Revision No - MyBioSource
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1. 7 ZN 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 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 in the kit Please thaw the buffer thoroughly and spin down briefly in the centrifuge before use It s better to use commercial kits for nuclei2. IYD Revision No ZJ0005 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Cryptosporidium Real Time PCR Kit User Manual 20 C MBS598073 Instrument I II YY For use with LightCycler1 0 2 0 instrument j 1 Intended Use Cryptosporidium real time PCR kit is used for the detection of Cryptosporidium in stool vomit or water 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 Cryptosporidium is a coccidian protozoan parasite that has gained much attention in the last 20 years as a clinically important human pathogen Electron microscopic examination of the intestinal mucosa led to the discovery that Cryptosporidium parvum was the infectious specie
3. kit is used for the detection of Cryptosporidium in stool vomit or water 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 Cryptosporidium is a coccidian protozoan parasite that has gained much attention in the last 20 years as a clinically important human pathogen Electron microscopic examination of the intestinal mucosa led to the discovery that Cryptosporidium parvum was the infectious species in humans In the early 1980s the strong association between cases of cryptosporidiosis and immunodeficient individuals such as those with AIDS acquired immunodeficiency syndrome brought Cryptosporidium to the forefront as a ubiquitous human pathogen Little is known about the pathogenesis of t
4. C 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 and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix the master mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 51 for SmartCycer I Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4pl 2 51 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 Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE Smart Cycler ll 93 C for 15sec 60 C for Imin died Fluorescence measured at 60 C oes 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 inter
5. alibration 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 a ca QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible O pe FE E S Result Analysis 25 35 Below the detection limit or negative PCR Instrument Target Nucleic Acid 2 lt 35 Positive and the software displays the quantitative value 35 40 25 35 Re test If it is still 35 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 IYD Revision No ZJ0005 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Cryptosporidium Real Time PCR Kit User Manual 20 C MBS598073 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 j 1 Intended Use Cryptosporidium real time PCR
6. ble centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Water samples 1 Take 3 ml water 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 100ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 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 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 DNA extraction kits are available You may 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 got in the HEX VIC JOE channel 9 3 Quantitation The kit can be used for quantitative or
7. c acid extraction 9 1 1 Stool or vomit samples 1 Take about 50mg samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100ul DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Water samples 1 Take 3 ml water 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 100ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 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 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 DNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For the DNA ext
8. epeated 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 e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 37 C for 2min 94 C for 2min 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 It s better to use commercial kits for nucleic acid extraction 9 1 1 Stool or vomit samples 1 Take about 50mg samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100ul DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a ta
9. he parasite and no safe and effective treatment has been successfully developed to combat cryptosporidiosis Unlike other intestinal pathogens Cryptosporidium can infect several different hosts can survive most environments for long periods of time due to its hardy cyst and inhabits all climates and locales Cryptosporidium real time PCR kit contains a specific ready to use system for the detection of the Cryptosporidium through polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Cryptosporidium DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Cryptosporidium DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 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 3Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml CPS Reaction Mix 1 vial 950ul 1 vial 12ul 1 vial 400u1 1 vial 30ul 1 vial 30u1 Analysis sensitivity 1X 10 copies ml LOQ 2X 10 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 dozen
10. hould be pipetted as follows ue ae ha XPCR system without 560nm channel be treated Reaction Mix Enyzme Mix intemal Control system withou nm channel may be treate with lul Molecular Grade Water instead of lul IC ve mu 1 The volumes of Reaction Mix and 18 4 pl Enzyme Mix per reaction multiply with Master Mix the number of samples which includes the number of the controls standards and 2 ul 18 pl sample prepared Molecular Grade Water Extraction DNA Master Mix is used as the negative control For ii T a reasons of unprecise pipetting always Reaction add an extra virtual sample Mix the Plate Tube master mix completely then spin down briefly in a centrifuge 2 Pipet 18ul Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 2ul 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 S5sec 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 C
11. nal 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 lt 35 QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value HEX VIC JOE Result Analysis Dae lt 35 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
12. qualitative real time PCR A positive control defined as 1x10 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 The step of 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 Aul Apt Aut 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 1x10 copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination Y 1X10 1X10f 1X10 1X 10i copisti 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35pl 0 4pl ipl 21 5 pl 0 4pl ipl Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4ul 22 9 Master Mix Master Mix 4ul 36yl 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 PCR Instrument OR XPCR system without HEX VI
13. r 2 vials 1 5ml CPS Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400u1 1 vial 30ul 1 vial 30ul Analysis sensitivity 1X 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 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 PCR Enzyme Mix Molecular Grade Water Internal Control CPS Positive Control 1 lt 10 copies ml e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 51 10001 e Sterile microtubes
14. raction 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 got in the 560nm channel 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control defined as 1x10 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 The step of 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 Aul Aud Aul To generate a standard curve on the real time system all four dilution standards should be iA j used and defined as standard with specification of the corresponding concentrations VO y y Attention A Mix thoroughly before next transfer 1X107 1X10f 1X10 1X10 copicsimi B The positive control 1 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 s
15. s in humans In the early 1980s the strong association between cases of cryptosporidiosis and immunodeficient individuals such as those with AIDS acquired immunodeficiency syndrome brought Cryptosporidium to the forefront as a ubiquitous human pathogen Little is known about the pathogenesis of the parasite and no safe and effective treatment has been successfully developed to combat cryptosporidiosis Unlike other intestinal pathogens Cryptosporidium can infect several different hosts can survive most environments for long periods of time due to its hardy cyst and inhabits all climates and locales Cryptosporidium real time PCR kit contains a specific ready to use system for the detection of the Cryptosporidium through polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Cryptosporidium DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Cryptosporidium DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 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 3Quantitation 4 Kit Contents DNA Extraction Buffe
16. s 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 PCR Enzyme Mix Molecular Grade Water Internal Control CPS Positive Control 1 lt 10 copies ml e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 7 Z 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 r
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