antiretroviral therapy someone who has AIDS typically dies within a year Principle of the Test Norgen s Urine Based HIV RT PCR Detection Kit constituents a ready to use system for the isolation and detection of HIV viral RNA using end point RT PCR The kit first allows for the isolation of total RNA including viral RNA from the urine samples using spin column chromatography based on Norgen s proprietary resin The viral RNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for HIV detection using the provided HIV Master Mix The HIV Master Mix contains reagents and enzymes for the specific amplification of a 297 bp region of HIV In addition Norgen s Urine Based HIV RT 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 HIV2. procedure The Isolation Control IsoC 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 The RT PCR components of the PPV RT PCR Detection Kit should remain at 200C until RNA is extracted and ready for RT PCR amplification It is important to work quickly during this procedure Obtain a 10 mL midstream urine sample Add 10 mL of RNA Lysis Solution directly to the urine Lyse cells by vortexing for 15 seconds Note RNA Lysis Solution contains resin and must be mixed well before every pipeting Add 10 mL of 96 100 ethanol provided by the user to the lysate Mix by vortexing for 10 seconds Add 10 uL of Isolation Control IsoC to the lysate Vortex for 10 seconds Centrifuge for 5 minutes at 2 000 RPM Discard the supernatant Add 500 uL RNA Wash Solution mix well by pipeting and then transfer the entire contents 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 Apply 500 uL of RNA 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 Repeat Step 6 Spin the column empty for 3 minutes at 14 000 RPM Discard the collection tube Transfer the spin column to a fresh
3. buffers is spilt clean with suitable laboratory 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 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 Itis 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 RNA from Urine Notes e awn gt oN e Do not spin down or filter the urine sample before proceeding
4. moving to the amplification detection area s RT PCR and gel electrophoresis 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 Supplies and equipment used for specimen preparation should not be used for pipetting or processing amplified RNA or other sources of target nucleic acids All amplification supplies and equipment should be kept in the amplification detection area at all times 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 e Do not substitute or mix reagents from different kit lots or from other manufacturers e Do not interchange reagent tube bottle caps as this may lead to contamination and compromise test results e Only use the protocol provided in this insert Alterations to the protocol and deviations from the times and temperatures specified may lead to erroneous results Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Urine Based
5. provided 2 The RT PCR products should be resolved on the 1X TAE 2 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Samples results are provided below Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of HIV at different concentrations The size of the HIV target amplicon corresponds to the 284 bp band represented by the provided DNA Marker M Isolation control PCR control gt S 2 gt L A E E E B d Figure 2 A representative 1X TAE 1 5 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X RT PCR 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 6 showed detection of both Isolation Control and PCR Control suggesting that the RNA isolation as well as the RT PCR reaction was successful Lane 7 and 8 showed only the detection of PCR Control suggesting that while the RT PCR was successful the isolation failed to recover even the spiked in Isolation control Table 5 Possible outcomes for the interpretation of PCR assay results Input Type Target reaction Control Reaction HIV Target Band IsoC band PCRC Band Interpretation 284 bp 499 bp 150 bp Positive Control X X X Valid Neg
6. with the isolation as this could negatively affect the isolation of HIV viral RNA e All centrifugation steps are performed at room temperature e A variable speed centrifuge 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 e Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of the RNA Wash Solution by adding 66 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated RNA Wash Solution This will give a final volume of 90 mL The bottle label contains a box to check to indicate that the ethanol has been added e Add 10 uL of B mercaptoethanol provided by the user to each 1 mL of RNA Lysis Solution required B mercaptoethanol is toxic and should be dispensed in a fume hood If precipitates are present in the RNA Lysis Solution it is highly recommended to warm up the RNA Lysis Solution at 60 C for 20 minutes and mix well until the solution becomes clear again It is important to work quickly during this procedure Elevated levels of bilirubin 215 mg dl and lipids 2800 mg dl and haemolytic samples do not influence the system Isolation Control IsoC An Isolation Control IsoC is supplied This allows the user to control the RNA isolation procedure For this assay add the Isolation Control IsoC to the lysate during the isolation
