Influenza Virus A&B Real Time RT-PCR Kit User Manual
Contents
1. Liferiver Revision No ZJ0008 Issue Date Jul 1 2012 C Influenza Virus A amp B Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C s RR 0097 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 pec rer Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net wal Shanghai 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 Influenza virus A amp B Real Time RT PCR Kit is used for the detection of Influenza virus A amp B virus in nasal and pharyngeal secretions by 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 pr2. Result Analysis
3. action tubes 4 Perform the following protocol in the instrument 45 C for 10min 95 C for 15min 95 C for 15sec 60 C for 1min Fluorescence measured at 60 C Selection of fluorescence channels FAM HEX VIC JOE Cal Red 610 ROX TEXAS RED IC 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 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 otherw the sample results is invalid HEX VICIJOE Cal Red 610 ROX TEXAS RED Molecular Grade Water a UNDET 35 Positive Control qualitative assay 1 lt 35 o lt 35 QS quantitative detection Correlation coefficient of QS curve lt 13 Data Analysis and Interpretation The following sample results are possible Ct value Cal Red 610 UNDET UNDET 25 35 Below the detection limit or negative and the software displays the quantitative value and the software displays the quantitative value 43 45 25 35 35 Re test If it is still 38 40 report as 1 UNDET E PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn A5cycles
4. ke 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 etiologic agents 9 Procedure 9 1 RNA Extraction Different brand RNA extraction kits are available You may use your own extraction systems the commercial kit based on the yield For the RNA extraction please comply with manufacturer s instructions The recommended Extraction kit is as follows 9 2 Internal Control It is necessary to add internal control IC in the reaction mix Internal Control IC allows the user determine and control the possibility of PCR inhibition Add the internal control IC lul rxn and the result will be shown in the Cal Red 610 ROX TEX RED 9 3 Quantitation The kit can be used for quantitative or qualitative real time RT PCR A positive control defined 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follow Molecular Grade Water is used for dilution Dilution is not needed for qualitative real time PCR detection Take positive control 1x10 copies ml as the starting high standard in the first tube Respectiv pipette 36ul Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul Aul 4
5. l FAM and influenza virus B fragment is performed in HEX VIC JOE with the fluorescent quencher BHQI1 In addition the kit contains a system to identify possible PCR inhibition by measuring the Cal Red 610 ROX TEXAS RED fluorescence of the internal control IC An external positive control 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents IFV A amp IFVB Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28 ul Molecular Grade Water 1 vial 400u1 Internal Control 1 vial 30u1 IFVA amp IFVB Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1X 10 copies ml LOQ 2X10 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 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 Requi
6. oportional 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 Influenza is a viral infection of the lungs characterized by fever cough and severe muscle aches In the elderly and infirm it is a major cause of disability and death often as a result of secondary infection of the lungs by bacteria Major outbreaks of influenza are associated with influenza virus type A or B Infection with type B influenza is usually milder than type A Type C virus is associated with minor symptoms Influenza virus A amp B real time RT PCR kit contains a specific ready to use system for the detection of the Influenza virus A amp B virus by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains a Super Mix for the specific amplification of Influenza virus A amp B virus RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Influenza virus A amp B virus 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 influenza virus A fragment is performed in channe
7. red 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 5 ul 1000 ul 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 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 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 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 smo
8. ul VY WV V F 1X107 1X108 1X10 1X 104 copiesmi To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 1x10 copies ml contains high concentration of the target DNA Therefore b 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 ul 1ul Super Mix Enzyme Mix Internal Control Syl 20ul Extraction RNA Master Mix Reaction Plate Tube PCR Instrument PCR system without Cal Red 610 ROX TEXAS RED channel may be treated with 1 Molecular Grade Water instead of 11 IC 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of sampl which includes the number of controls standards and sample prepared Molecular Grade Wa is used as the negative control For reasons of unprecise pipetting always add an extra virt 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 react plate tubes Separately add 5ul RNA sample positive and negative controls to different reacti 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 re
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