MBS598254
Contents
1. 9 1 3 Herpes secretion sample 1 Add 0 5ml normal saline to the herpes secretion sample and vortex vigorously 2 Transfer 50ul liquid into another tube add 50u DNA extraction buffer 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 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 used in 3 hours or store at 20 C for one month C DNA extraction kits are available from various manufacturers You can also use your own extraction systems or the commercial kit depending 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 channel CyS 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 35pl 0 4ul ipl 21 5 ul 0 4 ul tpl Reaction Mix Enyzme Mix Internal Control Reaction Mix Enyzme Mix intemal Control 36 4 22 9 yl Master Mix Master Mix 4ul 36yl 2 5 pl2. 22 54 Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube A This system is only for PCR Instrument Smart Cycler ll PCR Instrument OR PCR system without CY5 channel may be treated with 1ul 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 sample Mix completely then spin down briefly in a centrifuge 2 Pipet 36ul 22 5ul for Smart Cycler II Master Mix with micropipets of sterile filter tips to each Real time PCR reaction plate tubes Separately add 4p 2 5ul for Smart Cycler IDDNA 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 Selection of fluorescence channels 94 C for 2min Icycle HEX VICJOE CH 93 C for 15sec 60 C for 1min HEX VIC JOE Fluorescence measured at 60 C Cal Red 610 ROX TEXAS RED HSV1 A 5 4 If you use ABI Prism system please choose none as passive reference and quencher 40cycles 10 Threshold setting just above the
3. 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 9 1 1 Genital swab sample 1 Wash the genital swabs in 1 0ml normal saline and vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline and suspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50u 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 9 1 2 Serum sample 1 Pipet 50ul blood serum 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 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 can used for the PCR template
4. humans and vertebrates Herpes simplex virus HSV is one of the most common agents infecting humans of all ages The virus occurs worldwide and produces a variety of illnesses including mucocutaneous infections infections of the CNS and occasionally infections of the visceral organs It is also a sexually transmitted disease STD caused by the herpes simplex viruses type I HSV I and type II HSV II VZV is closely related to the herpes simplex viruses HSV sharing much genome homology VZV HSV2 HSV1 multiplex real time PCR kit contains a specific ready to use system for the detection of VZV HSV2 and HSV1 by polymerase chain reaction PCR in the real time PCR system The master contains reagents and enzymes for the specific amplification of 3 kinds of target genes Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified VZV DNA fragment HSV2 DNA fragment and HSV1 DNA fragment are performed in fluorimeter channel FAM HEX VIC JOE and Cal Red610 ROX TEXAS RED DNA extraction buffer is available in the kit In addition the kit contains a system to identify possible PCR inhibition by measuring the channel CY5 fluorescence of the internal control IC An external positive control contained 4 Kit Contents DNA Extraction Buffer VZV HSV2 HSV1 Reaction Mix PCR Enzyme Mix 1 vial 1 8ml 1 vial 950ul 1 vial 12ul 1 vial 400ul 1 vial 30ul 1 vial 30ul 1 vial 30ul Molecular G
5. own extraction systems or commercial kits UNDET UNDET HSV Type positive UNDET UNDET lt 38 HSV Type positive 38 40 25 35 Re test If it is still 38 40 report as 1 6 UNDET PCR Inhibition No diagnosis can be concluded e Avoid aerosols For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
6. Revision No ZJ0002 Issue Date Jul 1 2015 VZV HSV2 HSV1 Multiplex Real Time PCR Kit User Manual For Research Use Only Z y 20 C Z9 MBS598254 Instrument III IV For use with ABI Prism 7000 7300 7500 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 1 Intended Use VZV HSV2 HSV1 real time PCR Kit is used for the distinguishing of Varicella Zoster Virus Herpes Simplex Virus Typel and Type2 by real time PCR systems in samples like in serum herpes secretion or genital swabs and etc 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 to re open the reaction tube after the amplification 3 Product Description Varicella zoster virus VZV is one of eight herpesviruses known to infect
7. maximum level of molecular grade water 11 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Ct value potter S VZV Positive contol ss sd id HSVRSVI Positive contol es 3d 12 Data Analysis and Interpretation The following sample results are possible Ct value Result Analysis a eis 610 UNDET UNDET UNDET 25 35 Below the detection limit or negative UNDET UNDET VZV positive 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 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 Attention please thaw the buffer thoroughly and mix the buffer well before use because it contains insoluble particles You may use your
8. rade Water Internal Control IC VZV Positive Control HSV2 HSV1 Positive Control Analysis sensitivity 5X 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 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 Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Tube racks A I AN Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This
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MBS598254