MBS598152
Contents
1. Fluorescence measured at 60 C y AN 5 Z 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 Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE 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 Ct value fe yale OE Positive Control qualitative asa 35 OOO 13 Data Analysis and Interpretation The following sample results are possible Ct value HEX VIC JOE Result Analysis UNDET 25 35 Below the detection limit or negative 2 ao e O Positive and the software displays the quantitative value 3 38 40 25 35 Re test if it is still 38 40 report as 1 UNDET UNDET 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 PROCEDURES2. pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers You 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 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 HEX VIC JOE 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 1 lt 10 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 Aul 1X10 1X10 1X10 1X10 capiesmi To generate a standard curve on the real tim
3. EU C For Research Use Only In USA amp China Revision No ZJ0008 Issue Date Jul 1 2015 Coxsackie Virus Real Time RT PCR Kit User Manual 20 C MBS598152 Instrument III IV 25 For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCyclePiQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 LightCycler 480 Instrument a hand 1 Intended Use By using real time PCR systems coxsackie virus real time PCR kit is used for the detection of coxsackie virus in samples like nasal and pharyngeal secretions sputum provoked sputum stool C S F serum 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 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 Coxsackie virus is a cytolytic virus of the Picornav
4. ction 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 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 7 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 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 Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 10001 e Sterile microtubes e Wear separate coats and gloves in each area e Do not
5. e 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 control 1x10 copies ml 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 18ul 1 yl il Super Mix Enzyme Mix Internal Control Syl 204 Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without HEX VIC JOE channel may be treated with 111 Molecular Grade Water instead of 1ul 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 20ul Master Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 5ul RNA sample template positive and negative controls to different 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 95 C for 15sec 60 C for 1min
6. iridae family an enterovirus a group containing the polioviruses coxsackieviruses and echoviruses There are 61 non polio enteroviruses that can cause disease in humans of which 23 are Coxsackie A viruses 6 are Coxsackie B viruses Enteroviruses are the second most common viral infectious agents in humans after the rhinoviruses The most well known Coxsackie A disease is hand foot and mouth disease unrelated to foot and mouth disease a common childhood illness often produced by Coxsackie A16 Other diseases include acute haemorrhagic conjunctivitis A24 specifically herpangina and aseptic meningitis both Coxsackie A and B viruses The genome is single stranded positive sense RNA genome that is about 7500 nucleotides long The viral particle is about 30nm in diameter with icosahedral symmetry The Coxsackie Virus A24 real time RT PCR kit contains a specific ready to use system for the detection of the Coxsackie Virus A24 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 Coxsackie Virus A24 RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Coxsackie Virus A24 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
7. real time systems optical unit during the PCR The detection of amplified Coxsackie Virus A24 DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 In addition the kit contains a system to identify possible PCR inhibition by measuring the HEX VIC JOE 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 Type of reagent CoxV Super Mix 1 vial 480u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 4001 Internal Control 1 vial 30u1 CoxV Positive Control 1 lt 107copies ml 1 vial 30u1 Analysis sensitivity 5 X 10 copies ml LOQ 1X 10 1 X 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 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 e Cool all reagents during the working steps e Super Mix and Rea
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MBS598152