(UP) Real Time PCR Kit
Contents
1. IVD Revision No ZJO002 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Ureaplasma parvum UP Real Time PCR Kit User Manual 20 C MBS598251 Instrument I II 25 For use with LightCycler1 0 2 0 Instrument Eat 1 Intended Use Ureaplasma parvum real time PCR kit is used for the detection of Ureaplasma parvum in genital swabs or urine 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 Ureaplasma parvum has a circular chromosome consisting of 751 719 base pairs Its chromosome encodes 605 Open Reading Frames and 38 RNA genes Having a reduced genome it has a fast evolutionary rate Ureaplasma parvum has been associated with the cause of vari2. Issue Date Jul 1 2015 For Research Use Only In USA amp China Ureaplasma parvum UP Real Time PCR Kit User Manual 20 C REF issos Instrument III IV Z 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 eo 1 Intended Use Ureaplasma parvum real time PCR kit is used for the detection of Ureaplasma parvum in genital swabs or urine 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 Ureaplasma parvum has a circular chromosome consisting of 751 719 base pairs Its chromosome encodes 605 Open Reading Frames and 38 RNA g
3. IC 1 vial 30ul UP Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2 X 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 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 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 Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 7 Z warnings and Precaution Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay n
4. P Reaction Mix 1 vial 950ul PCR Enzyme Mix 1 vial 12ul Molecular Grade Water 1 vial 400ul Internal Control IC 1 vial 30u1 UP Positive Control 1 lt 10 copies ml 1 vial 30u1 Analysis sensitivity 1 X 10 copies ml LOQ 2 X 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 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 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 A Warnings and Precautio
5. eeds 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 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 supplied in the kit Attention please thaw the buffer thoroughly and mix the buffer well before use because it contains insoluble particles You may use your own extraction systems or commercial kits 9 1 1 Genital swabs sam
6. enes Having a reduced genome it has a fast evolutionary rate Ureaplasma parvum has been associated with the cause of various diseases It has been categorized as a mucosal parasite living within the genito urinary tracts It is a mycoplasma and pathogenic ureolytic mollicute which can cause male urethritis supperative arthritis adverse pregnancy outcomes chorioamnionitis surgical wound infections neonatal meningitis pelvic inflammatory diseases pyelonephritis and neonatal disease Ureaplasma parvum real time PCR kit contains a specific ready to use system for the detection of the Ureaplasma parvum by polymerase chain reaction in the real time PCR system The master contains reagents and ezmymes for the specific amplification of the Ureaplasma parvum DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Ureaplasma parvum DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and genital swabs samples are used for DNA extraction 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 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents Type of Reagent DNA Extraction Buffer 2 vials 1 5ml U
7. es Dilution of Standards 4ul 4u 4ul 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 y y Attention 1X105 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 Y 1X107 1x10 1 X 104 copiesimi 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as followd 35ul 0 4ul ipl 21 5 pl 0 4 tul Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control Deer i ONT 36 4 22 941 Master Mix Master Mix 4ul 36l 2 5 pl 22 5 Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube A This system is only for PCR Instrument OR PCR Instrument x dosacroa rise nssumen Smart Cycler N X PCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of 1 yul 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 36ul 22 5ul for SmartCyclerIT Master Mix wit
8. h micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 4p1 2 5p1 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 37 C for 2min 94 C for 2min 93 C for 15sec 60 C for Imin AOcycles Fluorescence measured at 60 C 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 Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE IC Ct value Ooo y ve CSC 25 35 Positive Control qualiaiveasay 35d SSSOS S 13 Data Analysis and Interpretation The following sample results are possible Ct value HEX VIC JOE Result Analysis L238 Positive and the software displays the quantitative value 3 25 test UNDET Re test If it is still 38 40 report as 1 UNDET UNDET PCR I
9. hould be used and defined as standard with specification of the corresponding concentrations y 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 PCR Protocol The Master Mix volume for each reaction should be pipetted as follow 17 pl 0 4pl ipl Reaction Mix Enzyme Mix internal Control in 18 4 pl Master Mix II 1X107 1X10 1X10 1X104 copicsim 2 ul 18 ul Extraction DNA Master Mix 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 181 Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Then separately add 2n DNA sample supernatant 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 prot
10. it contains insoluble particles You may use your own extraction systems or commercial kits 9 1 1 Genital swabs sample DNA extraction buffer is supplied 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 Wash the genital swabs in 1 0ml normal saline and vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline and suspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can used for PCR template 9 1 2 Urine sample 1 Take 1 5 ml sample to a tube Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly i
11. n 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 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 supplied in the kit Attention please thaw the buffer thoroughly and mix the buffer well before use because
12. n a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open for the vapor will volatilize into the air and may cause contamination in case the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C DNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kit based on the yield For RNA extraction please comply with 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 dilutions must be prepared first as follows Molecular Grade Water is used for dilution The step of 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 Molecular Grade Water into next three tubes Do three dilutions as the following figur
13. nhibition 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
14. ocol in the instrument 37 C for 2min 94 C for 2min 93 C for 5sec 60 C for 30sec AOcycles Selection of fluorescence channels Target Nucleic Acid 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 Crossing point value Molecular Grade Water 25 35 Positive Control qualitative assay Ee i QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible pete ah Result Analysis 25 35 Below the detection limit or negative 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 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 ZJ0003 EU
15. ous diseases It has been categorized as a mucosal parasite living within the genito urinary tracts It is a mycoplasma and pathogenic ureolytic mollicute which can cause male urethritis supperative arthritis adverse pregnancy outcomes chorioamnionitis surgical wound infections neonatal meningitis pelvic inflammatory diseases pyelonephritis and neonatal disease Ureaplasma parvum real time PCR kit contains a specific ready to use system for the detection of the Ureaplasma parvum by polymerase chain reaction in the real time PCR system The master contains reagents and ezmymes for the specific amplification of the Ureaplasma parvum DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Ureaplasma parvum DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and genital swabs samples are used for DNA extraction 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 3 Quantitation 4 Kit Contents Type of Reagent DNA Extraction Buffer 2 vials 1 5ml 1 vial 450ul 1 vial 12ul 1 vial 400ul UP Reaction Mix PCR Enzyme Mix Molecular Grade Water Internal Control
16. ple DNA extraction buffer is supplied 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 Wash the genital swabs in 1 0ml normal saline and vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline and suspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can used for PCR template 9 1 2 Urine sample 1 Take 1 5 ml sample to a tube Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 min
17. utes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open for the vapor will volatilize into the air and may cause contamination in case the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C DNA extraction kits are available from various manufacturers You may use your own extraction systems or the commercial kit based on the yield For RNA extraction please comply with 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 dilutions must be prepared first as follows Molecular Grade Water is used for dilution The step of 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 Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards 4ul 4u 4ul To generate a standard curve on the real time system all four dilution standards s
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