Pneumocystis Carinii Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJ0002 Issue Date Jul 1 2012 Pneumocystis Carinii Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C AD 0265 02 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument eal 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 Pneumocystis Carinii real time PCR kit is used for the detection of pneumocystis carinii in bronchial lavage sample or lung section sample from rats or other animals 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 is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of2. 35ul 0 4ul 1p Reaction Mix Enzyme Mix Internal Control 21 5 ul 0 4 ul ipl ee Reaction Mix Enzyme Mix Internal Control 36 4 E aae Master Mix 22 9 ae ee Master Mix 4ul 36 2 5 ul 22 5ul Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube X This system is only for PCR Instrument OR PCR Instrument mart Cycler Il XPCR system without HEX VIC JOE channel may be treated with 1p Molecular Grade Water instead of Il IC 1 The volumes of Super 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 36ul 22 5u1 for SmartCycler IT Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Separately add 4ul 2 5ul for SmartCycler II 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 93 C for 15sec 60 C for 1min Fluorescence measured at 60 C 5 A If you use ABI Prism system please choose none as passive reference and quencher 10 Threshold setting just above
3. Contents Type of reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml P Carinii Reaction Mix 1 vial 950u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30u1 P Carinii 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 e Cool all reagents during the working steps e Super Mix and Reaction Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Vortex mixer 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 PCR Enzyme Mix Molecular Grade Water e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5u
4. he 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 HEX VIC JOE 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR standard dilution must be prepared first as follows Molecular Grade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR 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 4ul dul 4ul Y WY V Y 1X107 1X10 1X10 1X 104 copiestm To generate a standard curve on the real time system all four dilution standards should be used and defined as standards with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive control 1x10 copies ml 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 be pipetted as follows pip
5. l 1000p1 e Sterile microtubes 7 Warni 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 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 Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C 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 9 1 1 Bronchial lavage sample 1 Take 400ul sam
6. ple in a tube and centrifuge the tube at 13000rpm for 2min Remove the supernatant and keep the sediment for processing 2 Add 100ul DNA extraction buffer in the tube sediment 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 2 Lung section sample 1 Wash the lung tissue with sterile saline for several times 2 Take 50mg sample in a tube add 1ml sterile saline and grind the tissue into homogenate 3 Transfer the homogenate to a 1 5ml tube and centrifuge the tube at 13000rpm for 5min Remove the supernatant and keep the sediment for processing 4 Add 100ul DNA extraction buffer in the tube sediment close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 5 Incubate the tube for 10 minutes at 100 C 6 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 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 t
7. the accumulating product without having to re open the reaction tube after the amplification 3 Product Description At first the name pneumocystis carinii was applied to the organisms found in both rats and humans as it was not yet known that the parasite was host specific Since 2011 the name pneumocystis carinii only applies to the pneumocystis species that is found in rats or other animals Pneumocystis is a source of opportunistic infection which can cause a lung infection with a weak immune system Pneumocystis carinii real time PCR kit contains a specific ready to use system for the detection of the pneumocystis carinii by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the pneumocystis carinii DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified pneumocystis carinii DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and bronchial lavage sample or lung section sample is used for the extraction of the DNA In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE fluorescence of the internal control IC An external positive control 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit
8. the maximum level of molecular grade water 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 internal control and QS curve must be performed correctly otherwise the sample results is invalid Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE Ct value HEX VIC JOE Molecular Grade Water UNDET 5 35 Positive Contol qualiatve may S35 SSS QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Ct value ena o UNDET 25 35 Below the detection limit or negative Positive and the software displays the quantitative value Re test if it is still 38 40 report as 1 apes S 38 40 25 35 UNDET UNDET PCR Inhibition no diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn
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