Enterovirus 71& Coxsackie Virus A16 Real Time RT-PCR
Contents
1. 4 Perform the following protocol in the instrument 45 C for 10min 95 C for Smin Selection of fluorescence channels EV71 HEX VIC JOE CA16 95 C for 15sec 60 C for 1min s0cvcles Fluorescence measured at 60 C y 5 ZN 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 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 and QS curve must be performed correctly otherwise the sample results is invalid HEX VIC JOE Molecular Grade Water UNDET UNDET Positive Control qualitative assay QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible ie Result Analysis UNDET Below the detection limit or negative 2 lt 48 Channel FAM EV71 positive Channel HEX VIC JOE CA16 Positive and the software displays the quantitative value 48 50 Re test if it is still 48 50 report as 12. Liferiver Revision No ZJ0002 Issue Date Jul 1 2012 Enterovirus 71 amp Coxsackie Virus A16 Real Time RT PCR Kit User Manual 20 C Z y For In Vitro Diagnostic Use Only QR 0234 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 LightCycler 480 Instrument rw 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 By using real time PCR systems Enterovirus 71 amp Coxsackie Virus A16 Real Time RT PCR Kit is used for the detection of Enterovirus 71 and Coxsackie Virus A16 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 produ
3. 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 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 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 e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5u1 10001 e Sterile microtubes De Aswan 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 sen
4. ct 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 Enterovirus 71 EV71 and Coxsackie Virus A16 is the major causative agents for hand foot and mouth disease HFMD is sometimes associated with severe central nervous system diseases In 1997 in Malaysia and Japan and in 1998 in Taiwan there were HFMD epidemics involving sudden deaths among young children and EV71 was isolated from the HFMD patients including the fatal cases The nucleotide sequences of each EV71 isolate were determined and compared by phylogenetical analysis EV71 strains from previously reported epidemics belonged to genotype A 1 while those from recent epidemics could be divided into two genotypes A 2 and B Coxsackieviruses are nonenveloped viruses with linear single stranded RNA Coxsackieviruses are divided into group A and group B viruses based on early observations of their pathogenicity in mice Group A coxsackieviruses were noted to cause a flaccid paralysis which was caused by generalized myositis while group B coxsackieviruses were noted to cause a spastic paralysis due to focal muscle injury and degeneration of neuronal tissue At least 23 serotypes 1 22 24 of group A and 6 serotypes 1 6 of group B are recognized The Enterovirus 71 amp Coxsackie Virus A16 real time RT PCR kit contains a specific ready to use syst
5. em for the detection of the EV71 and CA16 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 EV71 and CA16 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 PCR polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit during the PCR The detection of amplified EV 71 DNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 The detection of amplified CA16 DNA fragment is performed in fluorimeter channel HEX VIC JOE with the fluorescent quencher BHQ1 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 2 Quantitation 4 Kit Contents 1 EV71 amp CA16 Super Mix 1 vial 530ul 2 RT PCR Enzyme Mix 1 vial 8 5ul 3 Molecular Grade Water 1 vial 400u1 4 EV71 amp CA16 Positive Control 1x10 copies ml 1 vial 30ul Analysis sensitivity 1 X 10 copies ml LOQ 2X 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
6. o next three tubes Do three dilutions as the following figures Dilution of Standards 4 H l 4u l 4u l Y WY VY Y 1X107 1X10 1X10 1X 104 copiesimi 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 control 1x10 copies ml contains high concentration of the target DNA Therefore be careful during the dilution in order to avoid contamination 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 19 74l 0 3yl Super Mix Enzyme Mix 5ul 20 l Extraction RNA Master Mix Reaction Plate Tube PCR Instrument 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 Sul 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
7. sitivity 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 Wear separate coats and gloves in each area e Do not 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 9 2 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 int
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