MBS598092 - MyBioSource
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1. 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 Quickly prepare 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 8 Sample Collection Storage and transportation e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C PCR Enzyme Mix Molecular Grade Water Internal Control P vivax Positive Control 1 lt 10 copies ml 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 the commercial kit2. 1 5ml P vivax Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400ul 1 vial 301 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X10 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 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 5ul 10001 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 e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only
3. 1 Pipet 100u1 whole blood or one two mosquito es sample to a 0 5ml tube add 100u1 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 Attention A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or store at 20 C for one month C Different brand DNA extraction kits are available You can also use your own extraction systems or the commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 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 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
4. 500 million new cases every year with 1 5 to 2 7 million deaths worldwide Malaria is a potentially fatal tropical disease that is caused by a parasite known as Plasmodium Four kinds of malaria parasites can infect humans P falciparum P vivax P ovale and P malariae It is spread through the bite of an infected female mosquito P vivax is less virulent than Plasmodium falciparum which is the deadliest of the four and is seldom fatal P vivax is found mainly in the United States Latin America and in some parts of Africa P vivax can cause death due to splenomegaly a pathologically enlarged spleen but more often it causes debilitating but non fatal symptoms The Plasmodium vivax real time PCR Kit contains a specific ready to use system for the detection of the Plasmodium vivax through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Plasmodium vivax DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Plasmodium vivax 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 1 lt 10 copies ml is supplied which a
5. Revision No ZJO002 Issue Date Jul 1 2015 For Research Use Only In USA amp China Plasmodium Vivax Real Time PCR Kit User Manual 20 C MBS598092 Instrument HI IV 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 Lec ner 1 Intended Use The Plasmodium vivax real time RT PCR kit is used for the detection of Plasmodium vivax in whole blood or mosquito 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 Malaria is one of the leading causes of disease and death in the world It is estimated that there are 300
6. 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 the commercial kit 1 Pipet 100ul whole blood or one two mosquito es sample to a 0 5ml tube add 100ul1 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 Attention A During the incubation make sure the tube is not open as the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or store at 20 C for one month C Different brand DNA extraction kits are available You can also use your own extraction systems or the commercial kit based on the yield For the DNA extraction please comply with the manufacturer s instructions 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 l 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 Molecul
7. 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 1X107 1X10 1X10 1K 104 tapiesmi 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 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 ul 0 4ul ipl Reaction Mix Enzyme Mix Internal Control 18 4 wl Master Mix 2 ul 18 ul Extraction DNA Master Mix iaa ae Reaction Plate Tube l PCR Instrument PCR system without 560nm channel may be treated with 1ul Molecular Grade Water instead of 1 ul 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 18ul Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 2u DNA sam
8. J IYD Revision No ZJ0002 EU k Issue Date Jul 1 2015 For Research Use Only In USA amp China Plasmodium Vivax Real Time PCR Kit User Manual 20 C MBS598092 Instrument I II Z 25 For use with LightCycler1 0 2 0 Instrument EAL 1 Intended Use The Plasmodium vivax real time RT PCR kit is used for the detection of Plasmodium vivax in whole blood or mosquito 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 Malaria is one of the leading causes of disease and death in the world It is estimated that there are 300 500 million new cases every year with 1 5 to 2 7 million deaths worldwide Malaria is a potentially fatal tropical disease that is caused by a paras
9. ar 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 Ayl 4ul 1X107 1X10 1X10 1X 1D copiers 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 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 35pl 0 4yl ipl 21 5 pl 04 ipl Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4yl 22 9 pl Master Mix Master Mix 4ul 36 2 5 ul 22 5 ul Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube amp This system sa O aan is only for nsuumon R SS Smor Cycler Il XPCR system without HEX VIC JOE channel may be treated with 1ul 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 the controls standards a
10. ator and Freezer e Tube racks 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 Quickly prepare 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 PCR Enzyme Mix Molecular Grade Water Internal Control P vivax Positive Control 1 lt 10 copies ml e Wear separate coats and gloves in each area 8 Sample Collection Storage and transportation 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
11. ite known as Plasmodium Four kinds of malaria parasites can infect humans P falciparum P vivax P ovale and P malariae It is spread through the bite of an infected female mosquito P vivax is less virulent than Plasmodium falciparum which is the deadliest of the four and is seldom fatal P vivax is found mainly in the United States Latin America and in some parts of Africa P vivax can cause death due to splenomegaly a pathologically enlarged spleen but more often it causes debilitating but non fatal symptoms The Plasmodium vivax real time PCR Kit contains a specific ready to use system for the detection of the Plasmodium vivax through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Plasmodium vivax DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Plasmodium vivax 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
12. llow 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 P vivax Reaction Mix 1 vial 950ul 1 vial 12ul 1 vial 400ul 1 vial 30u1 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X10 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 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 5ul 10001 e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refriger
13. nd 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 36ul 22 5ul for SmartCycer II Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tube Then separately add 4ul 2 5yl for SmartCycer IT 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 93 C for 15sec 60 C for 1min Fluorescence measured at 60 C y 5 r 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 FAM HEX VIC JOE Molecular Grade Water UNDET 25 35 Target Nucleic Acid HEX VIC JOE Positive Control qualitative assay ae QS quantitative detection Correlation coefficient of QS c
14. ple 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 Icycle leycle 93 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 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 Channel Crossing point value Control 560nm Molecular Grade Water 25 35 40cycles Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible 2e lt 38 Positive and the software displays the quantitative value Blank For further questions or problems please contact our technical support ___ FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES EU C
15. urve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value HEX VIC JOE Result Analysis 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 Ma UNDET 2 s Positive and the software displays the quantitative value 38 25 UNDET FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
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