Coxsackie Virus A16 Real Time RT
Contents
1. Liferiver Revision No ZJ0007 Issue Date Jul 20 2012 C Coxsackie Virus A16 Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only 20 C QR 0207 01 e ee ZJ Bio Tech Co Ltd For use with LightCycler1 0 2 0 Instrument www liferiver com cn Tel 86 21 34680596 E Lec er Obelis S A trade liferiver com cn Fax 86 21 34680595 Boulevard G n ral Wahis 53 2 floor No 15 Building No 188 Xinjunhuan road 1030 Brussels BELGIUM Tel 32 2 732 59 54 PuJiang Hi tech Park Shanghai China Fax 32 2 732 60 03 E Mail mail obelis net 1 Intended Use By using real time PCR systems Coxsackie Virus A16 real time PCR kit is used for the detection of 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 product Monitoring the fluorescence intensities in real time2. allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description 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 Coxsackie Virus A16 real time RT PCR kit contains a specific ready to use system for the detection of the Coxsackie Virus Al6 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 A16 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 A16 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 Coxsackie Virus Al6 DNA fragment is performed in fluorimeter chann
3. before next transfer B The positive control 1 10 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 134 1 pl ipl Super Mix Enzyme Mix intemal Control opl 15pl Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without channel 560nm may be treated with 1ul Molecular Grade Water instead of 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 15ul Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Separately add 5u 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 95 C for 15min Target Nucleic Acid 95 C for 5sec 60 C for 30sec A0cvel Fluorescence measured at 60 C eee 10 Threshold setting Choose Arit
4. el 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 1 lt 10 copies ml contained allows the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents i CA16 Super Mix RT PCR Enzyme Mix 1 vial 350ul 1 vial 28ul Molecular Grade Water 1 vial 400ul Internal Control 1 vial 30ul CA16 Positive Control 1x10 copies ml 1 vail 30ul 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 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 fil
5. es 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 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 RT PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the channel 560nm 9 3 Quantitation The kit can be used for quantitative or qualitative real time 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 dul dpl dul 1x107 1x10 1X10 1X10fcopiesm 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
6. hmetic 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 Crossing point value Channel Control Molecular Grade Water 25 35 Positive Controlquahiaiveasayy 35 QS quantitative detection Correlation coefficient of QS curve lt 0 98 13 Data Analysis and Interpretation The following sample results are possible Crossing point value 25 35 Below the detection limit or negative Result Analysis 2 s8 Positive and the software displays the quantitative value 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
7. ter 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 1000p1 e Sterile microtubes 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 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 tub
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