Hangzhou Bioer Technology Co.,Ltd
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1. 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 reverse 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 An Internal Control is supplied This allows the user both to control the RNA isolation procedure and to check for possible PCR inhibition For this application add 1 u I the Internal Control to the isolation per sample Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn ys Bioer Ee Technology Hangzhou Bioer Technology Co Ltd Ingredients Ingredients Volume Quantity 1 Lysis Buffer 4 8ml 1 Bottle s 2 Virus Wash Buffer I 15 6ml 1 Bottle s 3 RNA Wash Buffer II 5 2ml 1 Bottle s 4 Extraction Wash Buffer III 24ml 1 Bottle s 5 Reagent Elution Buffer 2 4ml 1 Bottle s 6 Magnetic Beads 480ul 1 Tube s 7 Proteinase K 480ul 1 Tube s 8 Acryl Carrier 96ul 1 Tube s 9 RT PCR MIX 720ul 1 Tube s 10 Mn2 96ul 1 Tube s 11 HCV Probe Mix 1 96ul 1 Tube s 12 Internal control 48ul 1 Tube s 13 Negative control 200ul 1 Tube s 14 Posi2. Manual 48T Cat BSBO2M1D For HCV PCR Fluorescence Quantitative Detection With Internal control and Magabeads Extraction Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn ys Bioer Ms 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 1 2 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 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
3. mixture into the corresponding Real time PCR tube 2 Add 20 ul of the corresponding HCV RNA template Specimen controls Add 20 ul of the standard control 1 to 4 Cap each tube mix gently and centrifuge for a short time 3 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 Place the PCR tube in the Real Time PCR Detection Instrument Set reaction procedure as following 90 C 30 sec 61 C 20 min 95 C 1 min 95C 15 sec JH5cycles 60 C 1 min 5 Select the fluorescent channel of instrument for testing Choose F1 FAM and F2 HEX channels to collect fluorescent signals Before running Line Gene Series Real time PCR detection system set fluorescent signals detecting at 60 C for 10 seconds and then adjust gain to make the F1 FAM and F2 HEX background between 20 30 Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn ys Bioer Ee Technology Result analysis and judgments Hangzhou Bioer Technology Co Ltd 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 2 The Correlation of standard curve m
4. ecimens 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 ys Bioer Ms Technology Hangzhou Bioer Technology Co Ltd Protocol 1 Sample extraction Add 10 4 ml ethanol to Wash Buffer I add 20 8ml ethanol to Wash Buffer II Mix gently for 2 mins 1 1 Add 10 ul Proteinase K 2ul Acryl Carrier 100ul Lysis Buffer If precipitates have formed Place the bottle in a warm water bath lt 60 and shake the bottle approximately every 10 minutes until the precipitates are dissolved and 100ul of serum sample include Specimen Positive Control and Negative Control into the 1 5ml centrifuge tube mix thoroughly and incubate at 55 C for 10mins 1 2 Add 120 ul of ethanol and 10 ul of magnetic beads mix well before use Mix gently for 10mins at room temperature centrifuge the tubes for a short while 1 3 Put it on magnetic rack for 1 minute Then discard the supernatant 1 4 Add 500 ul of Wash buffer I and vortex for 15 seconds Centrifuge the tube for a short while Put it on magnetic rack for 1 minute Then discard the clarified supernatant 1 5 Add 500 ul of Wash buffer II and vortex for 15 seconds Centrifuge the tube for a short while Put it on magnetic rack for 1 minute Then discard the clarified supernatant Meanwhile Open the cap and keep the 1 5ml centrifuge tube still on the magnetic rack 1 6 Add 500 u
5. l of Wash buffer III Do not rush the magnetic beads down Then discard the clarified supernatant at once 1 7 Add 50ul of Elution buffer and vortex for 30 seconds 1 8 Incubate at 55 for 5 minutes Waving the tube light ly twice during this time in order to dissolve the RNA Centrifuge the tube for a short while And then put it on magnetic rack for 2 minutes and keep the supernatant to an RNase free tube for use later 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 40 ul standard reaction when preparing the reaction mixtures Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn a Bioer Ess Technology 1 Reagent prepare Note Thaw out the reagents at room temperature Before preparing RT PCR reagents mix gently and centrifuge all reagents for a few seconds Hangzhou Bioer Technology Co Ltd Make RT PCR reagents according to the quantity of sample and controls as below n tests add an extra blank control The volumes per tube are mentioned below Component RTPCRMIK Mnze HOVProbemic ene Dosage test 15 0 pl 2 0ul 2 0ul 1 0ul Dosage n 1 x15 pl n 1 x 2 0 ul n 1 x 2 0 ul n 1 pl Mix gently centrifuge for a short time and pipette 20ul the
6. n 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 at1 24 C Following centrifugation of the CPD CPDA 1 or CP2D 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 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 12 False positive results may occur if cross contamination of sp
7. orse clone therefore am The hepatitis C Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn
8. tive control 200ul 1 Tube s PCR Standard control 1 15 Reagent 5 0x10 copies ml PRUNI A 16 ae control 2 100l Tube 5 0x10 copies ml 17 ee control 3 100l 1 Tube s 5 0x10 copies ml 18 aoe control 4 100l 1 Tube s 5 0x10 copies ml Applied instrument Line Gene Series Real time PCR detection system Storage and period of validity Except for Proteinase K and Acryl Carrier store at 20 C Virus RNA Extraction Reagent store at room temperature PCR Reagent 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 Ethanol Nuclease free aerosol preventive pipette tips 2 Sterile centrifuge Eppendorf tube for preparing 0 2 ml real time PCR tube 3 Vortex shaker dry bath centrifuge and magnetic rack etc Address 1192 Bin An Rd Hi Tech Binjiang District Hangzhou 310053 China http www bioer com cn ys Bioer Ms 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 seve
9. ust be s 0 97 3 The Ct value of the Internal control must be lt 36 4 The Ct value of the positive control must be lt 35 Note a This kit is for research use only b Before you begin you should read this user s manual carefully Lysis buffer and wash buffer I will crystallize if the room temperature is low You must place the bottle in a warm water bath at 37 C and shake the bottle approximately eyery 5mins until the crystallization disappear c Prepare appropriate aliquots of the kit solutions and keep them separate from other reagents in the laboratory 2 The use of nuclease free lab ware e g pipettes pipette tips reactions vials as well as Ls Wearing gloves when performing the assay f To avoid cross contamination of samples and reagents use fresh aerosol preventive pipette tips for all pipetting steps o To avoid carry over contamination transfer the required solutions for one experiment into a fresh tube instead of directly pipetting from stock solutions z Do not touch the surface of the capillaries Always wear gloves when handling the capillaries i To minimize risk of carry over contamination it is worthwhile to physically separate the workplaces for RNA preparation References 1 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 2 Alter H 1991 Descartes before the h
10. ys Bioer Ee Technology Hangzhou Bioer Technology Co Ltd virus in current perspective Annals of Internal Medicine 115 644 649 3 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 4 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 5 Holland PV 1996 Viral infections and the blood supply editorial N Engl J Med 334 1734 35 6 Kleinman SH Busch MP 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 87774553 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 ys Bioer Ms Technology Hangzhou Bioer Technology Co Ltd PCR RT PCR Kit User s
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