MBS598154 - MyBioSource
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1. EU C For Research Use Only In USA amp China Y A Revision No ZJ0005 Issue Date Jul 1 2015 Enterovirus Real Time RT PCR Kit User Manual MBS598154 Instrument I I For use with LightCycler1 0 2 0 Instrument p 1 Intended Use By using real time PCR systems Enterovirus real time PCR kit is used for the detection of Coxsackie Virus in samples like nasal and pharyngeal secretions sputum provoked sputum stool C S F 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 Enteroviruses are small viruses that are made of ribonucleic acid RNA and protein In addition to the three different polioviruses there are 61 non polio enteroviruses that can cause disease in humans 29 Coxsackieviruses 23 Coxsac2. NA 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 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 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 Type of reagent EV Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control 1 vial 30ul EV Positive Control 1x 10 copies ml 1 vial 30u1 Analysis sensitivity 5 X 10 copies ml LOQ 1X 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 k
3. ROCEDURES
4. ailable in the kit and bronchial lavage sample or lung section sample is used for the extraction of the DNA 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 1 lt 10 copies ml contained allows the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents EV Super Mix 1 vial 3501 RT PCR Enzyme Mix 1 vial 281 Molecular Grade Water 1 vial 400u1 Internal Control 1 vial 30ul EV Positive Control 1x 10 copies ml 1 vial 30ul 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 e Cool all reagents during the working steps e Super Mix and Reaction Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Vortex mixe
5. arately add Sul 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 Selection of fluorescence channels Target Nucleic Acid 95 C for 15min 95 C for Ssec 60 C for 30sec 40cycl Fluorescence measured at 60 C a ea 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 internal control must be performed correctly otherwise the sample results is invalid 530nm Positive Controlqualitaive assay 35 SSS QS quantitative detection 13 Data Analysis and Interpretation The following sample results are possible Crossing point value RESMI ANALYSIS 38 Molecular Grade Water nk Below the detection limit or negative P Blank _ 25 sa S ositive and the software displays the quantitative value 7 25 35 Re test if it is still 38 40 report as 1 RT PCR Inhibition no dia
6. e Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 95 C for 15sec 60 C for 1min Fluorescence measured at 60 C y AN gt A 5 L If you use ABI Prism system please choose none as passive reference and quencher Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE 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 Positive Control qualitative assay oe re QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Ct value HEX VIC JOE Result Analysis AM UNDET Below the detection limit or negative 38 test 1 UNDET 5 Positive and the software displays the quantitative value 5 gt TEE 7 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 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC P
7. f the accumulating product without having to re open the reaction tube after the amplification 3 Product Description Enteroviruses are small viruses that are made of ribonucleic acid RNA and protein In addition to the three different polioviruses there are 61 non polio enteroviruses that can cause disease in humans 29 Coxsackieviruses 23 Coxsackie A viruses and 6 Coxsackie B viruses 28 echoviruses and 4 other enteroviruses Enteroviruses can be found in the respiratory secretions e g saliva sputum or nasal mucus and stool of an infected person Other persons may become infected by direct contact with secretions from an infected person or by contact with contaminated surfaces or objects such as a drinking glass or telephone Parents teachers and child care center workers may also become infected by contamination of the hands with stool from an infected infant or toddler during diaper changes The Enterovirus real time RT PCR kit contains a specific ready to use system for the detection of the Enterovirus 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 RNA including CA16 CA4 CA5 CA7 CA9 CA10 CB2 CB5 CB13 echoviruses and EV71 The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Enterovirus RNA is transcribed into cDNA Afterwards a thermostable D
8. gnosis can be concluded For further questions or problem please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES EU C Revision No ZJ0006 Issue Date Jul 1 2015 For Research Use Only In USA amp China Enterovirus Real Time RT PCR Kit User Manual 20 C MBS598154 Instrument IIT TV Z 25 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 od ond 1 Intended Use By using real time PCR systems Enterovirus real time PCR kit is used for the detection of Enterovirus 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 o
9. it label e Repeated thawing and freezing gt 3x should be avoided e Cool all reagents during the working steps 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 7 warnings and Precaution e 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 Real time PCR system e Real ti
10. kie A viruses and 6 Coxsackie B viruses 28 echoviruses and 4 other enteroviruses Enteroviruses can be found in the respiratory secretions e g saliva sputum or nasal mucus and stool of an infected person Other persons may become infected by direct contact with secretions from an infected person or by contact with contaminated surfaces or objects such as a drinking glass or telephone Parents teachers and child care center workers may also become infected by contamination of the hands with stool from an infected infant or toddler during diaper changes The Enterovirus real time RT PCR kit contains a specific ready to use system for the detection of the Enterovirus 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 RNA including CA16 CA4 CA5 CA7 CA9 CA10 CB2 CB5 CB13 echoviruses and EV71 The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Enterovirus 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 DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is av
11. llowing 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 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 18ul 1 yl il 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 111 Molecular Grade Water instead of 1ul 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 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 th
12. me PCR reaction tubes plates e Pipets 0 5ul 100041 e Sterile microtubes 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 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 Nucleic Acid Isolation Kit 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 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 1 lt 10 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 fo
13. r 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 Ts Warnings and Precaution e 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 smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and tran
14. sport 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 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 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 to determine and control the possibility of RT PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the channel 560nm 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 134l 1 yl il Super Mix Enzyme Mix Internal Control Spl 15l Extraction RNA Master Mix Reaction Plate Tube l PCR Instrument XPCR system without channel 560nm may be treated with lu 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 15ul Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Sep
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