Bosphore® HCV Genotyping Kit v1 USER MANUAL
Contents
1. Fischer D Miethke M Nicolaus S Zadak M Babiel R J Clin Virol 2007 38 326 33 7 JViral Hepat 2007 14 213 20 Code MBO8v3f 11 Date July 2011 8 Zeuzem et al J Viral Hepat 16 75 90 9 Strader et al Hepatology 2004 39 1147 1171 10 Davis et al Hepatology 2003 38 645 652 11 Fried M W et al 2002 N Engl J Med 347 975 982 12 Manns M P J G McHutchison S C Gordon V K Rustgi M Shiffman R Reindollar Z D Goodman K Koury M Ling and J K Albrecht 2001 Lancet 358 958 965 13 Kamal et al Gastroeneterology 2006 130 632 638 14 Lau et al J Infect Dis 1995 171 281 289 15 Simmonds et al Hepatology 2005 42 962 73 16 Mellor et al J Clin Microbiol 34 417 423 14 SYMBOLS Use by Lot Batch o Catalog number Temperature limitation Caution consult accompanying documents Manufacturer lt 5 E gt E E In Vitro Diagnostic Medical Device 15 CONTACT INFORMATION Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 490 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore areregistered trademarks of Anatolia Tani ve Biyoteknoloji A S Code MBO8v3f 12 Date July 20112. WHO Standard NIBSC Code 06 100 HCV 1b RNA HCV 1a RNA HCV3 RNA and HCV1 1 c d e f g h i j k RNA and each positive control should be used with its specific Detection Mix Detection Mix 1 2 3 and 4 respectively It must be included in the PCR to test the efficiency of the PCR exclusively 9 5 Preparing the RT PCR Make sure that all the kit components are thawed before use Refer to the table below for preparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing master mixes for more than 5 samples an extra 1096 should be added to the total sample number PCR Mix 20 ul RT mix 04 ul Detection Mix 5 6 ul Internal Kontrol 0 1 ul Sample RNA 14 ul Negatif Pozitif Control Total Volume 40 ul It is not necessary to include the internal control in the reaction if it has already been added during the extraction step Code MBO8v3f 6 Date July 2011 Pipette 26 ul of the master mix into the PCR tubes or strips and add 14 ul of RNA sample positive or negative control Close the tube cap Make sure that the solution in each tube is at the bottom of the tube Centrifuge if necessary 9 6 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore HCV Genotyping Kit v1 is composed of two steps firstly a reverse transcription step and secondly Real Time PCR steps an initial denaturation for activati
3. as the PCR product is accumulated the point at which the signal rises above background level and becomes distinguishable is called the threshold cycle Cr There is a linear relationship between the log of the starting amount of a template and its threshold cycle thus starting amount of unknown templates can be determined using standard curves constructed using C values of the known starting amounts of target templates Bosphore HCV Genotyping Kit v1 employs multiplex PCR and an internal control is incorporated into the system in order to control the isolation procedure and to check for possible PCR inhibition HCV RNA cDNA and an internal control are co amplified in a single reaction using sequence specific primers The fluorescent signal generated by the HCV amplification is detected by a probe labeled at the 3 end with FAM through the FAM channel The fluorescent signal generated by the internal control amplification is detected by a second probe labeled at the 5 end with a different reporter molecule Cy5 through the Cy5 channel 9 PROCEDURE 9 1 Sample Preparation Storage and Transport To isolate serum from the clinical specimen the blood sample should be collected into sterile vacutainers without any anticoagulant For venipuncture only sterile material should be used Attention EDTA or heparin containing vacutainers should not be used for sample collection The serum should be separated from blood within 6 hours after blo
4. Bosphore HCV Genotyping Kit v1 USER MANUAL For in vitro Diagnostic Use A NUNT TD iam LY W Document Code MBO8v3f Approval Date July 2011 cE 10 11 12 Contents Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Pathogen Method Procedure 9 1 Sample Preparation Storage and Transport 9 2 Interfering Substances 9 3 RNA Isolation 9 4 Kit Components 9 4 1 PCR Mix 9 4 2 RT Mix 9 4 3 Detection Mix 1 9 4 4 Detection Mix 2 9 4 5 Detection Mix 3 9 4 6 Detection Mix 4 9 47 Internal Control 9 4 8 Positive Control 9 5 Preparing the RT PCR 9 6 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting Specifications 12 1 Sensitivity Code MBO8v3f Date July 2011 10 11 11 12 2 Genotype Detection 12 3 Cross Reactivity 13 References 14 Symbols 15 Contact Information Code MBO8v3f Date July 2011 11 11 11 12 12 iii 1 PRODUCT DESCRIPTION Bosphore HCV Genotyping Kit v1 detects and characterizes the genotype of Hepatitis C Virus in human serum encompassing 4 major and most predominant HCV genotypes 1 1a 1b 2 3 4 The analytic sensitivity is 100 IU ml A region within the 5 UTR is amplified and fluorescence detection is accomplished using the FAM filter An internal control has been integrated into the kit in order to check PCR inhibition The
5. HCV RNA NAT assays NIBSC Code 06 100 The dilutions were tested in different runs in replicates The results were analyzed by probit method 12 2 Genotype Detection Efficiency of detecting and quantitating different genotypes were ensured both by sequence comparison analysis and a Real Time PCR assay using Worldwide HCV Performance Panel WWHV302 M Seracare The following genotypes were tested and found positive Panel Member Genotype HCV FAM 1 1b 2 la 3 1b 4 2a 2c 6 3b 8 3a 10 4 11 4 12 5a 14 6a 12 3 Cross Reactivity To eliminate potential cross reactivity both assay design evidence and experimental studies were employed Primer and probe sequences were checked for possible homology to other known pathogen sequences by sequence comparison analysis using database alignment Samples of HIV HDV HBV with known high positivity were tested and found negative 13 REFERENCES 1 K E Nelson C Williams and N Graham Infectious Disease Epidemiology Theory and Practice July 15 2000 p 1923 926 2 Theodore Sy and M Mazen Jamal Epidemiology of Hepatitis C Virus HCV Infection Int J Med Sci 2006 3 2 p 41 46 3 Anonymous Hepatitis C Fact Sheet No 164 2000 World Health Organization 4 Simmonds et al Hepatology 21 570 83 1995 5 Klenerman et al 2009 PloS Med 6 6 e1000096 doi 10 137 1 journal pmed 1000096 6 Sizmann D BoeckC Boelter J
