Pneumocystis Carinii Real Time PCR Kit User Manual For In Vitro
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 AD 0265 01 For use with LightCycler1 0 2 0 Instrument vi aial 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 of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description At first the name Pneumocystis carinii was2. 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 PCR Protocol The Master Mix volume for each reaction should be pipetted as follow 17 ul 0 4ul 1ul Reaction Mix Enzyme Mix Internal Control a ee 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix gs eal Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1ul 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 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 C for 30sec Fluorescence measured at 60 C 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method
3. 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 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 system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer P Carinii Reaction Mix PCR Enzyme Mix 2 vials 1 5ml 1 vial 450u1 1 vial 12ul 1 vial 400u1 1 vial 30ul 1 vial 30ul Molecular Grade Water Internal Control IC P Carinii Positive Control Analysis sensitivity 1 X 10 copies ml Note An
4. als 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 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 thoroughly and spin down briefly in the centrifuge before use 9 1 1 Bronchial lavage sample 1 Take 400ul sample in a tube and centrifuge the tube at 13000rpm for 2min Remove the supernatant and keen the sediment for nrocessing 2 Add 100ul DNA extraction buffer in the tube sedime
5. alysis 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 Trypsin Digestive Solution e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Refrigerator and Freezer 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 1000u1 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 materi
6. nt 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 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
7. 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid 560nm Molecular Grade Water 25 35 12 Data Analysis and Interpretation The following results are possible Crossing point value 560nm 25 35 Below the detection limit or negative Selection of fluorescence channels Target Nucleic Acid Result Analysis Positive 5 25 35 Re test if it is still 35 40 report as 1 Blank 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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