Enterovirus Real Time RT-PCR Kit User Manual For In Vitro
Contents
1. Liferiver Revision No ZJ0004 Issue Date Jul 1 2012 Enterovirus Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only QR 0206 01 For use with LightCycler1 0 2 0 Instrument Eo PEP Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 Fax 32 2 732 60 03 E Mail mail obelis net CE Ws 1 wal Shanghai 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 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 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 ac2. 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 Different brands of RNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For RNA extraction kit please comply with manufacturer s instructions The recommended extraction kit is as follows 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 RT PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the channel 560nm 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 134l 1 yl 1ul Super Mix Enzyme Mix Internal Control Spl 15l Extraction RNA Master Mix Reaction Plate Tube l PCR Instrument XPCR system without channel 560nm may be treated with 1u 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
3. 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 5ul 10001 e Sterile microtubes 7 Z N 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
4. cumulating product without having to re open the reaction tube after the amplification 3 Product Description Enteroviruses are small viruses that are made of ribonucleic acid RNA and protein In addition to the three different polioviruses there are 61 non polio enteroviruses that can cause disease in humans 29 Coxsackieviruses 23 Coxsackie A viruses and 6 Coxsackie B viruses 28 echoviruses and 4 other enteroviruses Enteroviruses can be found in the respiratory secretions e g saliva sputum or nasal mucus and stool of an infected person Other persons may become infected by direct contact with secretions from an infected person or by contact with contaminated surfaces or objects such as a drinking glass or telephone Parents teachers and child care center workers may also become infected by contamination of the hands with stool from an infected infant or toddler during diaper changes The Enterovirus real time RT PCR kit contains a specific ready to use system for the detection of the Enterovirus 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 Enterovirus RNA including CA16 CA4 CA5 CA7 CA9 CA10 CB2 CB5 CB13 echoviruses and EV71 The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Enterovirus RNA is transcribed into cDNA Afterwards a thermostable DNA polym
5. erase 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 Enterovirus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and bronchial lavage sample or lung section sample is used for the extraction of the DNA 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 1x10 copies ml contained allows the determination of the gene load For further information please refer to section 9 2 Quantitation 4 Kit Contents EV Super Mix 1 vial 350ul RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control 1 vial 30ul EV Positive Control 1x10 copies ml 1 vial 30u1 Analysis sensitivity 5 X 10 copies ml LOQ 1X10 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 e All reagents should be stored at 20 C Storage at 4 C is
6. lysis and Interpretation The following sample results are possible Crossing point value Below the detection limit or negative Positive and the software displays the quantitative value Molecular Grade Water Result Analysis 25 38 40 25 35 Re test if it is still 38 40 report as 1 RT PCR Inhibition no diagnosis can be concluded For further questions or problem please contact our technical support at trade liferiver com cn aa
7. 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 15ul Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Separately add 5ul 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 Selection of fluorescence channels i Target Nucleic Acid 95 C for 15min 95 C for 5sec 60 C for 30sec devcs Fluorescence measured at 60 C y 10 Threshold setting Choose 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 and internal control must be performed correctly otherwise the sample results is invalid 560nm Positive Control qualitative assay 35 QS quantitative detection 13 Data Ana
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