Chlamydia Pneumoniae Real Time PCR Kit User Manual
Contents
1. Liferiver Revision No ZJ0007 Issue Date Jul 1 2012 Chlamydia Pneumoniae Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C 3s RD 0099 01 For use with LightCycler1 0 2 0 Instrument rw NN 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 By using real time PCR systems chlamydia pneumoniae real time PCR kit is used for the detection of chlamydia pneumoniae in samples like nasal and pharyngeal secretions and swabs sputum provoked sputum bronchial lavage lung biopsy pleural effusion 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 fluorescence intensities during Real Time allows the detection of the accumulating product without having to re open the reaction tube after the amplification2. 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 transport of etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit Attention please thaw the buffer thoroughly and mix the buffer well before use because it contains insoluble particles You may use your own extr
3. the 560nm Channel 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 ul 0 4ul 1p Reaction Mix Enzyme Mix Internal Control ee 18 4 i Master Mix 2 ul 18 ul Extraction DNA Master Mix oo Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1ul Molecular Grade Water instead of 1u1 IC 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of 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 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 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 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
4. 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 Cua e Rc Po e ee Positive Control qualitative assay lt 35 12 Data Analysis and Interpretation The following sample results are possible Crossing point value 530nm 25 35 Below the detection limit or negative Result Analysis a lt 38 f Positive 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
5. 3 Product Description Chlamydia pneumoniae is a species of chlamydiae bacteria that infects humans and is a major cause of pneumonia The full genome sequence for Chlamydia pneumoniae was published in 1999 Chlamydia pneumoniae also infects and causes disease in Koalas emerald tree boa Corallus caninus iguanas chameleons frogs and turtles Chlamydia pneumoniae is a common cause of pneumonia around the world Chlamydia pneumoniae is typically acquired by otherwise healthy people and is a form of community acquired pneumonia Chlamydia Pneumoniae real time PCR Kit contains a specific ready to use system for the detection of the Chlamydia Pneumoniae by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of Chlamydia Pneumoniae DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Chlamydia Pneumoniae 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 extraction 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 4 Kit Con
6. action systems or commercial kits 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 2 NaOH solution Add 2mL 25mmol L CaCl2 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 100u1 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 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 and keep the pellet 2 Add 100u DNA extraction buffer to the pellet close the tube then vortex for 10 sec
7. onds 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 0 5ml 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 as 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 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
8. tents DNA extraction buffer 2 vials 1 5ml CP Reaction Mix 1 vial 450u1 PCR Enzyme Mix 1 vial 12ul Molecular Grade Water 1 vial 4001 Internal Control IC 1 vial 30ul CP Positive control 1x10 copies ml 1 vial 30ul 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 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
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