HCMV Real Time PCR Kit User Manual For In Vitro
Contents
1. Liferiver Revision No ZJO008 Issue Date Jul 1 2012 HCMV Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C OD 0022 02 25 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 ual 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 HCMYV real time PCR kit is used for the detection of CMV in serum plasma leukocyte or urine by using real time PCR systems High sensitivity lower detection line 10 copies ml LOQ 2X 10 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 High specificity test result will be positive only to human cytomegalovirus Short operating time 2 and a half hours totally Good stability kept for 12 months at 20 C CV lt 5 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogeni2. disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 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 for 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 HEX VIC JOE 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR A positive control defined as 1x10 copies ml is supplied in the kit For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution Dilution is not needed for performance of qualitative real time PCR Take positive control 1
3. c 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 to re open the reaction tube after the amplification 3 Product Description Human cytomegalovirus HCMV is a beta human herpesvirus with a genome size of 230 Kbp coding more then 70 viral proteins It is a widespread opportunistic human herpesvirus that causes severe diseases in immune compromised individuals such as organ transplant recipients and AIDS patients HCMV real time PCR kit contains a specific ready to use system for the detection of the human cytomegalovirus by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of HCMV DNA Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified HCMV DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in t
4. he kit and blood serum samples are used for DNA extraction 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 Contents Type of reagent Presentation 25rxns DNA Extraction Buffer 1 vial 1 8ml HCMV Reaction Mix 1 vial 950u1 1 vial 12pl 1 vial 400u1 1 vial 30ul 1 vial 30ul PCR Enzyme Mix Molecular Grade Water Internal Control IC HCMV Positive Control 1x10 copies ml 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended 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 Trypsin digestive Solution e Vortex mixer e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 pl 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 e Desktop microcentrifuge for eppendorf type
5. le Mix completely then spin down briefly in a centrifuge Pipet 36ul 22 51 for SmartCyclerII Master Mix with micropipets of sterile filter tips to each of the Real time PCR reaction plate tubes Separately add 4ul 2 5ul for SmartCyclerII 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 Icycle 94 C for 2min 93 C for 15sec 60 C for Imin AOcycles 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 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 Channel Ct value Control HEX VIC JOE Molecular Grade Water UNDET 25 35 Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value U lt 38 3 2 xr Selection of fluorescence channels Ta
6. rget Nucleic Acid HEX VIC JOE Result Analysis 25 35 Below the detection limit or negative T a e Positive and the software displays the quantitative value 25 35 Re test If it is still 38 40 report as 1 UNDET UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn
7. roughly and spin down briefly in the centrifuge before use It s better to use commercial kits for nucleic acid extraction 9 1 1 Serum or plasma sample 1 Pipet 50ul serum or plasma to a 0 5ml tube add 50ul 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 the template of the PCR 9 1 2 Leukocyte sample 1 Isolate Leukocyte from 1ml non heparin anticoagulant protocol in detail is omitted here 2 Take the liquid with Leukocyte into a new 1 5ml tube centrifuge the tube at 2000rpm for 10 minutes and carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer and close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template 9 1 3 Urine sample 1 Take 1 5 ml sample to a tube Centrifuge the tube at 13000rpm for 2 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline then vortex vigorously Centrifuge the tube at 15000rpm for 5 minutes carefully remove and discard supernatant from the tube without
8. tubes RCF max 16 000 x g 7 A Warnings and Precaution 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 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 tho
9. x10 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 4ul 4ul To generate a standard curve on the real time system all four dilution standards should be used and defined as standard with specification of the corresponding concentrations t Vv V Y Attention 1X107 1X10 1X10 1X 104eopiesim A Mix thoroughly before next transfer 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 follows 35yl 0 4 ipl 21 5 pl 0 4 ul ipl Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix internal Control 36 4 22 9 Master Mix Master Mix 4ul 36yl 2 5 pl 22 5 Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube This system l l is only for PCR Instrument OR PCR Instrument Smart Cycler Il X PCR system without HEX VIC JOE channel may be treated with 11 Molecular Grade Water instead of 1p 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 samp
Download Pdf Manuals
Related Search