Influenza Virus B Real Time RT-PCR Kit User Manual For In Vitro
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Influenza Virus B Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only RR 0053 01 For use with LightC ycler1 0 2 0 Instrument ec rer 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 Vos 1 il 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 Influenza virus B real time RT PCR kit is used for the detection of Influenza virus B in nasal and pharyngeal secretions by using 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 Ct is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities during Real Time allows the detection of the accumulating product without having to re ope2. PCR Reverse Transcription Polymerase Chain Reaction in the real time PCR system The master contains a Super Mix for the specific amplification of the Influenza virus B RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the Influenza virus B 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 Influenza virus B DNA fragment is performed in fluorimeter channel 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 defined as 10 copies ml is supplied which allow the determination of the gene load For further information please refer to section 9 3 Quantitation 4 Kit Contents IFVB Super Mix 1 vial 350u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal Control IC 1 vial 30u1 IFVB Positive Control 1 X 10 copies ml 1 vial 30ul Analysis sensitivity 5X 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
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 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 Trypsin digestive Solution e Real time PCR reaction tubes plates e Pipets 0 5 ul 1000 ul e Sterile microtubes e Biohazard waste container e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g 7 Warnings and Precaution 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 tub
4. es 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 Real time PCR system e Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves powderless e Refrigerator and Freezer 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 Different brand RNA extraction kits are available 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 lul rxn and the result will be shown in the 560nm Channel 9 3 Q
5. n the reaction tube after the amplification 3 Product Description Influenzavirus B is a genus in the virus family Orthomyxoviridae The only species in this genus is called Influenza B virus only known to infect humans and seals This limited host range is apparently responsible for the lack of Influenzavirus B caused influenza pandemics in contrast with those caused by the morphologically similar Influenzavirus A as both mutate by both genetic drift and reassortment The Influenza B virus capsid is enveloped while its virion consists of an envelope a matrix protein a nucleoprotein complex a nucleocapsid and a polymerase complex It is sometimes spherical and sometimes filamentous Its 500 or so surface projections are made of hemagglutinin and neuraminidase The Influenza B virus genome is 14648 nucleotides long and consists of eight segments of linear negative sense single stranded RNA The multipartite genome is encapsidated each segment in a separate nucleocapsid and the nucleocapsids are surrounded by one envelope Firm diagnosis is by means of virus isolation and serology The virus can be isolated from the nose or a throat swab This is used to infect cells in culture or eggs Hemadsorption may be used to detect infected cells Polymerase chain reaction PCR test are being developed to detect viral RNA The Influenza virus B real time RT PCR kit contains a specific ready to use system for the detection of the Influenza virus B using RT
6. tection limit or negative P52 AOcycles Positive and the software displays the quantitative value 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 Doe e _
7. uantitation The kit can be used for quantitative or qualitative real time RT PCR For performance of quantitative real time PCR Standard dilutions must prepare first as follows Molecular Grade Water is used for dilution Dilution is not needed for qualitative real time PCR detection Take positive control 1x10 copies ml as the starting high standard in the first tube Respectively pipette 36ul Molecular Grade Water into next three tubes Do three dilutions as the following figures Dilution of Standards Aul Aul 4ul Y WV V 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 standard with specification of the corresponding concentrations 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 4 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 Sul 15 l Extraction RNA Master Mix Reaction Plate Tube i 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 standards and sample prepared Molecular Grade Water is
8. 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 of the 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 Icycle Icycle 95 C for 5sec 60 C for 30sec Fluorescence measured at 60 C 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 internal control and QS curve must be performed correctly otherwise the sample results is invalid 530nm Molecular Grade Water Positive Contol qualtaiveassayy 35 QS quantitative detection 13 Data Analysis and Interpretation The following sample results are possible Crossing point value Result Anal 530nm 560nm mae an 25 35 Below the de
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