Legionella Pneumophila Real Time PCR Kit User Manual
Contents
1. Liferiver Revision No ZJO008 Issue Date Jul 1 2012 Legionella Pneumophila Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C s RD 0057 01 m Shanghai ZJ Bio Tech Co Ltd For use with LightCycler1 0 2 0 Instrument www liferiver com cn Tel 86 21 34680596 Eo rer 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 By using real time PCR systems legionella pneumophila real time PCR kit is used for the detection of legionella pneumophila in samples like nasal and pharyngeal secretions and swabs sputum provoked sputum bronchial lavage lung biopsy and etc 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 fluo2. 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 Sputum sample 1 Trypsin digestive Solution preparation Add 10g trypsin to 200ml sterile purified water and mix thoroughly Adjust the PH value to 8 0 with 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 2 NaOH solution Add 2mL 25mmol L CaCl mix thoroughly and store at 4 C Please incubate at 37 C for 10 minutes before use 2 Estimate the volume of the sputum and add partes aequales of the trypsin digestive solution then vortex vigorously Set at room temperature for 30 minutes Transfer 0 5ml mixture to a new tube Centrifuge the tube at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 1 0ml normal saline Resuspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 4 Repeat step 3 5 Add 50ul DNA extraction buffer closed the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 6 Incubate the tube for 10 minutes at 100 C 7 Centrifuge the tu
3. 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 aad Crossing point value Molecular Grade Water 25 35 Positive Control qualitative assay 2a o S QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Result Analysis 2 lt 38 Positive and the software displays the quantitative value 38 40 25 35 Re test If it is still 38 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
4. be at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Fluid samples Pleural effusion and etc 1 Take 400u1 3ml for water sample sample in a tube centrifuge the tube at 13000rpm for 2min and remove the supernatant and keep the pellet 2 Add 100u1 DNA extraction buffer to the pellet 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 10 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 3 Tissue and swabs sample 1 Wash the sample lung biopsy or swabs in 1ml normal saline and vortex vigorously Centrifuge at 13000rpm for 2 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100u1 DNA extraction buffer to the tube closed the tube then vortex for 10 seconds 3 Incubation the tube for 10 minutes at 100 C 4 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the extracted DNA and can be used for the template of the PCR Attention A During the incubation make sure the tube is not open Since 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 stored at 20 C for one month C DNA extraction kits are available from various manufacturers You may use you
5. be pipetted as follows X PCR system without 560nm channel may be 174l 0 44 3 pl treated with lul Molecular Grade Water instead of Reaction Mix Enzyme Mix Internal Control lul IC ie 1 The volumes of Reaction Mix and 18 4 ul Enzyme Mix per reaction multiply Master Mix with the number of samples which includes the number of controls 2ul 18 ul standards and sample prepared Extraction DNA Master Mix Molecular Grade Water is used as the ay e negative control For reasons of unprecise pipetting always add an igan extra virtual sample Mix the master Plate Tube i mix completely then spin down briefly in a centrifuge PCR Instrument 2 Pipet 18ul Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Then separately add 2ul DNA sample supernatant positive and negative controls to different plates 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 i 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 C
6. r 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 7 Warnings and Precaution 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 Prepare quickly 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 e Avoid aerosols 8 Sample Collection Storage and transport 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
7. r 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 Channel 9 3Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR standard dilutions must be prepared first as follows Molecular Grade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR detection 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 E MER i real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations y y y y Attention A Mix thoroughly before next transfer 1X107 1X10 1X10 1X104copiesmi B The positive control 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
8. raction 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 1x10 copies ml allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml L pneumophila Reaction Mix 1 vial 450ul 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul L pneumophila Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1X 10 copies ml LOQ 2 X 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 mixe
9. rescence 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 Legionella pneumophila is a motile rod shaped gram negative aerobic bacterium It requires complex nutritional requirements such as high cysteine levels and low sodium levels to grow Legionella pneumophila have always been found in non marine aquatic environments such as lakes and ponds The optimum growth temperature range for this bacteria is 20 45 degrees Celsius The organism has been found to possess the ability to survive in tap water at room temperature for over a year Legionella bacteria are transmitted to the lungs of human beings through a process called aerosilisation Legionella pneumophila real time PCR Kit contains a specific ready to use system for the detection of the legionella pneumophila by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the legionella pneumophila DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified legionella pneumophila DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and samples e g nasal and pharyngeal secretions sputum provoked sputum bronchial lavage lung biopsy are used for DNA ext
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