Enteropathogenic E. Coli (EPEC) Real Time PCR Kit User Manual
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Enteropathogenic E Coli EPEC Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C 3s DD 0091 01 atal Shanghai ZJ Bio Tech Co Ltd For use with LightCycler1 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 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 with2. encher 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 Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml EPEC Reaction Mix A 1 vial 450u1 EPEC Reaction Mix B 1 vial 450u1 PCR Enzyme Mix 1 vial 22u1 Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 55ul EPEC Positive Control 1 vial 60ul Analysis sensitivity 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 Real time PCR system e Desktop microcentrifuge
3. 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 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 Tra
4. ime 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 Target Nucleic Acid 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 ae a Crossing point value 530nm Molecular Grade Water 25 35 Positive Control qualitative assay Ea Fee 12 Data Analysis and Interpretation The following results are possible B Channel meaeucn Result Analysis Mix value Below the detection limit or Positive and it is of typical strains Positive and it is of atypical PB Blank strains 4 A amp B 35 40 oe If it is still 35 40 report as or B s4 530nm amp 560nm A Blank PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn 4 530
5. nm
6. nsportation 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 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 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 va
7. out 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 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 qu
8. por 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 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 ul 0 4ul 1ul Reaction Mix Enyzme Mix Internal Control 18 4 pl Master Mix 2 ul 18 ul Extraction DNA Master Mix DN Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1ul 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 18u1 Master Mix with micropipets of sterile filter tips to each Real t
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