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1. General overview of transfusion transmitted infections In Petz LD Swisher S Kleinman SH Spence R Strauss RG eds 1995 Clinical practice of transfusion medicine 3rd ed New York Churchill Livingstone 809 21 TECHNICAL SUPPORT For technical support please dial phone number 86 571 87774567 5211 or 87774575 by fax to 86 571 87774303 or by email to reagent bioer com cn CONTACT INFORMATION OF MANUFACTURER HANGZHOU BIOER TECHNOLOGY CO LTD Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China Website www bioer com cn TEL 86 571 87774575 FAX 86 571 87774565 Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 8 8 YW Bioer Mul Technology Hangzhou Bioer Technology Co Ltd PCR RT PCR Kit User s Manual 20T Cat BSBO2S1A For HCV PCR Fluorescence Quantitative Detection Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 1 8 PH Bioer BIOER Technology Preparation of the master mixes Hangzhou Bioer Technology Co Ltd Note The volumes indicated below are based on a single 50ul standard reaction Prepare HCV RT PCR MIX by multiplying the amount in the Volume column by the number of reactions to be cycled plus one additional reaction It is recommended preparing a mixture containing RT PCR MIX Mn2 and HCV Probe Mix in a 1 5ml tube first before dividi
2. 2 The Correlation of standard curve must be s 0 98 3 The Ct value of the positive control must be lt 35 Note 1 This kit is for research use only Before you begin you should read this user s manual carefully 3 Prepare appropriate aliquots of the kit solutions and keep them separate from other reagents in the laboratory 4 The use of nuclease free lab ware e g pipettes pipette tips reactions vials as well as Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 7 8
3. P2D samples at 800 1600xg for 20 minutes plasma may be stored at 1 6 C for an additional 7 days from the date the plasma was removed from the red blood cells Plasma separated from the cells may be stored at 18 C for up to one month 4 ACD A or 4 sodium citrate anticoagulated apheresis plasma can be stored at 1 6 C for up to 6 hours followed by subsequent storage at 18 C for up to one month 5 Do not freeze whole blood 6 Heparin has been shown to inhibit PCR Use of heparinized specimens is not recommended 7 Warm pooled or individual donor specimens to room temperature before using 8 Covered Archive Plates may be stored at 2 8 C for up to 7 days from the date the plasma was removed from the red blood cells 9 No adverse effect on assay performance was observed when plasma specimens were subjected to three freeze thaw cycles 10 Thaw frozen specimens at room temperature before using 11 The user should validate other collection and storage conditions If specimens are to be shipped they should be packaged and labeled in compliance with applicable federal and international regulations covering the transport of clinical specimens and etiologic agents 10 12 False positive results may occur if cross contamination of specimens is not adequately controlled during specimen handling and processing Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 4 8 YW Bioer Mul
4. Pj Bioer Ege Technology 5 6 Hangzhou Bioer Technology Co Ltd Wearing gloves when performing the assay To avoid cross contamination of samples and reagents use fresh aerosol preventive pipette tips for all pipetting steps To avoid carry over contamination transfer the required solutions for one experiment into a fresh tube instead of directly pipetting from stock solutions Do not touch the surface of the capillaries Always wear gloves when handling the capillaries To minimize risk of carry over contamination it is worthwhile to physically separate the workplaces for RNA preparation References Choo Q L Weiner A J Overby L R et al 1990 Hepatitis C Virus The major causative agent of viral non a non b hepatitis British Medical Bulletin 46 423 441 Alter H 1991 Descartes before the horse clone therefore am The hepatitis C virus in current perspective Annals of Internal Medicine 115 644 649 Dodd RY 1994 Adverse consequences of blood transfusion quantitative risk estimates In Nance ST ed Blood supply risks perceptions and prospects for the future Bethesda American Association of Blood Banks1 24 Schreiber GB Busch MP Kleinman SH Korelitz JJ 1996 The risk of transfusiontransmitted viral infections The Retrovirus Epidemiology Donor Study N Engl J Med 334 1685 90 Holland PV 1996 Viral infections and the blood supply editorial N Engl J Med 334 1734 35 Kleinman SH Busch MP
5. Technology Hangzhou Bioer Technology Co Ltd Protocol 1 Specimen Control and External Standard Control Preparation 1 1 Add 200ul of BIOZOL total RNA Extraction reagent to a 0 5ml centrifuge tube 1 2 Prepare 50ul of serum sample include Specimen 6 Positive Control and 5 Negative Control into the 0 5ml centrifuge tube and vortex 1 3 Add 50ul of Chloroform into the centrifuge tube and invert the tube 10 times to mix Do not vortex 1 4 Centrifuge at 12 000g for 15min at 4 C 1 5 Transfer the aqueous phase to a new 0 5ml centrifuge tube RNase free important Don t touch the middle phase 1 6 Add 200ul of isopropyl alcohol invert the tube 10 times to mix 1 7 Centrifuge at 12 000 g for 15 minutes at 4 C 1 8 Spill the supernatant and add 400ul of 75 ethanol into the centrifuge tube invert the tube 5 times to mix 1 9 Centrifuge at 12 000 g for 5 minutes at 4and completely remove the supernatant again Spill the supernatant and fast spin the tube again then use a new pipette tips to discard the supernatant 1 10 Airs dry the RNA deposition 5 minutes and add 25ul of 4 DEPC H2O to dissolve 2 PCR reaction mixtures prepare Define the experimental protocol before preparing the solutions Calculate the number of reactions needed plus one additional reaction for the HCV RT PCR MIX Proceed as described below for a 50 ul standard reaction when preparing the reaction mixtures Address 1192 Bin An Rd Hi Tec
