(EV71) Real Time RT-PCR Kit
Contents
1. polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Enterovirus 71 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 1 lt 10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents l EV71 Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28 ul Molecular Grade Water 1 vial 400u1 EV71 Positive Control 1 x10 copies ml 1 vial 30u1 Analysis sensitivity 1 X 10 copies ml LOQ 2X 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 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 e Cool all reagents during the working s2. possible Crossing point value Resit Analys Below the detection limit or negative Molecular Grade Water Positive 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 z IYD Revision No ZJ0008 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Enterovirus 71 EV71 Real Time RT PCR Kit 20 C User Manual MBS598153 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 so faer 1 Intended Use By using real time PCR systems Enterovirus 71 real time PCR kit is used for the detection of Enterovirus 71 in samples like nasal and pharyngeal secretions sputum provoked sputum stool C S F serum and etc 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 fluoresce
3. d 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 151 Master Mix with micropipets in sterile filter tips to each real time PCR reaction plate tubes Separately add 5ul RNA sample 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 Selection of fluorescence channels Target Nucleic Acid 95 C for 5sec 60 C for 30sec 40cycl Fluorescence measured at 60 C ee 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 and QS curve must be performed correctly otherwise the sample results is invalid Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are
4. fore 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 19 ul 1 ul Super Mix Enzyme Mix Sul 204 Extraction RNA Master Mix Reaction Plate Tube PCR Instrument 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 20ul Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 5ul 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 95 C for 15min Selection of fluorescence channels Target Nucleic Acid 95 C for 15sec 60 C for Imin 40cvycl Fluorescence measured at 60 C ee A 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 concentrat
5. ion of standard controls at the end of run and a standard curve will be automatically formed 12 Quality control Negative control positive control and QS curve must be performed correctly otherwise the sample results is invalid Molecular Grade Water UNDET Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Ct value FAM Result Analysis UNDET Below the detection limit or negative Positive and the software displays the quantitative value 38 40 Re test if it is still 38 40 report as 1 For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
6. n 1998 in Taiwan there were HFMD epidemics involving sudden deaths among young children and EV71 was isolated from the HFMD patients including the fatal cases The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis EV71 strains from previously reported epidemics belonged to genotype A 1 while those from recent epidemics could be divided into two genotypes A 2 and B The Enterovirus 71 real time RT PCR kit contains a specific ready to use system for the detection of the Enterovirus 71 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 the Enterovirus 71 RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Enterovirus 71 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 Enterovirus 71 DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQI 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 EV71 Super Mix 1 vial 3801 RT PCR E
7. nce 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 reaction tube after the amplification 3 Product Description Enterovirus 71 EV71 one of the major causative agents for hand foot and mouth disease HFMD is sometimes associated with severe central nervous system diseases In 1997 in Malaysia and Japan and in 1998 in Taiwan there were HFMD epidemics involving sudden deaths among young children and EV71 was isolated from the HFMD patients including the fatal cases The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis EV71 strains from previously reported epidemics belonged to genotype A 1 while those from recent epidemics could be divided into two genotypes A 2 and B The Enterovirus 71 real time RT PCR kit contains a specific ready to use system for the detection of the Enterovirus 71 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 the Enterovirus 71 RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Enterovirus 71 RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR
8. nzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 EV71 Positive Control 1 x10 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 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 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 pl 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 AN 7 Warnings and Precaution e Carefully read this instruction before starting the p
9. rocedure 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 and 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 brands of RNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For RNA extraction kit please comply wi
10. teps e Super Mix and Reaction 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 AN 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 glo
11. th manufacturer s instructions The recommended extraction kit is as follows 9 2 Quantitation The kit can be used for quantitative or qualitative real time RT PCR detection For performance of quantitative real time PCR standard dilutions must be prepared firstly as follows Molecular Grade Water is used as the dilution Dilution is not needed for performance of qualitative real time PCR detection Take positive control 1 lt 10 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 Apl Aul S WW WV Y 1X107 1X10 1X10 1X 104 copiesim 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 contains high concentration of the target DNA Therefore be careful of the dilution in order to avoid contamination 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 144 1 yl Super Mix Enzyme Mix Syl 15 l Extraction RNA Master Mix Reaction Plate Tube PCR Instrument 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 use
12. ves 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 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 9 2 Quantitation The kit can be used for quantitative or qualitative real time RT PCR For performance of quantitative 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 4ul Aul Aul 1x10 1X10 1X10 1X10 capiesmi 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 1 lt 10 copies ml contains high concentration of the target DNA There
13. z IYD Revision No ZJ0008 EU k Issue Date Jul 1 2015 For Research Use Only In USA amp China Enterovirus 71 EV71 Real Time RT PCR Kit 20 C User Manual MBS598153 Instrument I II For use with LightCycler1 0 2 0 Instrument Feed Coed 1 Intended Use By using real time PCR systems Enterovirus 71 real time PCR kit is used for the detection of Enterovirus 71 in samples like nasal and pharyngeal secretions sputum provoked sputum stool C S F serum and etc 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 reaction tube after the amplification 3 Product Description Enterovirus 71 EV71 one of the major causative agents for hand foot and mouth disease HFMD is sometimes associated with severe central nervous system diseases In 1997 in Malaysia and Japan and i
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