DATA SHEET HIV-2 Real Time RT-PCR Kit
Contents
1. 3 GENTAUR DATA SHEET HIV 2 Real Time RT PCR Kit Cat No SR 0021 02 For use with ABI Prism 7000 7300 7500 7900 Smart Cyclerll iCycler iQ 4 iQ 5 Rotor Gene6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480real time PCR systems For In Vitro Diagnostic Use Only User Manual 1 Intended Use HIV 2 real time RT PCR kit is used for the detection of HIV 2 in serum by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the 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 amplificaton 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 in2. Mix 1 vial 28u1 Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30ul HIV 2 Positive Control 1x10 TU ml 1 vial 30ul Analysis sensitivity 52 103IU ml LOQ 18 1048 18 108IU ml 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label 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 Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e RNA extraction kit e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5ul 1000ul e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium 7 Warnings and Precaution 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 b
3. C in the reaction mix Internal Control IC allows the user to determine and control the possibility of PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the HEX VIC JOE Attention It is necessary to dilute the internal control supplied in the kit by 10 times with molecular grade water before detection and close the tube immediately then vortex for 10 seconds Because of transportation with carbon dioxide ice there may be white precipitate in tubes of internal control and positive control but it will disappear in a few minutes when it is incubated at room temperature Besides the white precipitate have no effection on the detection result 9 3 Quantitation The kit can be used for quantitative or qualitative real time RT PCR A positive control defined as 1x10 IU ml is supplied in the kit For performance of quantitative real time PCR standard dilutions must be prepared first as follows Molecular Grade Water is used for dilution The step of dilution is not needed for performance of qualitative real time PCR Take positive control 1x10 IU ml as the starting high standard in the first tube Respectively pipette 36ul of Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards 4ul spi Y V V I 1X107 1X10 1X10 1X104 lUjmi To generate a standard curve on the real time system all four dilution standards should be used and d
4. e 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 the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 isolation of the RNA DNA and 2 amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Avoid aerosols 8 Sample Collection Storage and transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction Different brand RNA Extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For the RNA extraction please comply with the manufacturer s instructions The recommended extraction kit is as follows Nucleic Acid solatio Ki RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini extraction Kit 50 QIAGEN 9 2 Internal Control and Positive Control It is necessary to add internal control I
5. efined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 1x10 IU ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 4 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 181 1 yl 1l Super Mix Enzyme Mix Internal Control 5ul 20ul Extraction RNA Master Mix Reaction Plate Tube PCR Instrument Gentaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium X lt PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of 1 IC 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge 2 Pipet 20ul Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add Sul RNA sample positive 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 reaction tubes 4 Perform the following protocol in the instrument 45 C
6. 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 If you use ABI Prism system please choose none as passive reference and quencher 10 Baseline setting just above the maximum level of molecular grade water 11 Calabration 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 The Ct value of molecular grade water and positive control in FAM channel shows UNDET and lt 35 respectively The Ct value of internal control in HEX VIC JOE channel shows 25 33 Correlation coefficient of standard curve should be lt 0 98 otherwise the result is invalid 13 Data Analysis and Interpretation The following results are possible 1 The Ct value in channel FAM shows lt 38 The result is positive The sample contains HIV 2 RNA 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 2 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 Ge
7. humans is now pandemic Two species of HIV infect humans HIV 1 and HIV 2 HIV 1 is more virulent It is easily transmitted and is the cause of the majority of HIV infections globally HIV 2 is less transmittable and is largely confined to West Africa HIV 2 real time RT PCR kit contains a specific ready to use system for HIV 2 detection through Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains Super Mix for the specific amplification of HIV 2 RNA including subtype A G The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the HIV 2 RNA is transcribed into cDNA Then a thermostable DNA polymerase is used to amplify the specific gene fragments by polymerase chain reaction PCR Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified HIV 2 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 An external positive control defined as 1x10 IU ml is supplied It allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents Ref Type of reagent Presentation 25rxns HIV 2 Super Mix 1 vial 480u1 RT PCR Enzyme
8. ntaur Molecular Products Voortstraat 49 1910 Kampenhout Belgium
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