33740 - Protocol (48 reactions)
Contents
1. e Using a lower volume of sample RNA than recommended may affect the sensitivity of the HIV Limit of Detection e To avoid any contamination while preparing the RT PCR assay follow the order outlined in Tables 1 2 and 3 below to prepare the Negative Control Detection Assay and Positive Control 1 Prepare the RT PCR Negative Control Table 1 2 Prepare the RT PCR HIV Assay Table 2 3 Prepare the RT PCR Positive Control Dilution Series Table 3 4 Prepare the RT PCR Positive Control Assay Table 4 e To further avoid contamination add the components to the PCR tubes in the order shown in the tables below ie 1 Nuclease free water 2 Master Mix 3 Primer Set and 4 the Sample RNA or Positive Control 1 For each RT PCR set prepare one no template control RT PCR as shown in Table 1 below Table 1 RT PCR Negative Control Preparation RT PCR Components Volume Per RT PCR Reaction Nuclease Free Water 8 uL Total Volume 20 uL 2 Prepare the RT PCR reaction for sample detection as shown in Table 2 below The recommended amount of sample RNA to be used is 2 5 uL However a volume between 1 and 5 uL of sample RNA may be used as template Adjust the final volume of the RT PCR reaction to 20 uL using the Nuclease Free Water provided Table 2 RT PCR HIV Assay Preparation RT PCR Components Volume Per RT PCR Reaction Nuclease Free Water 5 5 uL Sample RNA 2 5 uL Total Volume 20 uL2. 3 For each RT PCR set prepare a Positive Control dilution series as shown in Table 3 below Table 3 RT PCR Positive Control Dilution Series Preparation 3 Volume of Nuclease Free Volume of PosC of Different nN Copies par mL Water Concentration 2x 104 Original PosC No dilution Original PosC No dilution Required Required 2x 10 18 uL 2 uL of PosC 2 x 10 2 x 10 18 uL 2 uL of PosC 2 x 10 2x10 18 uL 2 uL of PosC 2 x 10 2x10 18 uL 2 uL of PosC 2 x 10 4 Using the Positive Control dilution series prepared above prepare positive control PCRs as shown in Table 4 below Table 4 RT PCR Positive Controls Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 3 uL Total Volume 20 uL NOTE Set up one reaction for each of the PosC dilution C HIV RT PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 5 below 2 Run one step RT PCR Table 5 HIV Assay Program One Step RT PCR Cycle Step Temperature Duration Cycle 1 Step 1 50 C 25 min Cycle 2 Step 1 95 C 5 min Step 1 94 C 15 sec Cycle 3 40x Step 2 60 C 30 sec Step 3 72 C 45 sec Cycle 4 Step 1 72 C 5 min Cycle 5 Step 1 4 C eo D HIV RT PCR Assay Interpretation e For real time analysis use the analysis software of the thermocycler to generate a standard curve using the Ct values of the Positive Control
3. Circulating RNA and Exosomal Purification Kit Cat 42800 Plasma Serum RNA Purification Kit Cat 55000 If using a different spin column based sample preparation procedure that includes ethanol based wash buffers a column drying step consisting of centrifugation for 10 minutes at 14 000 x g 14 000 RPM using a new collection tube is highly recommended prior to the elution of the RNA This will help to prevent the carry over of any ethanol into the purified RNA as ethanol is known to be a strong inhibitor of PCR Ensure that any traces of ethanol from the sample preparation steps are eliminated prior to the elution of the RNA B RT PCR Assay Preparation Notes Before Use e Before use suitable amounts of all RT PCR components should be completely thawed at room temperature mixed by gentle vortexing or by pipetting and centrifuged briefly e Work quickly on ice e The amount of 2X Real Time RT PCR Master Mix provided is enough for up to 96 RT PCR reactions 48 sample RT PCR 32 positive control standard curve RT PCR and 16 no template control RT PCR e For every RT PCR run one reaction containing the non diluted HIV Positive Control and one reaction as no template control must be included for proper interpretation of results e For quantitative interpretation a dilution series of the positive control RNA should be generated instruction provided below e The recommended minimum number of RNA samples tested per RT PCR run is 6
