HIV-1 Real Time RT-PCR Kit User Manual For In Vitro
Contents
1. 2 amplification detection of amplification products yield For the RNA extraction please comply with the e Pipets vials and other working materials should not circulate manufacturer s instructions The recommended extraction kit is as among working units follows e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area Nucleic Acid Isolation Kit Cat Number e Avoid aerosols RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini 52904 QIAGEN 8 Sample Collection Storage and transport extraction Kit 50 e Collected samples in sterile tubes 9 2 Internal Control e 1 1 1 m o Specimens can be extracted immediately or frozen at 20 C to It is necessary to add internal control IC in the reaction mix Internal 80 C E l control IC allows the user to determine and control the possibility of e Transportation of clinical specimens must comply with local PCR inhibition regulations for the transport of etiologic agents 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18ul 1 ul ul X PCR system without HEX VIC JOE channel may be treated with lul Super Mix Enzyme Mix Internal Control Molecular Grade Water instead of 1ul IC Oa ee 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the Sul 20ul number of samples which includes the number of controls standards and Extraction RNA Master Mix sample prepared2. fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description Human immunodeficiency virus HIV is a retrovirus that can lead to acquired immunodeficiency syndrome AIDS Infection with HIV occurs by the transfer of blood semen vaginal fluid pre ejaculate or breast milk HIV infection in humans is now pandemic As of January 2006 the Joint United Nations Programme on HIV AIDS UNAIDS and the World Health Organization WHO estimate that AIDS has killed more than 25 million people since it was first recognized on December 1 1981 making it one of the most destructive pandemics in recorded history HIV 1 real time RT PCR kit contains a specific ready to use system for HIV 1 detection for sub genotype A H through Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains a Super M
3. Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix in ee completely then spin down briefly in a centrifuge 2 Pipet 20ul Master Mix with micropipets of sterile filter tips to each of the Reaction Real time PCR reaction plate tubes Separately add Sul RNA sample Plate Tube supernatantor QS1 QS2 QS3 QS4 and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the PCR Inst reaction tubes peau 4 Perform the following protocol in the instrument 45 C for 10 min 1 cycle 95 C for 15 min 1 cycle 95 C for 15 sec 60 C for 60sec 40 cycles Fluorescence is measured at 60 C FAM and HEX VIC JOE channels should be chosen 5 Aif you use ABI Prism system please choose none as passive reference and quencher 10 Threshold setting just above the maximum level of molecular grade water 11 Calibration for quantitative detection Input each concentration of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Ct value FAM Target Nucleic Acid HEX VIC JOE IC Molecular Grade Water UNDET 25 35 QS 1 QS2 QS3 QS4 lt 38 and Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible 1 The Ct value in chan
4. ix for the specific amplification of HIV 1 RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the HIV 1 RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction PCR Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified HIV 1 DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 In addition the kit can be used for identification of possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC Four quantitation standards are supplied allows the determination of the gene load 4 Kit Contents __ Ref Cap Color Type of reagent Presentation _25rxns HIV 1 Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28ul 3 Molecular Grade Water 1 vial 400u1 4 Internal Control 1 vial 30ul 5 Pink HIV 1 QS1 5x10 IU ml 1 vial 20ul 6 Purple HIV 1 QS2 5x10 TU ml 1 vial 20ul T Orange HIV 1 QS3 5x10 IU ml 1 vial 20ul 8 Yellow HIV 1 QS4 5x10 TU ml 1 vial 20ul QS Quantitation Standard e Sterile filter tips for micro pipets e Tube racks 5 Storage e Desktop microcentrifuge for eppendorf type tubes RCF max e All reagents should be stored at 20 C Storage at 4 C is not 16 000 x g recommended e All reagents can be used until the e
5. nel FAM shows lt 38 The result is positive The sample contains HIV 1 RNA The sample contains HCV RNA Quantitative value of samples is automatically reported according to the standard curve Quantitative Value Data Analysis and Suggestion lt 1x10 IU ml HIV 1 RNA Positive its concentration lower than 10 IU ml 10 2x10 IU ml HIV 1 RNA Positive the quantitative value for recommendation only 2x10 10 IU ml HIV 1 RNA Positive the quantitative value is valid gt 10 IU ml 1 HIV 1 RNA Positive but the quantitative value for recommendation only 2 Re test the sample after dilute the sample by several times making the quantitative value within 2 X 10 10 IU ml 2 The Ct value in channel FAM shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative 3 In channel FAM no signal is detected at the same time a HEX VIC JOE signal from the Internal Control appears The sample does not contain any HIV 1 RNA It can be considered negative 4 Neither in channel FAM nor in channel HEX VIC JOE is a signal detected A diagnostic statement can not be made Inhibition of the RT PCR reaction For further questions or problems please contact our technical support at trade liferiver com cn
6. oO feri ty Revision No ZJ0007 Issue Date Jul 1 2012 weriver HIV 1 Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only IVD SR 0020 02 A B For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 aed Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P M Option2 Chromo4 LightC ycler 480 Instrument atal Shanghai ZJ Bio Tech Co Ltd 2 25 www liferiver com cn Tel 86 21 34680596 trade liferiver com cn Fax 86 21 34680595 one floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use HIV 1 real time RT PCR kit is used for the detection of HIV 1 in serum or plasma by using real time PCR systems Its characteristics High sensitivity lower detection line 10 IU ml LOQ 210 10 IU ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the RNA extraction kits recommended the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher High specificity test result will be positive only to HIV genotype I for sub genotype A H Short operating time 2 and a half hours totally Good stability kept for 12 months at 20 C CV lt 5 2 Principle of Real Time PCR The principle of the real time detection is based on the
7. xpiration date indicated on the kit label Warnings and Precaution e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Real time PCR reaction tubes plates Vortex mixer e Pipets 0 5 ul 1000 ul e Cryo container e Disposable gloves powderless e Sterile microtubes e Biohazard waste container e Refrigerator and freezer Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly 9 Procedure the tubes before use 9 1 RNA Extraction e Prepare quickly the Reaction mix on ice or in the cooling block Different brand RNA Extraction kits are available You may use e Set up two separate working areas 1 isolation of the RNA DNA your own extraction systems or the commercial kit based on the and
Download Pdf Manuals
Related Search