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1. 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 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 Crossing point value Control 30nm 560nm Molecular Grade Water Blank 25 35 335 Correlation coefficient of QS curve lt 0 98 Positive Control qualitative assay QS quantitative detection 13 Data Analysis and Interpretation The following results are possible crossing p _ Result Analysis 1 Blank 25 35 Below the detection limit or negative 2 lt 35 Positive and the software displays the quantitative value 3 35 40 25 35 Re test if it is still 35 40 report as 1 4 Blank Blank PCR Inhibition no diagnosis can be concluded For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
2. Revision No ZJ0002 Issue Date Dec 11 2015 Zika Virus ZIKV Real Time RT PCR Kit User Manual Real Time RT PCR Kit User Manual 20 C 25 For Research Use Only MBS598109 For use with LightCycler1 0 2 0 Instrument 1 Intended Use Zika Virus ZIKV Real Time RT PCR Kit is used for the detection of Zika virus in serum or plasma 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 Zika virus is enveloped and icosahedral with a non segmented single stranded positive sense RNA genome It is most closely related to the Spondweni virus and is one of the two viruses in the Spondweni virus clade The virus was first isolated in 1947 from a rhesus monkey in the Zika Forest of Uganda Africa
3. and was isolated for the first time from humans in 1968 in Nigeria Common symptoms of infection with the virus include mild headaches maculopapular rash fever malaise conjunctivitis and arthralgia In 2009 it was proved that Zika virus can be sexually transmitted between humans The Zika Virus ZIKV real time RT PCR Kit contains a specific ready to use system for the detection of the Zika Virus using RT PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains a Super Mix for the specific amplification of the Zika Virus RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Zika Virus RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of PCR polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified Zika Virus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 In addition the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC An external positive control defined as 1x10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents Ref Type of
4. er Tube racks 7 warnings and Precaution Carefully read this instruction before starting the procedure For in vitro diagnostic use only This assay needs to be carried out by skilled personnel Clinical samples should be regarded as potentially infectious materials and should be prepared in laminar flow hood This assay needs to be run according to Good Laboratory Practice Do not use the kit after its expiration date Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test Once the reagents have been thawed vortex and centrifuge briefly the tubes before use Prepare quickly the Reaction mix on ice or in the cooling block Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products Pipets vials and other working materials should not circulate among working units Use always sterile pipette tips with filters Wear separate coats and gloves in each area eep ee Do not pipette by mouth Do not eat drink smoke in laboratory Avoid aerosols 8 Sample Collection Storage and transport Collected samples in sterile tubes Specimens can be extracted immediately or frozen at 20 C to 80 C Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers You
5. may use your own extraction systems or the commercial kit based on the yield For the RNA extraction please comply with the manufacturer s instructions The recommended extraction kit is as follows Nucleic Acid Isolation Kit Cat Number Manufacturer RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini extraction Kit 50 52904 QIAGEN 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 560nm 9 3 Quantitation The kit can be used for quantitative or qualitative real time RT PCR For performance of quantitative real time PCR standard dilution must be prepared first as follows Molecular Grade Water is used for dilution 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 Aul 4ul Aul 1X10 4X10 1X105 1K 104 capies To generate a standard curve on the real time system all four dilution standards should be used and defined as standards with specification of the corresponding concentrations Attention A Mix thoroughly before next transfer B The positive c
6. ontrol contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 4 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 13pl 1p ipl Super Mix Enzyme Mix Internal Control Spl 15pl Extraction RNA Master Mix Reaction Plate Tube PCR Instrument PCR system without 560nm channel may be treated with 14l Molecular Grade Water instead of 1 1 IC 1 The volumes of Super 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 15pl Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 5pl RNA 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 45 C for 10min Icycle Selection of fluorescence channels 95 C for 15min Icycle 530nm Target Nucleic Acid 95 C for 5sec 60 C for 30sec Adevele 560nm IC Fluorescence measured at 60 C eyeres 10 Threshold setting Choose
7. reagent Presentation 25rxns 1 ZIKV Super Mix 1 vial 350ul 2 RT PCR Enzyme Mix 1 vial 28pl 3 Molecular Grade Water 1 vial 400u1 4 Internal Control IC 1 vial 30pl ZIKV Positive Control 110 copies ml 1 vial 30ul Analysis sensitivity 5X 10 copies ml LOQ 1X 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 RNA extraction kits recommended 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 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 Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay Cool all reagents during the working steps Super Mix should be stored in the dark 6 Additionally Required Materials and Devices Biological cabinet Real time PCR system Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g Vortex mixer RNA extraction kit Real time PCR reaction tubes plates Cryo container Pipets 0 5 yl 1000 ul Sterile filter tips for micro pipets Sterile microtubes Disposable gloves powderless Biohazard waste container Refrigerator and freez

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