7. 1 7 mL Elution tube Apply 100 uL of RNA Elution Solution to the column and centrifuge for 2 minutes at 2 000 RPM followed by 3 minute at 14 000 RPM C HIV RT PCR Assay Preparation Notes Before use suitable amounts of all RT PCR components should be completely thawed at room temperature gently vortexed and centrifuged briefly The amount of HIV 2X RT PCR Master Mix provided is enough for up to 32 RT PCR reactions 24 sample RT PCR 4 positive control RT PCR and 4 no template control RT PCR For each sample one RT PCR reaction using the HIV 2X Detection RT PCR Mastermix and one RT PCR reaction using Control 2X RT PCR Mastermix should be set up in order to have a proper interpretation of the results For every RT PCR run one reaction containing HIV Positive Control HIV PosC and one reaction as no template control Nuclease Free Water must be included for proper interpretation of results The recommended minimum number of RNA samples tested per RT PCR run is 6 Using a lower volume from the sample than recommended may affect the sensitivity of PPV Limit of Detection 1 Prepare the RT PCR reaction for sample detection Set 1 using HIV 2X Detection RT PCR Mastermix and the RT PCR reaction for control detection Set 2 using Control 2X RT PCR Mastermix as shown in Table 1 below The recommended amount of sample RNA to be used is 1 2 uL Ensure that one HIV detection reaction and one control reaction is prepared for each RNA sampl
8. HIV RT PCR Detection Kit the HIV 2x RT PCR Mastermix Control 2X RT PCR Mastermix Isolation Control IsoC and the HIV Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Based HIV RT 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 Biosafety level 2 practices are recommended for works involving clinical samples from patients who are suspected of having HIV infection Ensure the appropriate containment equipment and facilities are used for activities involving cultures or potentially infectious clinical materials 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 guanidine salts and should be handled with care Guanidine salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these
9. Isolation Control IsoC or the PCR control PCRC does not reduce the detection limit of the analytical HIV PCR The kit is designed to allow for the testing of 24 samples Kit Components Component Contents RNA Lysis Solution 3 x90 mL RNA Wash Solution 24 mL RNA Elution Solution 6 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 IsoC Isolation Control PosC Positive Control The positive control is an in vitro transcribed HIV RNA fragments The isolation control is a cloned PCR product Customer Supplied Reagents and Equipment Disposable Latex gloves Centrifuge with a swinging bucket rotor capable of 2000 x g Benchtop microcentrifuge Micropipettes with an accuracy range between 1 10 uL 10 100 uL and 100 1000 uL Laminar flow hood for extractions Vortex Sterile nuclease free aerosol barrier micropipettor tips Microcentrifuge tube rack PCR tubes 96 100 ethanol B mercaptoethanol 50 mL conical tubes Storage Conditions and Product Stability The Positive Control HIV PosC red cap and Isolation Control IsoC orange cap should be stored at 70 C If needed make aliquots of the controls according to the volume used in the protocol 10 uL of HIV PosC or 10 uL of HIV IsoC prior to freezing The HIV 2X RT PCR Mastermix green cap and the Control 2X RT PCR Ma
10. ative Control X Valid Sample X X Positive Sample X Negative Sample X Re Test Sample Re Test Sample X Negative Sample X Positive Sample X Positive Sample Re Test For results obtained that are not covered in Table 3 above please refer to the Troubleshooting Section E Specificity The specificity of Norgen s Urine Based HIV RT PCR Detection Kit is first and foremost ensured by the selection of the HIV 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 Furthermore the specificity of the HIV specific primers were tested against most of the known sexually transmitted pathogens F Linear Range e The linear range of Norgen s Urine Based HIV RT PCR Detection Kit was determined by analyzing a dilution series of HIV 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 HIV RT PCR Detection Kit on 1X TAE 1 7 Agarose gels e The linear range of Norgen s Urine Based HIV RT PCR Detection Kit has been determined to cover concentrations from 10 VP ul to at least 8 x 10 VP ul e Under the conditions of Norgen s Urine RNA Isolation procedure Norgen s Urine Based HIV RT PCR detection Kit covers a linear range from 1000VP mL urine to at least 8 x 10 VP mL urine G Frequently Asked Q
11. ction 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 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 P133700 5
