Enteric Adenovirus Real Time PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJ0009 Issue Date Jul 1 2012 Enteric Adenovirus Real Time PCR Kit User Manual C For In Vitro Diagnostic Use Only 20 C 3s DD 0043 02 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 LightC ycler 480 Instrument Eo pep 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 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 Enteric Adenovirus real time PCR kit is used for the detection of Adenovirus in stool sputum gargle or blood samples by 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 amou2. 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 E ADV Reaction Mix 1 vial 950u1 1 vial 12ul 1 vial 400u1 Internal Control IC 1 vial 30ul E ADV Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X10 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 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 Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable
3. A 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 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 DNA extraction kits are available You may 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 PCR inhibition Add the internal control IC 1ul rxn and the result will be got in the HEX VIC JOE channel 9 3 Quantitation The kit can be used for quantitative or qualitative real time PCR For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution The step of 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 4ul Aul 4ul To generate a standard curve on the real time pS ali _ system all four dilution standards should be used and defined as stan
4. dard with specification of the corresponding concentrations Attention y y A Mix thoroughly before next transfer 1X105 1X104 copies ml 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 Y t 1X107 1X10 OR X PCR 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 the 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 n the number of reaction Mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5ul for SmartCycer I Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tube Then separately add 4ul 2 5ul for SmartCycer II 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 Selection of fluorescence channels Target Nucleic Acid 93 C for 15sec 60 C for 1min Fluor
5. escence measured at 60 C HEX VIC JOE AOcycles 5 Ar 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 Se Ct value iin FAM _ _ HEXNIC JOE 25 35 Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following results are possible Ct value HEX VIC JOE U T 25 35 Below the detection limit or negative Result Analysis 35 25 35 Re test If it is still 35 40 report as 1 T UNDET PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn we Loe s35 Positive and the software displays the quantitative value
6. gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g PCR Enzyme Mix Molecular Grade Water e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes 7 N 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 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 transportation e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e T
7. nt 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 adenoviruses are classified into 47 serotypes and six subgenera A F with different tropisms In recent years adenovirus type 40 Ad40 and 41 Ad41 of subgenus F have been shown to be causative agents in enteric infections which is second in importance only to rotaviruses as a cause of infantile gastroenteritis Infection with EAds occurs worldwide and has been associated with 4 17 of cases of diarrhoea in children AD40 and Ad41 primarily affect young children less than 2 years of age and occur throughout the year Enteric Adenovirus real time PCR kit contains a specific ready to use system for the detection of enteric adenovirus by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of the Adenovirus DNA including type 1 2 and 5 Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified Adenovirus DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and stool sputum gargle nasopharyngeal swab or blood samples are used for DNA extraction In addition the kit contains a system to identify possible
8. ransportation 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 9 1 1 Sputum sample 1 Trypsin digestive Solution preparation 37 C for 2min Icycle 94 C for 2min Icycle Add 10g trypsin to 200ml sterile purified water and mix thoroughly Adjust the PH value to 8 0 with 2 NaOH solution Add 2mL 25mmol L CaCl mix thoroughly and store at 4 C Please incubate at 37 C for 10 minutes before use 2 Estimate the volume of the sputum and add partes aequales of the Trypsin digestive Solution then vortex vigorously Set at room temperature for 30 minutes Transfer 0 5ml mixture to a new tube Centrifuge the tube at 13000rpm for 5 minutes carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 1 0ml normal saline Resuspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 4 Repeat step 3 5 Add 50u1 DNA extraction buffer close the tube then resuspend the pellet with vortex vigorously Spin down briefly in a table centrifuge 6 Incubate the tube for 10 minutes at 100 C 7 Centrifuge the tube at 13000rpm for 10 minutes The supe
9. rnatant contains DNA extracted and can be used for PCR template 9 1 2 Blood sample 1 Take 2ml anticoagulation and transfer the plasma layer and buffy coat layer to another tube after it is natural stratified 2 Add 50u DNA extraction buffer into the tube and 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 5 minutes The supernatant contains DNA extracted and can be used for PCR template 9 1 3 Stool sample 1 Take about 50mg 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 50u1 DNA extraction buffer closed 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 9 1 4 Gargle 1 Take 1ml sample in a tube centrifuge the tube at 13000rpm for 2min and remove the supernatant and keep the pellet 2 Add 50ul DNA extraction buffer to the pellet 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 DN
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