4. HIV infects primarily vital cells in the human immune system such as helper T cells CD4 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 people will progress to AIDS within 10 years of HIV infection however others will progress much sooner while some will take much longer Treatment with anti retrovirals increases the life expectancy of people infected with HIV In 2013 around 12 9 million people living with HIV 37 of the total had access to antiretroviral therapy Product Description Norgen s HIV Quantitative RT PCR Kit is a research use only diagnostic test for the detection of HIV specific RNA transcripts The kit could be used for quantification of HIV RNA using end point RT PCR gel electrophoresis based or real time RT PCR detected via SYBR Green with the Primer Set and the Master Mix provided with the kit via amplification of a 142 nt region of the HIV RNA genome In addition the kit contains a quantified Positive Control PosC 20 000 copies per uL that can be used for construction of a dilution series for HIV RNA quantification Norgen s HIV Quantitative RT PCR Kit was developed and validated to be used with the following PCR instruments e Qiagen Rotor Gene Q e BioRad iCycler e BioRad CFX96 Touch Kit Components Component Product 33740 48 Samples 2X Real Time
5. 6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2014 Norgen Biotek Corp PI33740 1
6. Dilution Series The standard curve can then be used to determine the starting quantity of the sample of interest e For the analysis of the end point RT 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 provided e The RT 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 Valid Test Run e Positive Sample A sample is determined to be positive only when o Sample lanes shows the 142 bp band corresponding to the HIV target amplicon o Positive Control shows the 142 bp band o Positive Control shows the 142 bp band even it is diluted to as little as 20 copies per uL o Negative Control shows no bands e Negative Sample A sample is determined to be negative only when o Sample lanes contain no bands o Positive Control shows the 142 bp band o Negative Control shows no bands Invalid Test Run e A test run is invalid if o The run has not been completed o Positive Control does not show the 142 bp band o Negative Control shows any amplification M 10 10 105 104 10 102 10 109 Nes mM 2000 1500 1000 750 500 300 150 Figure 1 A representative 1X TAE 2 agarose gel showing the amplification of HIV The size of the HIV target amplicon corresponds to the 142 np bp band represented by the provided DNA Marker M No amplification of the target is obs
7. RT PCR Master Mix 1 mL HIV Primer Set Mix 300 uL HIV Positive Control 100 uL Nuclease Free Water 1 25 mL DNA Ladder 200 uL Product Insert 1 Storage Conditions and Product Stability The HIV Quantitative RT PCR Kit is shipped on dry ice The components of the kit should be frozen upon arrival If one or more of the components is not frozen when the kit is received or if any of the components have been compromised during shipment please contact Norgen Biotek for assistance All kit components should be stored at 20 C upon arrival Repeated thawing and freezing gt 2 x of the Master Mix and Positive Control should be avoided as this may affect the performance of the assay If the reagents are to be used only intermittently they should be frozen in aliquots These reagents should remain stable for at least 1 year when stored at the specified conditions Customer Supplied Reagents and Equipment Appropriate End point or Real Time PCR Instrument RNA Purification Kit o The kit is compatible with all RNA purification kits that yield high quality inhibitor free DNA o Recommended Purification Kit Norgen Biotek s purification kits for RNA isolation including Total RNA Purification Kit Cat 17200 Plasma Serum Circulating RNA and Exosomal Purification Kit Cat 42800 Plasma Serum RNA Purification Kit Cat 55000 Disposable powder free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filte
8. eagents and add it to the reaction mix in a spatially separated facility e Dispose of unused kit reagents and human specimens according to local provincial or federal regulations e Do not substitute or mix reagents from different kit lots or from other manufacturers Do not use components of the kit that have passed their expiration date e The presence of PCR inhibitors may cause false negative or invalid results e Potential mutations within the target regions of the HIV genome covered by the primers in this kit may result in failure to detect the presence of the pathogen e Good laboratory practice is essential for the proper performance of this kit Ensure that the purity of the kit and reactions is maintained at all times and closely monitor all reagents for contamination Do not use any reagents that appear to be contaminated e Ensure that appropriate specimen collection transport storage and processing techniques are followed for optimal performance of this test Instructions for Use A Sample Preparation Purified RNA is the starting material for Norgen s HIV Quantitative RT PCR Kit The quality of the RNA template will have a major impact on the performance of the diagnostic test The user must ensure that the method used for RNA purification is compatible with RT PCR technology We recommend the use of Norgen s purification kits for RNA isolation including Norgen s Total RNA Purification Kit Cat 17200 Plasma Serum
