MBS598220 - MyBioSource
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1. are the most common pathogens which cause bronchiolitis and pneumonia among infants and young children RSV is the leading cause of hospitalization and lower respiratory disease in children under five years of age Rhinovirus is a species in the genus Enterovirus of the Picornaviridae family of viruses hMPV is a negative single stranded RNA virus of the family Paramyxoviridae The genomic organisation of hMPV is analogous to RSV HRV RSV HMPV multiplex real time RT PCR kit contains a specific ready to use system for the detection of HRV RSV and HMPV by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains a Super Mix for the specific amplification of these three kinds of virus RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the virus RNA is transcribed into cDNA Afterwards a thermostable DNA polymerase is used to amplify the specific gene fragments by means of polymerase chain reaction PCR Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified HRV fragment RSV fragment and HMPV fragment are performed in channel FAM HEX VIC JOE and Cal Red610 ROX TEXAS RED with the fluorescent quencher BHQ1 In addition the kit contains a system to identify possible PCR inhibition by measuring the CY5 fluorescence of the internal control IC An external positive control contained2. 4 Kit Contents Ref Type of reagent Presentation 25rxns 1 HRV RSV hMPV Super Mix 1 vial 480p1 2 RT PCR Enzyme Mix 1 vial 28p1 3 Molecular Grade Water 1 vial 400u1 4 HRV RSV hMPV Internal Control 1 vial 30p1 5 HRV RSV hMPV Positive Control 1 vial 304 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 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 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 ul 1000 ul Sterile filter tips for micro pipets Sterile microtubes Disposable gloves powderless Biohazard waste container Refri
3. Revision No ZJ0002 Issue Date Jul 1 2015 HRV RSV HMPV Multiplex Real Time RT PCR Kit User Manual For Research Use Only Z y 20 C 25 MBS598220 Instrument 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 HRV RSV HMPV multiplex Real Time RT PCR Kit is used for distinguishing of human rhinovirus respiratory syncytial virus and human metapneumo virus in nasal and pharyngeal secretions 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 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 Respiratory syncytial virus RSV human rhinovirus HRV and human metapneumovirus HMPV
4. gerator and freezer Tube racks 7 A 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 a 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 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 Different brand RNA extraction kits are available You may use you
5. ive 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 lcycle FAM HRV 95 C for 15sec 60 C for Imin A5cycles HEX VIC JOE HMPV Fluorescence measured at 60 C y Cal Red 610 ROX TEXAS RED RSV cys IC 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Channel Ct value Control FAM HEX VIC JOE Cal Red 610 CYS Molecular Grade Water UNDET UNDET UNDET 25 40 Positive control 335 335 lt 35 12 Data Analysis and Interpretation The following sample results are possible Ct value FAM HEX CalRed 610 CY5 So l UNDET UNDET UNDET 25 40 Below the detection limit or negative 2 lt 43 UNDET UNDET Human rhinovirus positive 3 UNDET lt 43 UNDET Human metapneumo virus positive 4 UNDET UNDET lt 43 Respiratory syncytial virus positive 5 43 45 25 40 Re test If it is still 43 45 report a
6. r 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 channel Cy5 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18yl 1 yl ipl Super Mix Enzyme Mix Internal Control 5yl 20ul Extraction RNA Master Mix Reaction Plate Tube l PCR Instrument XPCR system without CY5 channel may be treated with 1ul Molecular Grade Water instead of 1 ul 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 201 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 negat
7. s 1 For further questions or problems please contact our technical support FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES
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