Rift Valley Fever Virus (RVFV) Real Time RT
Contents
1. Liferiver Revision No ZJO003 Issue Date Jul 1 2012 Rift Valley Fever Virus RVFV Real Time RT PCR Kit User Manual 20 C z y For In Vitro Diagnostic Use Only AR 0116 02 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument rw ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan Road PuJiang Hi tech Park Shanghai China 1 Intended Use Rift Valley Fever Virus RVFV real time RT PCR kit is used for the detection of RVFV in serum sample by using the 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 is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to re open the re2. RT PCR The first step is a reverse transcription RT during which the RVFV RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified RVFV DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC An external positive control defined as 1x10 copies ml is supplied which allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents RVFV Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30ul RVFV Positive Control 1x10 copies ml 1 vial 30u1 Analysis sensitivity 5X 10 copies ml LOQ 1X10 1X 10 copies 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 5 Storage e All reagents should be store
3. action tube after the amplification 3 Product Description Rift valley fever RVF is a viral zoonosis that primarily affects animals but also has the capacity to infect humans Infection can cause severe disease in both animals and humans leading to high rates of disease and death The disease also results in significant economic losses due to death and abortion among RVF infected livestock The vast majority of human infections result from direct or indirect contact with the blood or organs of infected animals The virus can be transmitted to humans through the handling of animal tissue during slaughtering or butchering assisting with animal births conducting veterinary procedures or from the disposal of carcasses or fetuses Certain occupational groups such as herders farmers slaughterhouse workers and veterinarians are therefore at higher risk of infection The virus infects humans through inoculation for example via a wound from an infected knife or through contact with broken skin or through inhalation of aerosols produced during the slaughter of infected animals The aerosol mode of transmission has also led to infection in laboratory workers RVFV real time RT PCR kit contains a specific ready to use system for the detection of RVFV by RT PCR reverse transcription polymerase chain reaction in the real time PCR system The master contains a Super Mix for the specific amplification of the RVFV RNA The reaction is done in one step real time
4. al 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 template positive and negative controls to different 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 for 10min Selection of fluorescence channels j Target Nucleic Acid 95 C for 15sec 60 C for 1min Fluorescence measured at 60 C 5 A If 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 Negative control positive control internal control and QS curve must be performed correctly otherwise the sample results is invalid HEX VIC JOE Ct value po tale O Positive Control qualiaiveasayy 35 PSS QS quantitative detection 13 Data Analysis and Interpretation The following sample results are possible Ct value HEX VIC JOE UNDET 25 35 Below the detection limit or negative 25 Result Analysis Positive and the software displays the quantitativ
5. d 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 Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000u1 e Sterile microtubes 7 A Warnings and Precaution e 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 the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate
6. e value 1 2 Re test if it is still 38 40 report as 1 For further questions or problems please contact our technical support at trade liferiver com cn
7. ve real time PCR standard dilution must be prepared first as follows Molecular Grade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR Take positive control 1x10 copies 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 4 H l 4u l Au l Y WY V Y 1X107 1X10 1X10 1X10fcopies mi To generate a standard curve on the real time system all four dilution standards should be used and defined as standards with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 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 18ul 1 pl 1 ul Super Mix Enzyme Mix Internal Control 5ul 20ul Extraction RNA Master Mix Reaction Plate Tube i PCR Instrument X PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of Il 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 virtu
8. 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 Do not pipette by mouth Do not eat drink smoke in laboratory 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 RNA extraction kits are available from various manufacturers 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 Isolation Kit Cat Number RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini extraction Kit 50 52904 QIAGEN 9 2 Internal Control It is necessary to add internal control IC 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 9 3 Quantitation The kit can be used for quantitative or qualitative real time RT PCR For performance of quantitati
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