User Manual For In Vitro Diagnostic Use Only AR-0103-02
Contents
1. Liferiver Revision No ZJ0005 Issue Date Jul 1 2012 Porcine Reproductive and Respiratory Syndrome Virus PRRSV Real Time RT PCR Kit User Manual 20 C For In Vitro Diagnostic Use Only AR 0103 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 NN 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 PRRSV real time RT PCR kit is used for the detection of PRRSV in serum plasma or animal tissue samples 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 having2. 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 95 C for 15min 95 C for 15sec 60 C for 1min device Fluorescence measured at 60 C y 5 A If you use ABI Prism system please choose none as passive reference and quencher Selection of fluorescence channels Target Nucleic Acid HEX VIC JOE 10 Threshold setting just above the maximum level of molecular grade water 11 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Conon ee SE HEX VIC JOE Molecular Grade Water UNDET 25 35 Positive Control qualitative assay eae he 13 Data Analysis and Interpretation The following sample results are possible Ct value Lo HEX VIC JOE Result Analysis UNDET 25 35 Below the detection limit or negative pe ese e foie SC 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 at trade liferiver com cn
3. 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 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 transport of etiologic agents 9 Procedure 9 1 RNA Extraction RNA extraction kits are available from various manufacturers You may use your own extraction syste
4. e PRRSV Super Mix 1 vial 480u1 North America type PRRSV Super Mix 1 vial 480p1 RT PCR Enzyme Mix 1 vial 54ul Molecular Grade Water 1 vial 400ul Internal Control 1 vial 55ul PRRSV Positive Control 1 vial 60ul Analysis sensitivity 5 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 Z 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 tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and freezer e Tube racks
5. ms 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 Cat Number RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech 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 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 18ul 1 pl 1 ul Super Mix Enzyme Mix Internal Control Syl 20ul Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without HEX VIC JOE channel 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 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
6. to re open the reaction tube after the amplification 3 Product Description PRRSV is a member of the family Arteriviridae genus Arterivirus The name of this recently established family is derived from the disease caused by its type species equine arteritis virus PRRS occurs in most major pig producing areas throughout the world The reproductive failure is characterized by abortions stillbirths and the birth of weak piglets that often die soon after birth of respiratory disease and secondary infections Older pigs may demonstrate mild signs of respiratory disease sometimes complicated by secondary infections PRRSV real time RT PCR kit contains a specific ready to use system for the detection of the porcine reproductive and respiratory syndrome virus by reverse transcription polymerase chain reaction RT PCR in the real time PCR system The master contains two Super Mixes for European type and North America type of PRRSV RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the PRRSV 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 PRRSV virus cDNA fragment is performed in fluorimeter channel FAM with the fluorescent quencher BHQ1 4 Kit Contents European typ
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