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RD-0181-01

1. Nucleic Acid 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
2. OD sveriver vision No ZJ0001 1e Date Jul 1 2012 C s ebsiella pneumoniae Kpn Real Time PCR Kit User Manual r In Vitro Diagnostic Use Only 20 C 3s EF RD 0181 01 ruse with LightCycler 1 0 2 0 Instrument m Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 Obelis S A trade liferiver com cn Fax 86 21 34680595 ilevard G n ral Wahis 53 2 floor No 15 Building No 188 Xinjunhuan Road 10 Brussels BELGIUM 32 2 732 59 54 PuJiang Hi tech Park Shanghai China 32 2 732 60 03 Aail mail obelis net ntended Use bsiella pneumoniae real time PCR kit is used for the detection of Klebsiella pneumoniae in samples nasal and pharyngeal secretions sputum bronchial lavage lung biopsy pleural effusion and etc Principle of Real Time PCR gt principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR ction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the ncher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent ial generated by the cleaved reporter dye which is monitored real time by the PCR detection system gt PCR cycle at which an increase in the fluorescence signal is detected initially Ct is proportional to amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows detection of the accumulating product without having to re
3. e It s better to use commercial kits for nucleic acid extraction 1 Sputum sample rypsin digestive Solution preparation q 10g trypsin to 200ml sterile purified water and mix thoroughly Adjust the PH value to 8 0 with NaOH solution Add 2mL 25mmol L CaCl mix thoroughly and store at 4 C Please incubate at 37 C 10 minutes before use 4stimate the volume of the sputum and add partes aequales of the trypsin digestive solution then vortex orously Set at room temperature for 30 minutes Transfer 0 5ml mixture to a new tube Centrifuge the e at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing pellet 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 S down briefly in a table centrifuge 6 Incubate the tube for 10 minutes at 100 C 7 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 2 Fluid samples nasal and pharyngeal secretions and etc 1 Take 1ml sample in a tube centrifuge the tube at 13000rpm for 2min and remove the supernatant anc keep the pellet 2 Add 100u DNA extraction buffer to the pellet close the tube then vortex for 10 second
4. ecular Grade Water e Tube racks 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 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 transport of etiologic agents gt rocedure DNA Extraction A extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the trifuge before us
5. lt it Contents DNA Extraction Buffer 2 vials 1 5ml Kpn Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul GAS Positive Control 1x10 copies ml 1 vial 30ul alysis sensitivity 1 X 10 copies ml LOQ 210 1X 10 copies ml te Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods other factors If you use the DNA extraction buffer in the kit the analysis sensitivity is the same as it lares However when the sample volume is dozens or even hundreds of times greater than elution ume by some concentrating method it can be much higher 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 dditionally Required Materials and Devices e Biological cabinet e Real time PCR system e Trypsin digestive Solution e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 ul e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g PCR Enzyme Mix Mol
6. open the reaction tube after the olification gt roduct Description bsiella pneumoniae is a Gram negative non motile encapsulated lactose fermenting facultative erobic rod shaped bacterium found in the normal flora of the mouth skin and intestines K umoniae can cause the disease Klebsiella pneumonia They cause destructive changes to human lungs ammation and hemorrhage with cell death necrosis that sometimes produces a thick bloody mucoid tum currant jelly sputum Typically these bacteria gain access after a person aspirates colonizing pharyngeal microbes into the lower respiratory tract bsiella pneumoniae real time PCR kit contains a specific ready to use system for the detection of bsiella pneumoniae by polymerase chain reaction PCR in the real time PCR system The master tains reagents and enzymes for the specific amplification of the Klebsiella pneumoniae DNA orescence is emitted and measured by the real time systems optical unit during PCR The detection of olified Klebsiella pneumoniae DNA fragment is performed in fluorimeter channel 530nm with the yrescent quencher BHQ1 DNA extraction buffer is available in the kit In addition the kit contains a tem to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control An external positive control 1x10 copies ml contained allows the determination of the gene load further information please refer to section 9 3 Quantitation
7. s 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 be used for PCR template 9 1 3 Tissue sample 1 Wash the sample lung biopsy in 0 5ml normal saline and vortex vigorously Centrifuge at 13000r for 2 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100ul 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 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 lt 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 your own extract systems or the commercial kit based on the yield For the DNA extraction please comply with 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 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 3Q
8. uantitation 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 follo 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 pipe 36ul of Molecular Grade Water into next th Dilution of Standards tubes Do three dilutions as the follow Aul Aul Au figures To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specificatio y y y y of the corresponding concentrations Attention 1X107 1X10 1X10 1X10fcopies m A Mix thoroughly before next transfer B The positive control 1x10 copies ml contains high concentration of the target DNA Therefore 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 17 ul 0 4ul ul Reaction Mix Enyzme Mix Internal Control a ee 18 4 ul Master Mix ki 2 ul 18 ul Extraction DNA Master Mix oa cal 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 sampl which includes the n
9. umber of controls standards and sample prepared Molecular Grade Wate used as the negative control For reasons of unprecise pipetting always add an extra virtual samt 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 react plate tubes Separately add 2ul DNA sample positive and negative controls to different react 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 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 adj the Noise band just above the maximum level of molecular grade water and adjust the threshold just un the minimum of the positive control 11 Calibration for quantitative detection Input each concentration of standard controls at the end of 1 and a standard curve will be automatically formed 12 Quality control Negative control positive control internal control and QS curve must be perforn correctly otherwise the sample results is invalid ee Crossing point value Molecular Grade Water Positive Conrolqualitiveassayy 5d 13 Data Analysis and Interpretation The following sample results are possible eae Selection of fluorescence channels Target

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RD 0181 01

RD-0181-01

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