12. e Adjust the final volume of the RT PCR reaction to 20 uL using the Nuclease Free Water provided Table 1 RT PCR Assay Preparation RT PCR Components Volume Per RT PCR Reaction Sample RNA 2 uL Nuclease Free Water 8 uL Total Volume 20 uL 2 For every RT PCR run prepare one positive control RT PCR as shown in Table 2 below Table 2 RT PCR Negative Control Preparation RT PCR Components Volume Per RT PCR Reaction Total Volume 20 uL 3 For every RT PCR run prepare one no template control RT PCR as shown in Table 3 below Table 3 RT PCR Positive Control Preparation RT PCR Components Volume Per RT PCR Reaction Nuclease Free Water 10 uL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate RT PCR reactions D One Step HIV RT PCR Assay Programming 1 Program the thermocycler according to the program shown in Table 4 below 2 Run RT PCR Table 4 HIV RT PCR Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 50 C 30 min Cycle 2 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 3 40x Step 2 64 C 30 sec Step 3 72 C 45 sec Cycle 4 Step 1 72 C 5 min Cycle 5 Step 1 4 C 20 E HIV One Step RT PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 20 uL RT PCR reaction should be loaded on a 1X TAE 2 Agarose DNA gel along with 10 uL of Norgen s DNA Marker
13. 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 HIV RT PCR Detection Kit Product 33700 Product Insert Human immunodeficiency virus HIV is a lentivirus a member of the retrovirus family that causes acquired immunodeficiency syndrome AIDS a condition in humans in which the immune system begins to fail leading to life threatening opportunistic infections Infection with HIV occurs by the transfer of blood semen vaginal fluid pre ejaculate or breast milk Within these bodily fluids HIV is present as both free virus particles and virus within infected immune cells The four major routes of transmission are unsafe sex contaminated needles breast milk and transmission from an infected mother to her baby at birth vertical transmission Screening of blood products for HIV has largely eliminated transmission through blood transfusions or infected blood products in the developed world HIV infection in humans is considered pandemic by the World Health Organization WHO From its discovery in 1981 to 2006 AIDS has killed more than 25 million people HIV infects about 0 6 of the world s population In 2005 alone AIDS claimed an estimated 2 4 3 3 million lives of which more than 570 000 were children HIV infects primarily vital cells in the human immune system such as helper T cells CD4
14. mple e The sample tested can be considered positive At high HIV viral load the HIV amplicon will be predominant and the HIV PCR control as well as the HIV Isolation control may not amplify 7 How should it be interpreted if only the HIV PCR control and the HIV Isolation Control IsoC showed amplification e The sample tested can be considered negative 8 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 RNA isolation e Adding less volume may reduce your RNA yields Adding more may not affect the RNA yields EXCEPT if more Elution Buffer was added Eluting RNA in higher volumes of Elution Buffer will result in diluting your RNA 10 What If forgot to do a dry spin after my second wash e Your RNA elution will be contaminated with the Wash Solution This may dilute the RNA yield in your elution and it may interfere with your down stream applications 11 What If forgot to add the HIV Isolation control during the Isolation The Isolation must be repeated 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 HIV PCR Dete
15. stermix yellow cap should be stored at 20 C Make appropriate aliquots if needed All other kit components may be stored at room temperature The HIV 2X RT PCR Mastermix the Control 2X RT PCR Mastermix the Control 2X RT PCR Mastermix and Isolation Control IsoC should not undergo repeated freeze thaw a maximum freeze thaw of three times Do not use kit or reagents past their expiration dates Allow reagents to thaw at room temperature prior to use Kit reagents are stable through the end of the expiration month indicated on the packaging label when stored at the recommended temperatures For RT PCR o Allow reagents to thaw at room temperature prior to use o When thawed mix the components and centrifuge briefly o Work quickly on ice o After addition of RT PCR Mastermix use within one hour General 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 Workflow in the laboratory should proceed in a uni directional manner beginning in the pre amplification area s i e specimen collection and RNA extraction and
16. uestions 1 How many samples should be included per PCR run e Norgen s Urine Based HIV RT 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 HIV PCR control nor the HIV Isolation Control IsoC amplifies e If neither the HIV PCR control nor the HIV 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 HIV PCR control showed amplification but neither the HIV target nor the HIV 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 HIV Isolation Control IsoC was amplified in a sample e The sample tested can be considered as HIV negative 5 How should it be interpreted if only the HIV target and the HIV PCR control were amplified in a sample e The sample tested can be considered as HIV positive 6 How should it be interpreted if only the HIV target was amplified in a sa
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