Yellow Head Virus (YHV) Real Time RT
Contents
1. Liferiver Revision No ZJ0008 Issue Date Jul 1 2012 Yellow Head Virus YHV Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only AR 0201 01 For use with LightC ycler1 0 2 0 Instrument 20 C 23 aal 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 YHV real time RT PCR kit is used for the detection of Yellow Head Virus CYHV in gill or muscle samples of shrimp 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 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 Yellow head virus or YHV is a pleomorphic enveloped virus with single stranded RNA of2. Mix with micropipets of sterile filter tips to each of the real time PCR reaction plate tubes Separately add 5u1 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 DNA or RNA 95 C for S5sec 60 C for 30sec devdek 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 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Positive Control quatitative asa 35 12 Data Analysis and Interpretation The following results are possible Crossing point value Result Anal B 530nm 560nm a 25 35 Below the detection limit or negative a 38 _ Positive 38 40 25 35 Re test if it is still 38 40 report as 1 PCR Inhibition no diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn
3. positive polarity primarily localized in the cytoplasm of infected cells YHV is known to occur in wild shrimp populations but the extent of its distribution in wild populations is not known Viral replication seems to occur only in the cytoplasm without any sign of replication in the intact nuclei of infected cells A long filamentous form of the virus some over 800 nm in length perhaps a precursor to the enveloped rod shape form is present in the cytoplasm of many cells Viral envelopes appear to be acquired by passage of these provirions through the endoplasmic reticulum of the host cells Enveloped virions then cluster in cytoplasmic vesicles sometimes densely packed resembling paracrystalline arrays where they appear to divide into the smaller rod shaped units YHYV real time RT PCR kit contains a specific ready to use system for the detection of yellow head virus by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the YHV 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 YHV cDNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 In addi
4. rt of etiologic agents 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 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 560nm Attention A Mix thoroughly before next transfer B The positive control 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 13l 1 yl 1ul Super Mix Enzyme Mix Internal Control Spl 15 Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without 560nm channel may be treated with 1ul Molecular Grade Water instead of 11 IC 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls 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
5. tion the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC 4 Kit Contents Type of reagent Presentation 25rxns YHV Super Mix RT PCR Enzyme Mix 1 vial 350u1 1 vial 28 ul 1 vial 400u1 1 vial 30ul 1 vial 30ul Molecular Grade Water Internal Control YHV Positive Control Analysis sensitivity 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 as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Real time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e RNA extraction kit e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 ul e Sterile filter
6. tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and freezer e Tube racks 7 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 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 e Transportation of clinical specimens must comply with local regulations for the transpo
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