MBS598302 - MyBioSource
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1. 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 Collect 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 DNA Extraction DNA extraction buffer is contained in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or commercial kits 1 Pipet 100ul sample to a 0 5ml tube add 1001 DNA extraction buffer close the tube and vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubation the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and is used for PCR template 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 PCR A positive control 1 10 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 fo2. 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 Anaplasma phagocytophilum formerly Ehrlichia phagocytophila a tick transmitted pathogen that infects several animal species including humans involved as accidental dead end hosts is the causative agent of human granulocytic anaplasmosis HGA It is a pathogen of veterinary importance responsible for tickborne fever of ruminants and for granulocytic anaplasmosis of horses and dogs Anaplasma phagocytophilum real time PCR Kit contains a pairs of primers and a probe for the detection of the HGA through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the HGA DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The det
3. IVD Revision No ZJ0002 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Anaplasma phagocytophilum Real Time PCR Kit User Manual 20 C REF rassogz02 Instrument I II Z 25 For use with LightCycler1 0 2 0 Instrument e col 1 Intended Use The Anaplasma phagocytophilum real time PCR kit is used for the detection of Anaplasma phagocytophilum in whole blood non anticoagulant or ticks samples 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 Anaplasma phagocytophilum formerly Ehrlichia phagocytophila a tick transmitted pathogen that infects several animal species including humans involved as accidental dead end hosts is the causative agent of hum
4. an granulocytic anaplasmosis HGA It is a pathogen of veterinary importance responsible for tickborne fever of ruminants and for granulocytic anaplasmosis of horses and dogs Anaplasma phagocytophilum real time PCR Kit contains a pairs of primers and a probe for the detection of the HGA through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the HGA DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified HGA DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit 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 DNA Extraction Buffer 2 vials 1 5ml HGA Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400ul 1 vial 30ul 1 vial 30ul Analysis sensitivity 110 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 DNA extraction buffer in the kit the analysis sensitivity is the same as it declares However
5. 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 Molecular Grade Water Positive Control gualitative assay 35 QS quantitative detection 13 Data Analysis and Interpretation The following results are possible Crossing point value l AAAS 2 2 Below the detection limit or negative lt Positive and the software displays the quantitative value 38 40 Re test If it is still 38 40 report as 1 5 5 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 ZJ0002 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Anaplasma phagocytophilum Real Time PCR Kit User Manual 20 C REF assogz02 Instrument III IV Z 5 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 Foo Cd 1 Intended Use The Anaplasma phagocytophilum real time PCR kit is used for the detection of Anaplasma phagocytophilum in whole blood non anticoagulant or ticks samples by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is
6. ection of amplified HGA DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit 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 DNA Extraction Buffer 2 vials 1 5ml HGA Reaction Mix 1 vial 950ul PCR Enzyme Mix 1 vial 12ul Molecular Grade Water 1 vial 400ul Internal control IC 1 vial 30u1 HGA Positive Control 1 lt 10 copies ml 1 vial 30ul Analysis sensitivity 1X 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 DNA extraction buffer in the kit 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 reage
7. is used for PCR template 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 560nm 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control 1 10 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 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 41 4ul 4ul Y VY V 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 during the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 pl 0 4yl 1ul Reaction Mix Enzyme Mix Internal Control 18 4 pl Mas
8. lecular 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 36pl 22 5ul for SmartCyclerII Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 4ul 2 5ul1 for SmartCyclerII DNA 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 93 C for 15sec 60 C for 1min Fluorescence measured at 60 C y 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 e e Puro a Positive Control qualiative assay 35 S 13 Data Analysis and Interpretation The following results are possible Ct value MEE HEX VIC JOE Result Analysis l UNDET 25 35 Below the detection limit or negative Selection of fluo
9. nts during the working steps e Reaction 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 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 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 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
10. r 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 4ul 4ul Y WV V Y 1X107 1K 108 1 X105 1X 104 copiessm 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 during the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35yl 0 4yl ipl 21 5 pl 0 4 pl ipl Reaction Mix Enzyme Mix internal Control Reaction Mix Enzyme Mix Internal Control 36 4ul 22 91 Master Mix Master Mix 4ul 36pl 2 5 22 5pl Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system is only tor PCR Instrument OR PCR Instrument Smart Cycler It lt PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of 1 yl IC 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Mo
11. rescence channels Target Nucleic Acid HEX VIC JOE 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 FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
12. 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 Collect samples in sterile tubes PCR Enzyme Mix Molecular Grade Water Internal control IC HGA Positive Control 1 lt 10 copies ml 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 DNA Extraction DNA extraction buffer is contained in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or commercial kits 1 Pipet 100ul sample to a 0 5ml tube add 1001 DNA extraction buffer close the tube and vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubation the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and
13. ter Mix 2ul 18 pl Extraction DNA Master Mix i tai Reaction Plate Tube PCR Instrument XPCR system without 560nm channel may be treated with 14 Molecular Grade Water instead of 11 IC 1 The volumes of Reaction 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 181 Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 2n1 DNA 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 37 C for 2min Selection of fluorescence channels Target Nucleic Acid 93 C for 5sec 60 C for 30sec Fluorescence measured at 60 C y 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
14. 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 Reaction 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 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 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
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