9. erved in with the Negative Control Amplification Figure 2 A representative RT qPCR baseline graph showing the successful amplification of a dilution series of HIV Positive Control E Specificity The specificity of Norgen s HIV Quantitative RT PCR 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 to all GenBank published sequences by sequence comparison analysis The specific detectability of all relevant strains has thus been ensured by a database alignment and by PCR amplification with the following commonly found pathogens Pneumocystis jirovecii Neisseria gonorrhoea Chlamydia trachomatis Norovirus West Nile Virus HIV F Linear Range e The linear range analytical measurement of Norgen s HIV Quantitative RT PCR Kit was determined by analysing a dilution series of a HIV quantification standard ranging from 1 x 10 copes l to 1 x 10 copies l e Each dilution has been tested in replicates n 4 using Norgen s HIV Quantitative RT PCR Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s HIV Quantitative RT PCR Kit has been determined to cover concentrations from 1 x 10 copies ul to at least 1 x 10 copies l of isolated DNA G Technical Support Contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll F
10. fy 3430 Schmon Parkway N O 2 G F N Thorold ON Canada L2V 4Y6 p Phone 905 227 8848 BIOTEK wie CORPORATION Fax 905 227 1061 Email techsupport norgenbiotek com HIV Quantitative RT PCR Detection Kit Product Insert Product 33740 Background Information 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 Since its discovery in 1981 AIDS has infected over 78 million people and 39 million people have died Currently an estimated 0 8 of adults aged 15 49 years worldwide are living with HIV In 2013 alone AIDS claimed an estimated 1 5 million lives and an estimated 2 1 million people were newly infected with HIV
11. ree at 1 866 667 4362 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com Product Use Restriction Good laboratory practice is essential for the proper performance of this kit Ensure that the purity of the kit and reactions is maintained at all times and closely monitor all reagents for contamination Do not use any reagents that appear to be contaminated Ensure that appropriate specimen collection transport storage and processing techniques are followed for optimal performance of this test The presence of PCR inhibitors may cause false negative or invalid results Potential mutations within the target regions of the HIV genome covered by the primers in this kit may result in failure to detect the presence of the pathogen The respective user is liable for any and all damages resulting from application of Norgen s HIV Quantitative RT PCR Kit for use deviating from the intended use as specified in the user manual All products sold by Norgen Biotek are subjected to extensive quality control procedures and are warranted to perform as described when used correctly Any problems should be reported immediately The kit contents are for laboratory use only and they must be stored in the laboratory and must not be used for purposes other than intended The kit contents are unfit for consumption Norgen Biotek Corp 3430 Schmon Parkway Thorold ON Canada L2V 4Y
12. rs PCR tubes Vortex mixer Agarose gel electrophoresis apparatus End Point PCR UV transilluminator with suitable gel documentation system End Point PCR Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s HIV Quantitative RT PCR Kit is tested against predetermined specifications to ensure consistent product quality Warnings and Precautions Follow universal precautions All patient specimens should be considered as potentially infectious and handled accordingly Ensure that a suitable lab coat disposable gloves and protective goggles are worn when handling specimens and kit reagents Use sterile pipette tips with filters Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values 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 e Do not use supplies and equipment across the dedicated areas of i specimen extraction ii reaction set up and iii amplification detection No cross movement should be allowed between the different areas Personal protective equipment such as laboratory coats and disposable gloves should be area specific e Store and extract positive material specimens controls and amplicons separately from all other r
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