Enterotoxigenic E. Coli (ETEC) Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Enterotoxigenic E Coli ETEC Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only DD 0092 01 For use with LightC ycler1 0 2 0 Instrument Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net CE asal Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan road PuJiang Hi tech Park Shanghai China 1 Intended Use ETEC real time PCR kit is used for the detection of ETEC 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 after2. 0u1 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 Ws 1 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 Extrac
3. ately 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 94 C for 2min cycle asda 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 and internal control must be performed correctly otherwise the sample results is invalid oa e J e ee ee a Positive Control qualitative ssa 35 13 Data Analysis and Interpretation The following results are possible een B h li ti A Positive and it is of producing heat stable enterotoxin lt 35 lt 35 Positive and it is of producing 7 heat labile enterotoxin Selection of fluorescence channels 35 40 Re test If it is still 35 40 report as 1 530nm ae s4 amp A amp B Blank PCR Inhibition No diagnosis can be concluded 560nm For further questions or problems please contact our technical support at trade liferiver com cn
4. 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 pl 0 4ul 14l Reaction Mix Enyzme Mix Internal Control as 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix Sie Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1u 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 2ul DNA sample positive and negative controls to different reaction plate tubes Immedi
5. it 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 ETEC Reaction Mix A 1 vial 450u1 ETEC Reaction Mix B 1 vial 450u1 PCR Enzyme Mix 1 vial 22u1 Molecular Grade Water 1 vial 400ul Internal Control IC 1 vial 55ul ETEC 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 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 5u1 100
6. the amplification 3 Product Description Enterotoxigenic Escherichia coli ETEC is an important cause of diarrhea in young children in the developing world as well as in travelers to these areas ETEC comprise a relatively small proportion of the species and have been etiologically associated with diarrheal illness of all age groups from diverse global locations The organism frequently causes diarrhea in infants in less developed countries and in visitors there from industrialized countries The etiology of this cholera like illness has been recognized for about 20 years ETEC spreads though contaminated water and food It causes diarrhea by producing different combinations of the heat labile LT or heat stable ST enterotoxins and 1 or more of at least 22 different colonization factors which contribute to the virulence of the pathogen Enterotoxigenic E Coli ETEC real time PCR kit contains a specific ready to use system for the detection of the Enterotoxigenic E Coli ETEC by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the ETEC DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified ETEC 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 k
7. tion 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 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
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