Toxoplasmosis Gondii Real Time PCR Kit User Manual For
Contents
1. Liferiver Revision No ZJO006 Issue Date Jul 1 2012 Toxoplasmosis Gondii Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C s ZD 0075 01 For use with LightCycler2 0 Instrument 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 Toxoplasma gondii real time PCR kit is used for the detection of toxoplasma gondii in blood C S F or stool 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 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 Toxoplasmosis is a disease provoked by the obligate intracellular protozoan toxoplasma gondii It i2. 9 1 2 Stool sample 1 Take about 30mg stool samples to a 1 5ml tube add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 100u DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 3 Incubate the tube for 10 minutes at 100 C 4 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 store at 20 C for one month C Different brand of DNA extraction kits are available The customer can 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 lul rxn and the result will be shown in the 560nm Channel 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 17 ul 0 4ul 1yl Reaction Mix Enzyme Mix Internal Co
3. 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 5 ul 1000 ul 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 Aarna 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 andshould 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 free
4. ntrol i 18 4 ul Master Mix 2ul 18 ul Extraction DNA Master Mix S Reaction Plate Tube l PCR Instrument XPCR system without 560nm channel may be treated with 1u Molecular Grade Water instead of 1ul 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 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 37 C for 2min Selection of fluorescence channels Target Nucleic Acid 94 C for 2min 93 C for 5sec 60 C for 30sec AOcycles 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 Quality control Negative control positive control internal co
5. ntrol must be performed correctly otherwise the sample results is invalid Molecular Grade Water Positive Control qualitative assay o ce 12 Data Analysis and Interpretation The following results are possible Crossing point value f Result Anal n 530nm 560nm eens aa 25 35 Below the detection limit or negative pa es f Positive 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
6. s found in a variety of mammal and bird hosts The most common intermediate host is the cat It is one of the most frequent causes of retinochoroiditis in humans with more than 60 percent of the United States population and up to 75 percent of the world s general population possessing some seropositive findings Toxoplasmosis gondii real time PCR kit contains a specific ready to use system for the detection of toxoplasmosis gondii by polymerase chain reaction in the real time PCR system The master contains reagents and enzyme for the specific amplification of toxoplasmosis gondii DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Toxoplasmosis gondii DNA fragment is performed in fluorimeter channel 530nm with the fluorescent 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 4 Kit Contents Type of Reagent Presentation 25rxns DNA Extraction Buffer TOXO Reaction Mix PCR Enzyme Mix 2 vials 1 5ml 1 vial 450u1 1 vial 12ul 1 vial 400u1 1 vial 30u1 1 vial 30ul Molecular Grade Water Internal Control IC TOXO Positive Control 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
7. zing 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 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 please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or commercial kits 9 1 1 Blood or S C F sample 1 Pipet 100u1 sample to a 0 5ml tube add 100ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 2 Incubate the tube for 10 minutes at 100 C 3 Centrifuge the tube at 13000rpm for 10 minutes The supernatant contains the DNA extracted and can be used for PCR template
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