HBV Quantitative Real Time PCR Kit User Manual For In
Contents
1. 4 Perform the following protocol in the instrument 37 C for 2 min 1 cycle 94 C for 2 min 1 cycle 93 C for 15 sec 60 C for 60 sec 40 cycles Fluorescence is measured at 60 C FAM and HEX VIC JOE channels should be chosen 5 ANE 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 Channel Control FAM Target Nucleic Acid HEX VIC JOE IC Molecular Grade Water UNDET 25 35 QS 1 QS82 Q83 QS4 lt 38 and Correlation coefficient of QS curve lt 0 98 _ 13 Data Analysis and Interpretation The following results are possible 1 The Ct value in channel FAM shows lt 38 The result is positive The sample contains HBV DNA Quantitative value of samples is automatically reported according to the standard curve Quantitative Value Data Analysis and Suggestion lt 5x10 IU ml HBV DNA Positive its concentration lower than 5x 10 IU ml 5x10 10 U ml HBV DNA Positive the quantitative value for recommendation only 10 10 IU ml gt 10 U ml HBV DNA Positive the quantitative value is valid 1 HBV DNA Positive but the quantitative value for recommendation only 2 Re test the sample after dilute the sample by several times2. contains a specific ready to use system for HBV detection for genotype A H through polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific HBV DNA amplification Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified HBV DNA fragment is performed in fluorimeter channel FAM 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 HEX VIC JOE fluorescence of the internal control IC Four quantitation standards are supplied allows the determination of the gene load 4 Kit Contents p Ref Cap Color Type of reagent Presentation _25rxns_ Blue DNA Extraction Buffer 1 vial 1 8ml Gray HBV Reaction Mix 1 vial 950ul Blue PCR Enzyme Mix 1 vial 12ul Yellow Molecular Grade Water 1 vial 400ul Orange Internal control IC 1 vial 30ul Pink HBV QS1 5x10 IU ml 1 vial 20ul Purple HBV QS2 5x10 TU ml 1 vial 20ul Orange HBV QS3 5x10 IU ml 1 vial 20ul Yellow HBV QS4 5x10 IU ml 1 vial 20ul QS Quantitation Standard 1 2 3 4 5 6 7 8 9 e For in vitro diagnostic use only 5 Storage e This assay needs to be carried out by skilled personnel 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
3. making the quantitative value within 10 10 IU ml 2 The Ct value in channel FAM shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative 3 In channel FAM no signal is detected at the same time a HEX VIC JOE signal from the Internal Control appears The sample does not contain any HBV DNA It can be considered negative 4 Neither in channel FAM nor in channel HEX VIC JOE signal is detected A diagnostic statement can not be made Inhibition of the PCR reaction For further questions or problems please contact our technical support at trade liferiver com cn
4. units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area 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 It s better to use commercial kits for nucleic acid extraction 1 Take 50ul serum or plasma add 50u DNA extraction buffer and 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 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 3 Centrifuge the tube at 13000rpm for 10 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 pleas
5. Issue Date Jul 1 2012 O y Revision No ZJ0007 miraria HBV Quantitative Real Time PCR Kit User Manual For In Vitro Diagnostic Use Only IVD HD 0002 02 A B For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 sae Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument wal Shanghai ZJ Bio Tech Co Ltd ji 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 HBV quantitative real time PCR kit is used for the detection of HBV in serum or plasma by using real time PCR systems Its characteristics High sensitivity lower detection line 500 IU ml LOQ 10 10 IU 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 hepatitis B virus Short operating time One 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 fluorogeni
6. 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 Chronic hepatitis B virus HBV infection affects over 350 million people worldwide and over 1 million die annually of HBV related chronic liver disease Although many individuals eventually achieve a state of nonreplicative infection the prolonged immunologic response to infection may lead to the development of cirrhosis liver failure or hepatocellular carcinoma HCC in up to 40 of patients HBV is an enveloped virus in the family Hepadnaviridae with partially double stranded DNA and 42nm in diameter Its antigenic components includes hepatitis B surface antigen HBsAg hepatitis B core antigen HBcAg and hepatitis B e antigen HBeAg Virus replicates in liver and sheds into blood in high concentration HBV is transmitted through blood and body fluid HBV real time PCR kit
7. e comply with 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 35yl 0 4ul ul 21 5 pl 0 4 pl ipl Reaction Mix Enzyme Mix Internal Control Reaction Mix Enzyme Mix Internal Control 36 4ul 22 9 Master Mix Master Mix 4ul 36pl 2 5 yl 22 5 l Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube A This system FCR Instrument OR is only for FCR Instrument Smart Cycler Il XPCR system without HEX VIC JOE channel may be treated with 1ul Molecular Grade Water instead of Il 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 36ul 22 51 for Smart Cycler II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Separately add 4ul 2 5ul for Smart Cycler II DNA sample QS1 QS2 QS3 QS4 and negative control 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
8. 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 Real time PCR reaction tubes plates Vortex mixer e Pipets 0 5 ul 1000 ul e Cryo container e Disposable gloves powderless e Sterile microtubes e Biohazard waste container e Refrigerator and freezer e Sterile filter tips for micro pipets e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Warnings and Precaution Carefully read this instruction before starting the procedure 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
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