MBS598197 - MyBioSource
Contents
1. Do not eat drink smoke in laboratory e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 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 Cat Number Manufacturer 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 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 1 lt 10 copies ml as2. 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 Ap l Au l 1X107 1X10 1X10 1X 104 copiessmt 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 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 13l 1 yl ipl Super Mix Enzyme Mix Internal Control Syl 154l Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without 560nm channel may be treated with 1u Molecular Grade Water instead of 11 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 20p1 Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 541 RNA sample supernatantor positive and n
3. ample results are possible Result Analysis l UNDET 25 35 Below the detection limit or negative or not H274Y mutation strain of influenza virus A 2 S38 Positive The sample is H274Y mutation strain of SE H1N1 2009 influenza virus A and the software displays the quantitative value For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
4. chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Influenza virus A DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQI In addition the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC An external positive control defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents H1N1 2009 H274Y Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 281 Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30pl H1N1 2009 H274Y Positive Control 1 x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 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 ma
5. e 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 541 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 Selection of fluorescence channels Target Nucleic Acid 95 C for 15sec 60 C for Imin 40cvel Fluorescence measured at 60 C ayes 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 Molecular Grade Water UNDET 25 35 HEX VIC JOE Positive Control qualitatve assay lt 5 O QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following s
6. efined 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 Au l 4u I 1X107 1X 10 171X105 1X104 copiessmi 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 1 lt 10 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 18ul 1 ul ipl Super Mix Enzyme Mix Internal Control Syl 204 Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without HEX VIC JOE channel may be treated with 141 Molecular Grade Water instead of 11 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 th
7. egative 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 Icycle 95 C for 15min Icycle Target Nucleic Acid 95 C for S5sec 60 C for 30sec Fluorescence measured at 60 C 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the Noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control 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 530nm Positive Control quatitative assay 35s 13 Data Analysis and Interpretation The following sample results are possible Crossing point value Blank 25 35 Below the detection limit or negative or not H274Y mutation strain of influenza virus A ll inal Selection of fluorescence channels AOcycles Result Analysis Positive The sample is H274Y mutation strain of H1N1 2009 influenza virus A and the software displays the quantitative value lt 3 38 40 25 35 Re test If i
8. ens 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 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 10001 e Sterile microtubes 7 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
9. nza A virus which causes influenza in birds and some mammals Strains of all subtypes of influenza A virus have been isolated from wild birds although disease is uncommon Some isolates of influenza A virus cause severe disease both in domestic poultry and rarely in humans Occasionally viruses are transmitted from wild aquatic birds to domestic poultry and this may cause an outbreak or give rise to human influenza pandemics The oral oseltamivir is widely used as one of the commercially available NA inhibitors which have been demonstrated clinical benefits in the prevention and treatment of seasonal influenza virus infections During the 2007 to 2008 influenza season a significant rise in the frequency of influenza A virus strains carrying the oseltamivir resistance NA mutation H274Y was reported worldwide in untreated patients Oseltamivir Resistant H274Y H1N1 2009 Influenza virus A real time RT PCR kit contains a specific ready to use system for detecting H274Y mutation of the Influenza virus A using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains a Super Mix for the specific amplification of H274Y mutation in NA gene 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 RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR polymerase
10. 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 Influenzavirus A is a genus of the Orthomyxoviridae family of viruses Influenzavirus A includes only one species Influenza A virus which causes influenza in birds and some mammals Strains of all subtypes of influenza A virus have been isolated from wild birds although disease is uncommon Some isolates of influenza A virus cause severe disease both in domestic poultry and rarely in humans Occasionally viruses are transmitted from wild aquatic birds to domestic poultry and this may cause an outbreak or give rise to human influenza pandemics The oral oseltamivir is widely used as one of the commercially available NA inhibitors which have been demonstrated clinical benefits in the prevention and treatment of seasonal influenza virus infections During the 2007 to 2008 influenza season a significant rise in the frequency of influenza A virus strains carrying the oseltamivir resistance NA mutation H274Y was reported worldwide in untreated patients Oseltamivir Resistant H274Y H1IN1 2009 Influenza virus A real time RT PCR kit contains a specific ready to use system for detecting H274Y mutation of the Influenza virus A using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains a Super Mi
11. t is still 38 40 report as 1 PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES Revision No ZJ0007 Issue Date Jul 1 2015 Oseltamivir Resistant H274Y H1N1 2009 Influenza A Virus Real Time RT PCR Kit User Manual 20 C For Research Use Only MBS598197 Instrument III IV 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 1 Intended Use Oseltamivir Resistant H274Y H1N1 2009 Influenza virus A real time RT PCR kit is used for the detection of the oseltamivir resistance NA mutations H274Y of H1N1 2009 Influenza virus A in nasal and pharyngeal secretions 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
12. 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 Ct value HEX VIC JOE 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 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 control d
13. x for the specific amplification of H274Y mutation in NA gene 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 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 Influenza virus A DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQI1 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 allow the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents H1N1 2009 H274Y Super Mix 1 vial 4801 RT PCR Enzyme Mix 1 vial 281 Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30ul H1N1 2009 H274Y Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 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 doz
14. y 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 AN 7 amp gt 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
15. z IYD Revision No ZJ0002 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Oseltamivir Resistant H274Y H1N1 2009 Influenza A Virus Real Time RT PCR Kit User Manual 20 C 25 MBS598197 Instrument I I For use with LightCycler1 0 2 0 instrument EA bad 1 Intended Use Oseltamivir Resistant H274Y H1N1 2009 Influenza virus A real time RT PCR kit is used for the detection of the oseltamivir resistance NA mutations H274Y of H1N1 2009 Influenza virus A in nasal and pharyngeal secretions 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 amplification 3 Product Description Influenzavirus A is a genus of the Orthomyxoviridae family of viruses Influenzavirus A includes only one species Influe
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