Pneumocystis Jirovecii Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJ0009 Issue Date Jul 1 2012 Pneumocystis Jirovecii Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only QD 0082 01 For use with LightCycler1 0 2 0 Instrument Ec ner 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 C Vs I aal 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 Jirovecii real time PCR kit is used for the detection of Pneumocystis Jirovecii in bronchial lavage sample or lung section sample from human 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 of the accumul2. 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 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 can Crossing point value Molecular Grade Water 25 35 Positive Control qualitative assay 85 ee QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Crossing point value 25 35 Below the detection limit or negative Selection of fluorescence channels Target Nucleic Acid Result Analysis 2 s35 Positive and the software displays the quantitative value 35 40 25 35 Re test if it is still 35 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
3. 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 please thaw the buffer thoroughlv and spin down brieflv in the centrifuge before use 9 1 1 Bronchial lavage sample 1 Take 400u1 sample 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 extrac
4. ating product without having to re open the reaction tube after the amplification 3 Product Description Pneumocystis jirovecii is a yeast like fungus of the genus Pneumocystis It is an important human pathogen among immunocompromised hosts 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 In 1976 the name Pneumocystis jirovecii or jiroveci was proposed to distinguish the organism found in humans from variants of Pneumocystis in other animals It is the most common opportunistic infection in persons with HIV infection P jirovecii is now one of several organisms known to cause life threatening opportunistic infections in patients with advanced HIV infection worldwide Pneumocystis Jirovecii real time PCR kit contains a specific ready to use system for the detection of the Pneumocystis Jirovecii by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Pneumocystis Jirovecit DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Pneumocystis Jirovecii DNA fragment is performed in fluorimeter channel 530nm 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 syste
5. d as standard with specification of the corresponding concentrations Attention y A Mix thoroughly before next A F 6 r 1X107 1X10 1X10 1X 104 copies mi 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 be pipetted as follow 17y 0 4yl 1ul Reaction Mix Enzyme Mix Internal Control aa TT 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix i el Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with lu Molecular Grade Water instead of 11 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 Separately add 2ul 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 5sec 60
6. e gloves powderless e Refrigerator and Freezer e Trypsin digestive Solution e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000p1 e Sterile microtubes e Biohazard waste container 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 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 and smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and Transport e Collected samples in sterile
7. m to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC An external positive control 1x10 copies ml contained allows the determination of the gene load 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer 2 vials 1 5ml 1 vial 450ul 1 vial 12ul 1 vial 400u1 P Jirovecii Reaction Mix PCR Enzyme Mix Molecular Grade Water Internal Control IC 1 vial 30ul P Jirovecii Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X 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 Super 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 Disposabl
8. ted 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 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 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR standard dilutions must be prepared firstly as follows Molecular Grade Water is used as the 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 Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards To generate a standard curve on Au l Anl 4 ul the real time system all four i on a dilution standards should be used and define
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