Enteroadhesive E. Coli (EAEC) Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Enteroadhesive E Coli EAEC Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C 3s DD 0094 01 atal Shanghai ZJ Bio Tech Co Ltd For use with LightCycler 1 0 2 0 Instrument www liferiver com cn Tel 86 21 34680596 ec ner Obelis S A trade liferiver com cn Fax 86 21 34680595 Boulevard G n ral Wahis 53 2 floor No 15 Building No 188 Xinjunhuan road 1030 Brussels BELGIUM Tel 32 2 732 59 54 PuJiang Hi tech Park Shanghai China Fax 32 2 732 60 03 E Mail mail obelis net 1 Intended Use Enteroadhesive E Coli EAEC real time PCR kit is used for the detection of Enteroadhesive E Coli EAEC 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 accumu2. 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 EAEC Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul EAEC Positive Control 1x10 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 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 m
3. 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 shown in the 560nm 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR 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 Y VY V Y 1X107 1X10 1X10 1X 104 copiesimi 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 du
4. ic 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 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 100u1 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
5. icrocentrifuge 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 10001 e Sterile microtubes 7 N 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 etiolog
6. lating product without having to re open the reaction tube after the amplification 3 Product Description Enteroaggregative E coli EAEC another DEC pathotype is an emerging pathogen that causes diarrhea in many parts of the world It is identified by the characteristic aggregative pattern of adherence AA in HeLa HEp 2 cells bacteria attach in aggregates to cell surfaces as well as around cells EAEC colonizes the intestinal mucosa forming a thick biofilm that favors prolonged colonization and induces malnutrition Actually this pathotype is heterogeneous regarding the presence of putative virulence genes and has recently been subgrouped into typical and atypical EAEC which carry and lack AggR a global regulator of EAEC virulence respectively Enteroadhesive E Coli EAEC real time PCR kit contains a specific ready to use system for the detection of the Enteroadhesive E Coli EAEC by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the EAEC DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified EAEC 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
7. ring the dilution in order to avoid contamination 9 4 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 yl 0 4ul ipl Reaction Mix Enyzme Mix Internal Control 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix a a Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1u1 Molecular Grade Water instead of 1ul 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 241 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 94 C for 2min Icycle 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 maxim
8. um 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 tng Crossing point value Molecular Grade Water 25 35 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 Crossing point value 25 35 Below the detection limit or negative Target Nucleic Acid 40cycles Result Analysis 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 at trade liferiver com cn
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