HCV Genotype Real Time RT-PCR Kit User Manual For In
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 HCV Genotype Real Time RT PCR Kit User Manual For In Vitro Diagnostic Use Only YY y 20 C 25 HR 0009 01 For use with LightCycler1 0 2 0 Instrument 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 HCV genotype real time RT PCR kit is used for the detection of HCV genotype 1 in serum or plasma 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 open the reaction tube after the amplification 3 Product Description Hepatitis C virus has at least six forms or genotypes HCV genotypes and subtypes are distributed va2. 5 C for S5sec 58 C for 30sec Fluorescence measured at 58 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 Quality control Negative control and positive control must be performed correctly otherwise the sample results is invalid ee a Crossing point value Gl ae 12 Data Analysis and Interpretation Negative or positive judgement __ Crossing point Value Negative or Positive The following results are possible Results Super Super Crossing point Value of Super Aa CW Negative lt 3 5 HCV Positive and Genotype I HCV Positive but not Genotype I HCV Positive but not Genotype I For further questions or problems please contact our technical support at trade liferiver com cn
3. ds 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 Cat Number RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini Extraction Kit 50 52904 QIAGEN 9 2 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 14 ul 1 ul Super Mix Enzyme Mix Sul 15yl Extraction RNA Master Mix Reaction Plate Tube l PCR Instrument 1 The volumes of Super 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 151 Super Mix with micropipets of sterile filter tips to several Real time PCR reaction tubes Separately add 5ul RNA sample positive control A positive control B and negative control to different reaction tubes Immediately close the 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 Selection of fluorescence channels Reaction Mix A 530nm HCV Reaction Mix B 530nm HCV genotype I 9
4. nel 530nm with the fluorescent quencher BHQ1 4 Kit Contents HCV Super Mix A 1 vial 380u1 HCV Super Mix B 1 vial 380u1 RT PCR Enzyme Mix 1 vial 54ul Molecular Grade Water 1 vial 400u1 HCV Positive control A 1 vial 60ul HCV Positive control B 1 vial 60ul Analysis sensitivity 1 X 10 IU 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 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 e Real time PCR system e Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable glove
5. riously in different parts of the world Genotypes 1 3 are widely distributed throughout the world Subtype la is prevalent in North and South America Europe and Australia Subtype 1b is common in North America and Europe and is also found in parts of Asia Genotype 2 exists in most developed countries and is less common than genotype 1 Some studies suggest that different types of HCV may be related to different transmission routes HCV genotype 1 is significantly associated with human immunodeficiency virus Genotype 1 is related to a poor response to treatment Genotyping can help doctor determine an appropriate hepatitis C treatment and how long treatment should be given HCV genotype real time RT PCR kit contains a specific ready to use system for the detection of HCV genotype 1 by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains Super Mix for the specific amplification of HCV RNA and HCV genotypel RNA Super Mix A is specific for HCV RNA Super Mix B is specific for HCV genotypel RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT HCV RNA is transcribed into cDNA Then a thermostable DNA polymerase is used to amplify the specific gene fragments by polymerase chain reaction PCR Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified HCV DNA fragment is performed in fluorimeter chan
6. s powderless e Refrigerator and Freezer 7 Aaina 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 45 C for 10min 95 C for 15min e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink and 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 bran
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