(EPEC) Real Time PCR Kit
Contents
1. IYD Revision No ZJ0008 EU C Issue Date Jul 1 2015 For Research Use Only In USA amp China Enteropathogenic E Coli EPEC Real Time PCR Kit User Manual 20 C y For use with LightCycler1 0 2 0 Instrument eS MBS598053 Instrument I II 1 Intended Use Enteropathogenic E Coli EPEC real time PCR kit is used for the detection of EPEC in stool 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 Enteropathogenic Escherichia coli EPEC is a leading cause of infantile diarrhea in developing countries It is defined as E coli belonging to serogroups epidemiologically implicated as pathogens but whose virulence mechanism is unrelated to the excret2. 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 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 pl 0 4yl ipl Reaction Mix Enyzme Mix Internal Control re 18 4 ul Master Mix 2 ul 18 pl Extraction DNA Master Mix jpm Reaction Plate Tube l PCR Instrument X PCR system without 560nm channel may be treated with 1 ul 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 18ul Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 2u 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 leycle leycle
3. 0 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 lt 35 QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible FAM BVE 88 UNDET Below the detection limit or 25 35 negative Positive and it is of typical strains py FAM Positive and it is of atypical B UNDET strains 544 FAM amp HEX VIC JOE Aon UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support PNA extraction buffer is supplied in BSR ES PAHA USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
4. 1 Take about 50mg stool 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 50ul 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 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 DNA extraction please comply with
5. 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid a See ei N Positive Control qualitative assay lt 35 12 Data Analysis and Interpretation The following results are possible Channel Reacuon Crossine port Result Analysis Mix value ee Below the detection limit or a 5600mm AOR 25 35 negative Positive and it is of typical strains 40cycles sam Positive and it is of atypical B Blank i strains 530nm 5 St AorB Blank 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 ZJ0008 EU Issue Date Jul 1 2015 For Research Use Only In USA amp China Enteropathogenic E Coli EPEC Real Time PCR Kit User Manual 20 C MBS598053 Instrument III IV YY 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 LightCycl
6. EPEC Reaction Mix A 1 vial 450ul EPEC Reaction Mix B 1 vial 450pl PCR Enzyme Mix 1 vial 22u1 Molecular Grade Water 1 vial 400ul Internal Control IC 1 vial 55ul EPEC Positive Control 1 vial 60u1 Analysis sensitivity 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 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 1000u1 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 Z Warnings and Precaution e Carefully
7. Internal Control IC 1 vial 55ul EPEC Positive Control 1x10 copies ml 1 vial 60ul Analysis sensitivity 5X 10 copies ml LOQ 1X 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 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 e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000p1 e Sterile microtubes Ve Z Warnings and Precaution e Carefully read this instruction before starting the pro
8. aster Mix 4ul 36yl 2 5 ul 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 gt PCR system without HEX VIC JOE channel may be treated with 111 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 n the number of reaction Mix completely then spin down briefly in a centrifuge 2 Pipet 36p1 22 51 for SmartCycer II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4pl 2 5u1 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 Icycle Selection of fluorescence channels Target Nucleic Acid 94 C for 2min Icycle 93 C for 15sec 60 C for Imin Fluorescence measured at 60 C Smart Cycler ll HEX VIC JOE AOcycles 5 Ar you use ABI Prism system please choose none as passive reference and quencher 1
9. cedure 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 in the centrifuge before use It s better to use commercial kits for nucleic acid extraction 9 1 1 Stool samples 1 Take about 50mg stool samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes careful
10. e frequency of these organisms has decreased but they continue to be an important cause of diarrhea The central mechanism of EPEC pathogenesis is a lesion called attaching and effacing A E which is characterized by microvilli destruction intimate adherence of bacteria to the intestinal epithelium pedestal formation and aggregation of polarized actin and other elements of the cytoskeleton at sites of bacterial attachment Enteropathogenic E Coli EPEC real time PCR kit contains a specific ready to use system for the detection of the EPEC by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the EPEC DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified EPEC DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and excreta or water samples are used for the extraction of the DNA 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 DNA Extraction Buffer 2 vials 1 5ml EPEC Reaction Mix A 1 vial 950ul EPEC Reaction Mix B 1 vial 950ul 1 vial 22ul 1 vial 400pl
11. er 480 Instrument 1 Intended Use Enteropathogenic E Coli EPEC real time PCR kit is used for the detection of EPEC in stool 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 Enteropathogenic Escherichia coli EPEC is a leading cause of infantile diarrhea in developing countries It is defined as E coli belonging to serogroups epidemiologically implicated as pathogens but whose virulence mechanism is unrelated to the excretion of typical E coli enterotoxins E coli are Gram negative rod shaped bacteria belonging the family Enterobacteriaceae Source s and prevalence of EPEC are controversial because foodborne outbreaks are sporadic In industrialized countries th
12. ion of typical E coli enterotoxins E coli are Gram negative rod shaped bacteria belonging the family Enterobacteriaceae Source s and prevalence of EPEC are controversial because foodborne outbreaks are sporadic In industrialized countries the frequency of these organisms has decreased but they continue to be an important cause of diarrhea The central mechanism of EPEC pathogenesis is a lesion called attaching and effacing A E which is characterized by microvilli destruction intimate adherence of bacteria to the intestinal epithelium pedestal formation and aggregation of polarized actin and other elements of the cytoskeleton at sites of bacterial attachment Enteropathogenic E Coli EPEC real time PCR kit contains a specific ready to use system for the detection of the EPEC by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the EPEC DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified EPEC DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and excreta or water samples are 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 4 Kit Contents Type of Reagent DNA Extraction Buffer 2 vials 1 5ml
13. ly remove and discard supernatant from the tube without disturbing the pellet 2 Add 100u1 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 50ul 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 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 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 allo
14. 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 supplied 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 samples
15. ws the user to determine and control the possibility of PCR inhibition Add the internal control IC 1 pl rxn and the result will be got in the HEX VIC JOE 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 Aul Aul To generate a standard curve on the real time system all four dilution standards should be i used and defined as standard with l specification of the corresponding i concentrations ji y Attention 6 5 i A Mix thoroughly before next transfer coll TAT re o PEU akai 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 follows 35ul 0 4pl ipl 21 5 pl 0 4pl ipl Reaction Mix Enzyme Mix internal Control Reaction Mix Enzyme Mix Internal Control 36 4ul 22 94 Master Mix M
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