(H1N1) Real Time RT-PCR Kit
Contents
1. f IYD Revision No ZJ0008 EU 8 Issue Date Jul 1 2015 For Research Use Only In USA amp China New Influenza A Virus H1N1 Real Time RT PCR Kit User Manual 20 C MBS598296 Instrument I II YY 25 For use with LightCycler1 0 2 0 Instrument 1 Intended Use New Influenza A virus H1N1 real time RT PCR kit is used for the detection of new reassortment Influenza A virus H1N1 by using real time PCR systems 2 Principle of Real Time RT PCR RT PCR Reverse Transcription Polymerase Chain Reaction is a technique in which an RNA strand is reverses transcribed into its DNA complement followed by amplification of the resulting DNA using a polymerase chain reaction PCR RT PCR can be used to examine gene expression level in cells and tissues clone the specific gene of cDNA sequences and test RNA viruses One Step RT PCR Kit adopts one tube system Because operator doesn t need to open the lid during the reaction process this user friendly improved version avoids cross contamination 3 Product Description Influenza A virus subtype HIN1 A HIN1 is a subtype of influenza A virus It is the most common cause of influenza flu in humans Some strains of HIN1 are endemic in humans including the strain s responsible for the 1918 flu pandemic which killed 50 100 million people worldwide Less virulent H1N1 strains still exist in the wild today worldwide causing a small fraction of all influenza like illness and a large fr2. 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 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 Real time PCR system 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 Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g 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 prep
3. RNA Mini extraction Kit 50 Respiratory specimens should be collected and transported in virus transport media Virus transport medium A Virus transportation medium use in collecting throat and nasal swabs 1 Add 10g veal infusion broth and 2g bovine albumin fraction V to sterile distilled water to 200ml 2 Add 0 8ml gentamicin sulfate solution S50mg ml and 3 2ml amphotericin B 250pg ml 3 Sterilize by filtration B Nasal wash medium Sterile saline 0 85 NaCl 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 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows Lung biopsy Post mortem lung or tracheal tissue Convalescent serum Manufacturer ZJ Biotech QIAGEN 1p ul 1p 1 The volumes of Super Mix and Super Mix Enzyme Mix Internal Control Enzyme Mix per reaction io p multiply with the number of Sul 20ul reaction which includes the Extraction RNA Master Mix number of controls standards and sample prepared Molecular ce Grade Water is used as the Reaction negative control For reasons of Plate Tube unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge Pipet 20l Master Mix with micr
4. 40 report as 1 UNDET UNDET PCR Inhibition No diagnosis can be concluded please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
5. action of all seasonal influenza In March and April 2009 hundreds of laboratory confirmed infections and a number of deaths were caused by an outbreak of a new strain of HIN1 The new Influenza A virus real time RT PCR kit contains a specific ready to use system for the detection of the new Influenza A virus H1N1 using RT PCR in the real time PCR system The primer and probe is designed for specifically detect new influenza virus H1N1 with new reassortment genome The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the virus 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 virus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 An external positive control is supplied which allow the determination of the gene load Internal control is available in the kit It can be used for monitoring the yield of the nucleic acid extraction and whether there existing inhibition in the sample or not 4 Kit Contents New H1N1 Super Mix 1 vial 350ul RT PCR Enzyme Mix 1 vial 281 Molecular Grade Water 1 vial 400u1 New HINI Positive Control 1 vial 30ul Internal Control 1 vial 30ul Analysis sensitivity 1 lt 10 copies ml
6. ally indicated Transtracheal aspirate Bronchoalveoar lavage Lung biopsy Post mortem lung or tracheal tissue Specimens for the laboratory diagnosis of avian influenza A should be collected in the following order of priority Nasopharyngeal aspirate Convalescent serum Acute serum 9 1 3 RNA extraction kits 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 QIAamp Viral RNA Mini extraction Kit 50 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 1 wl rxn and the result will be shown in the 560nm 9 3 RT PCR Protocol 1 Preparation of positive control and negative control It is necessary to dilute the positive control and 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 2 The Master Mix volume for each reaction should be pipetted as follows il Attention PCR system without 13 1 pl 560nm channel e g LightCycler1 0 Super Mix Enzyme Mix intemal Control can be treated with 1pl Molecular mie ae Grade Water ins
7. ared 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 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 9 1 1 Type of specimens A variety of specimens are suitable for the diagnosis of virus infections of the upper respiratory tract Nasal swab Nasopharyngeal aspirate Nasopharyngeal swab Throat swab In addition to swabs from the upper respiratory tract invasive procedures can be performed for the diagnosis of virus infections of the lower respiratory tract where clinic
8. ents 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 Real time PCR system e RNA extraction kit e Real time PCR reaction tubes plates Cryo container e Pipets 0 5 ul 1000 pl e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and freezer e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Tube racks 7 A Warnings and Precaution eCarefully 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
9. filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory For further questions or problems 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 9 1 1 Type of specimens A variety of specimens are suitable for the diagnosis of virus infections of the upper respiratory tract Nasal swab Nasopharyngeal aspirate Nasopharyngeal swab Throat swab In addition to swabs from the upper respiratory tract invasive procedures can be performed for the diagnosis of virus infections of the lower respiratory tract where clinically indicated Transtracheal aspirate Bronchoalveoar lavage Specimens for the laboratory diagnosis of avian influenza A should be collected in the following order of priority Nasopharyngeal aspirate Acute serum 9 1 2 RNA extraction kits 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 ME 0010 ME 0012 RNA Isolation Kit 52904 QIAamp Viral
10. g DNA using a polymerase chain reaction PCR RT PCR can be used to examine gene expression level in cells and tissues clone the specific gene of cDNA sequences and test RNA viruses One Step RT PCR Kit adopts one tube system Because operator doesn t need to open the lid during the reaction process this user friendly improved version avoids cross contamination 3 Product Description Influenza A virus subtype HIN1 A HIN1 is a subtype of influenza A virus It is the most common cause of influenza flu in humans Some strains of HIN1 are endemic in humans including the strain s responsible for the 1918 flu pandemic which killed 50 100 million people worldwide Less virulent H1N1 strains still exist in the wild today worldwide causing a small fraction of all influenza like illness and a large fraction of all seasonal influenza In March and April 2009 hundreds of laboratory confirmed infections and a number of deaths were caused by an outbreak of a new strain of HIN1 The new Influenza A virus real time RT PCR kit contains a specific ready to use system for the detection of the new Influenza A virus H1N1 using RT PCR in the real time PCR system The primer and probe is designed for specifically detect new influenza virus H1N1 with new reassortment genome The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the virus RNA is transcribed into cDNA Afterwards a thermostable DNA poly
11. merase 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 virus DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 An external positive control is supplied which allow the determination of the gene load Internal control is available in the kit It can be used for monitoring the yield of the nucleic acid extraction and whether there existing inhibition in the sample or not 4 Kit Contents 1 New H1N1 Super Mix 1 vial 480ul RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 New HINI Positive Control 1 vial 30ul Internal Control 1 vial 30ul Analysis sensitivity 5 lt 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 reag
12. nalysis and Interpretation The following sample results are possible Crossing point value ee 25 35 Below the detection limit or negative 2 lt 43 Positive New Influenza A virus H1 subtype s genome bears strong resemblance to the genome of strain A California 04 2009 If the results are obtained it is likely that a new reassortment of the New Influenza A virus has emerged 43 45 25 35 Re test If it 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 IVD Revision No ZJO005 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China New Influenza A Virus H1N1 Real Time RT PCR Kit User Manual 20 C MBS598296 Instrument III IV J 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 CI 1 Intended Use New Influenza A virus H1N1 real time RT PCR kit is used for the detection of new reassortment Influenza A virus H1N1 by using real time PCR systems 2 Principle of Real Time RT PCR RT PCR Reverse Transcription Polymerase Chain Reaction is a technique in which an RNA strand is reverses transcribed into its DNA complement followed by amplification of the resultin
13. opipets 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 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes Perform the following protocol in the instrument Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE PCR Instrument 2 3 4 45 C for 10min 95 C for 15min 95 C for 15sec 60 C for Imin Fluorescence measured at 60 C 45cycles 5 Arf 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid aua ie Pe Positive Control qualitative assay s85 12 Data Analysis and Interpretation The following sample results are possible Ct value HEX VIC JOE Result Analysis 1 UNDET 25 35 Below the detection limit or negative Positive New Influenza A virus H1 subtype s genome bears strong resemblance to the genome of strain A California 04 2009 If the results are obtained it is likely that a new reassortment of the New Influenza A virus has emerged ia 43 45 25 35 Re test If it is still 38
14. tead of 1m Internal Control Spl 15yl 3 The volumes of Super Mix Extraction RNA Master Mix and Enzyme Mix per reaction multiply with the number of i nn reaction which includes the Reaction number of controls standards Plate Tube and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix PCR Instrument completely then spin down briefly in a centrifuge 1 Pipet 151 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 2 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 3 Perform the following protocol in the instrument 95 C for 5sec 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Crossing point value Molecular Grade Water 25 35 Positive Control qualitative assay s85 12 Data A
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