6. e s Applied instrument Line Gene Series Real time PCR detection system Storage and period of validity Except for BIOZOL total RNA Extraction reagent store at 4 C the others store at 20 C The kit can be stored for up to 12 months if all components are kept in the manner above Additional required reagents and equipment 1 Chloroform isopropanol 75 ethanol Nuclease free aerosol preventive pipette tips 2 Sterile centrifuge Eppendorf tube for preparing 0 2 ml real time PCR tube 3 Pre cool the high speed centrifuge containing a rotor for 2 0 ml reaction tubes and rotor to 2 8 C Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China 3 8 http www bioer com cn Pj Bioer Mul Technology Hangzhou Bioer Technology Co Ltd Specimen Collection and Storage 1 EDTA CPD CPDA 1 CP2D ACD A and 4 Sodium Citrate may be used with this kit 2 Blood collected in EDTA may be stored at 2 30 C for up to 72 hours from time of draw followed by an additional two days at 2 8 C For storage longer than five days remove the plasma from the red blood cells by centrifugation at 800 1600xg for 20 minutes Following removal plasma may be stored at 2 8 C for an additional seven days Alternatively plasma may be stored at 18 C for up to one month 3 Blood collected in CPD CPDA 1 or CP2D may be stored for up to 72 hours ati 24 C Following centrifugation of the CPD CPDA 1 or C
7. h Binjiang District Hangzhou 310053 China http www bioer com cn 5 8 YW Bioer Mul Technology Hangzhou Bioer Technology Co Ltd Preface Hepatitis C Virus is considered to be the principal etiologic agent responsible for 90 95 of the cases of post transfusion non A and non B hepatitis 42 HCV is a single stranded positive sense RNA virus with a genome of approximately 10 000 nucleotides coding for 3 000 amino acids As a blood borne virus HCV can be transmitted by blood and blood products The global prevalence of HCV infection as determined by immunoserology ranges from 0 6 in Canada to 1 5 in Japan 2 Serological screening assays have greatly reduced but not completely eliminated the risk of transmitting viral infections by transfusion of blood products3 Recent studies indicate that nucleic acid based amplification tests for HCV RNA will allow detection of HCV infection earlier than the current antibody based tests Nucleic acid testing of whole blood donations has been in place in the United States since 1999 under Investigational New Drug Application Nucleic acid based tests can detect the units of virus donated by carriers who do not seroconvert or who lack antibodies to serological markers normally detected by immunological assays This fluorescence Detection Kit is adapted for many kinds of Real Time PCR Detection Instrument specifically adapted for Line Gene I amp II Real time PCR Detection System HCV RNA is rever
8. ng the mixture in separate PCR tube and adding the Detection RNA sample Mixes The volumes per tube are mentioned below Volume 25 0 ul 2 5ul 2 5ul Mix gently and pipette 30ul master mix into the corresponding Real time PCR tube 2 Add 20 ul of the corresponding HCV RNA template Specimen control e Add 20 ul of the standards control 1 to 4 Cap each tube and put the tube into a standard benchtop microcentrifuge Centrifuge at 700 x g for 5 s 3000 rpm in a standard benchtop microcentrifuge Note Place the centrifuge adapters in a balanced arrangement within the centrifuge 4 Place the PCR tube in the Real Time PCR Detection Instrument Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 6 8 PW Bioer Hangzhou Bioer Technology Co Ltd wiles Technology Ingredients Ingredients Volume Quantity Sample BIOZOL total RNA Extraction reagent 5ml 1 Tube s 1 RT PCR MIX 500ul 41 Tube s 2 Maer 50ul 4 Tube s 3 HCV Probe Mix 50ul 1 Tube s 4 DEPC H20 1ml 1 Tube s 5 Negative control 200ul 1 Tube s 6 Positive control 200ul 1 Tube s 7 Standard control 1 5 0 X 10 copies ml 100ul 1 Tube s 8 Standard control 2 5 0 x 10 copies ml 100ul 1 Tube s 9 Standard control 3 5 0 X 10 copies ml 100ul 1 Tube s 10 Standard control 4 5 0 X 10 copies ml 100ul 1 Tub
9. se transcribed and a specific fragment is amplified with specific primers in a one step RT PCR reaction The products are detected by using a specific Taqman MGB Probes This Taqman MGB Probes is labeled at the 5 end with FAM report dye and the 3 end by NFQ Non Fluorescent Quencher MGB Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn 2 8 YY Bioer Ma Technology Hangzhou Bioer Technology Co Ltd Cycle the samples as described in follows PCR amplification I 1 cycles Analysis mode None Segment 1 90 C 30s Segment 2 61 C 20min 5 Segment 3 95 C 1min PCR amplification II 50 cycles Analysis mode Single FAM amp HEX Segment 1 95 C 15s Segment 2 60 C 60s In the Edit Sample Screen define the site containing the HCV standards 1 4as standard and enter the respective concentrations provided with the kit in the lot specific information Make sure all other samples are 6 defined as unknown Note More detailed information on options of the Edit Sample Screen is given in the Real Time PCR Detection Instrument Operator s Manual Result analysis and judgments Perform data analysis as described in the Real Time PCR Detection Instrument Operator s Manual Analysis method Use of the Fit points method This renders the method independent from user born influences Quality Control 1 The Ct value of the Negative control must be

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