User Manual For In Vitro Diagnostic Use Only ED-0216
Contents
1. Liferiver Revision No ZJ0001 Issue Date Jul 1 2012 Plasmodium Malariae Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only ED 0216 01 For use withLightC ycler1 0 2 0 Instrument Eo rer 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 CE Vos 1 rw N 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 The Plasmodium malariae real time RT PCR kit is used for the detection of Plasmodium malariae in whole blood or mosquito samples 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 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 to2. 25 35 55 QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Crossing point value 530nm 25 35 Below the detection limit or negative Selection of fluorescence channels Target Nucleic Acid 40cycles 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
3. 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 RT 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 Aul Aul 4ul Y VY V Y 1X107 1X10 1X10 1X 104 copiesimi 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 17 ul 0 4ul 1 Reaction Mix Enzyme Mix Internal Control i 18 4 ul Master Mix 2 ul 18 ul Extraction DNA Master Mix al Reaction Plate Tube l PCR Instrument amp PCR system without 560nm channel may be treated with 1 Molecular Grade Water instead of 11 IC 1 The v
4. re open the reaction tube after the amplification 3 Product Description Malaria is one of the leading causes of disease and death in the world It is estimated that there are 300 500 million new cases every year with 1 5 to 2 7 million deaths worldwide Malaria is a potentially fatal tropical disease that is caused by a parasite known as Plasmodium Four kinds of malaria parasites can infect humans P falciparum P vivax P ovale and P malariae It is spread through the bite of an infected female mosquito P malariae is closely related to Plasmodium falciparum and Plasmodium vivax which are responsible for most malarial infection It is a so called benign malaria and is not nearly as dangerous as that produced by P falciparum or P vivax P malariae causes fevers that recur at approximately three day intervals a quartan fever longer than the two day tertian intervals of the other malarial parasites hence its alternate name quartan malaria The Plasmodium malariae real time PCR Kit contains a specific ready to use system for the detection of the Plasmodium malariae through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Plasmodium malariae DNA Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Plasmodium malariae DNA fragment is performed in fluorimeter channel 530nm with the fluo
5. olumes 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 of the 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 leycle cycle 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 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 Channel Crossing point value Control 530nm 560nm Molecular Grade Water Blank
6. ortation 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 contained in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or the commercial kit 1 Pipet 100u1 whole blood or one two mosquito es sample to a 0 5ml tube add 100ul DNA extraction buffer close the tube and vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubation the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and is 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 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 brand DNA extraction kits are available You can also 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
7. rescent quencher BHQ1 DNA extraction buffer is available in the kit 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 defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents DNA Extraction Buffer 2 vials 1 5ml P malariae Reaction Mix 1 vial 450u1 1 vial 12ul 1 vial 400u1 1 vial 30u1 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 1X10 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 microcen
8. trifuge 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 10001 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 PCR Enzyme Mix Molecular Grade Water Internal Control P malariae Positive Control 1x10 copies ml 7 AN Warming 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 Quickly prepare 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 8 Sample Collection Storage and transp
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