Avian Influenza Virus H5N7 Real Time RT
Contents
1. Liferiver Revision No ZJ0001 Issue Date Feb 19 2013 Avian Influenza Virus H5N7 Real Time RT PCR Kit User Manual C s For In Vitro Diagnostic Use Only 20 C RR 0153 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 ner 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 asal 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 Avian influenza virus H5N7 real time RT PCR kit is used for the detection of avian H5N7 virus in human nasal and pharyngeal secretions and bird fece 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 initial2. Ct value l O Ha HEX VIC JOE Result Analysis UNDET 25 35 Below the detection limit or negative 2 lt 38 Positive and the software displays the quantitative value 38 40 25 35 Re test If it is still 38 40 report as 1 UNDET UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn
3. c amplification of Avian influenza virus RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Avian influenza 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 Avian influenza virus 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 1 lt 10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents HSN7 Super Mix 1 vial 480ul RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30ul H5N7 Positive Control 1 x 10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2 X 10 1 X 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 elutio
4. control defined as 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows 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 Respectively pipette 36ul Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul dul 4ul Y WV V F 1X107 1X10 1X10 1X104 copies mi 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 tigi transfer B The positive control 1x10 copies 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 18 1 ul iyl Super Mix Enzyme Mix intemal Control Spl 20pl Extraction RNA Master Mix Reaction Plate Tube PCR Instrument gt lt PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of ll 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 Wat
5. e 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 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 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 Isolation Kit RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech 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 A positive
6. er 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 5ul RNA sample supernatantor 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 for 10min 95 C for 15min 95 C for 15sec 60 C for 1min Adevel Fluorescence measured at 60 C eee 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 Selection of fluorescence channels A Target Nucleic Acid HEX VIC JOE Channel Control 25 35 Positive Contol quahitatveasay 35 O Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible
7. ly 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 Highly pathogenic avian influenza HPAI caused by certain subtypes of influenza A virus in animal populations particularly chickens poses a continuing global human public health risk Direct human infection by an avian influenza A H5N1 virus was first recognized during the 1997 outbreak in Hong Kong Subsequently human infections with avian strains of the H9 and H7 subtypes have been further documented Avian influenza A H5 and H7 viruses can be distinguished as low pathogenic and high pathogenic forms on the basis of genetic features of the virus and the severity of the illness they cause in poultry influenza H9 virus has been identified only in a low pathogenicity form Each of these three avian influenza A viruses H5 H7 and H9 theoretically can be partnered with any one of nine neuraminidase surface proteins thus there are potentially nine different forms of each subtype e g H5N1 HSN2 HSN3 H5N7 H5N9 Avian influenza virus H5N7 real time RT PCR kit contains a specific ready to use system for the detection of the Avian H5N7 virus by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains a Super Mix for the specifi
8. n 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 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 1000p1 e Sterile microtubes T A 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 befor
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