6. V genotype 1 1 c d e f g h i j k and for internal control 9 4 7 Internal Control An internal control is included in the kit to control RNA isolation and PCR inhibition The internal control is a synthetic DNA molecule derived from human genome It is added into the serum proteinase K and carrier RNA mixture during DNA isolation to control the isolation efficiency and PCR inhibition The amount of IC that should be added during isolation is 5 ul per 400 ul serum sample Alternatively the internal control can be added directly into the PCR master mix to control the PCR inhibition exclusively For this purpose 0 1 ul of internal control should be added for each reaction into the master mix Caution It is not necessary to include the internal control in the reaction if it has already been added during the extraction step Lack of internal control amplification in the FAM negative samples may indicate a problem in isolation or PCR inhibition In this case isolation and PCR should be repeated In samples that contain a high viral load internal control can be suppressed and no increase of the signal is detected Please use the table below for the interpretation of internal control data HCV FAM Internal Control Cy5 Interpretation Sample positive Sample negative Sample positive Repeat the test 9 4 8 Positive Control The kit includes 4 positive controls calibrated with the International
7. amplification data of the internal control is detected with the Cy5 filter The internal control can be added either during RNA extraction or PCR step 2 CONTENT Bosphore HCV Genotyping Kit v1 is composed of Real Time RT PCR reagents and positive and negative controls Component REAGENT 100 Tests 50 Tests 25 Tests 1 dH20 1000 ul 1000 ul 500 ul 2 PCR Mix 4400 ul 2200 ul 1100 pl 3 RT Mix 44 ul 22 ul 11 ul 4 Detection Mix1 616 ul 308 ul 154 ul 5 Detection Mix2 616 ul 308 ul 154 ul 6 Detection Mix3 616 ul 308 ul 154 ul 7 Detection Mix4 616 ul 308 ul 154 ul 8 Internal Kontrol 560 ul 280 ul 140 ul 9 Positive Control 1 120 ul 60 ul 30 ul 10 Positive Control 2 120 ul 60 ul 30 ul 11 Positive Control 3 120 ul 60 ul 30 ul 12 Positive Control 4 120 ul 60 ul 30 ul 3 STORAGE Bosphore HCV Genotyping Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing 23x should be avoided since it may reduce sensitivity If the components are to be used in small amounts they should be frozen in aliquots While preparing the PCR the components should not be exposed to room temperature for more than 10 min and the detection mix components should not be exposed to light more than 1 2 min We recommend preparing the PCR on a cooling block and keeping the detection mixes within a closed container The components maintain their stability unt
8. anded RNA virus that is classified into six main genotypes 1 6 with more than one hundred different subtypes 1 Genotype 1 is the most common one and is the one with the least response to therapy Since HCV has a high tendency to mutate and doesn t initiate a severe response in human T lymphocytes of the immune system a white blood cell type it results a high rate of chronic infection The genetic heterogeneity of this virus which cannot be Code MBO8v3f 2 Date July 2011 grown by cell culture makes the diagnosis difficult lowers the response to treatment and also impedes the development of the vaccine against the disease 4 5 It has been observed that different HCV genotypes show different responses to antiviral therapy The duration and success rate of HCV medication PEG IFN and ribavirin mostly depend on the virus genotype The response rate to treatment in genotype 2 and 3 is higher than the one in genotype 1 and 4 70 8096 against 40 5096 in long term Moreover the successful treatment of genotype 2 and 3 takes approximately 6 month while it is 1 year for genotype 1 and 4 6 7 8 It has been reported that the response to treatment is 0 396 and ceasing the treatment should be considered if the HCV RNA level of the patients has not revealed at least 2 log decrease after the 12 weeks of treatment 9 10 It has been observed that duration of the treatment of acute infection of genotype 1 is shorter and the success rate is hig
9. d After thawing all components should be centrifuged briefly spin down for 3 5 seconds and mixed well to ensure homogeneity prior to use The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 209C PCR and nucleic acid isolation must be performed in different compartments Samples should be stored separately to avoid contact with the kit components Pathogen information should be reviewed to be aware of the health related risks Serum samples should be handled with extreme caution suitable class microbiological safety cabinet should be used Physical contact with pathogens should be avoided by wearing lab coats and gloves no allowance for eating or drinking within the workspace prevention of unauthorized individuals access to the working area All the pathogenic wastes produced during the nucleic acid isolation step including the serum samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCTUSELIMITATIONS All the components may exclusively be used for in vitro diagnostics This product should be used in accordance with this user manual by personnel specially trained to perform in vitro diagnostic procedures 7 PATHOGEN Causative Agents The hepatitis C virus is a hepacivirus of the Flaviviridae family of viruses that causes Hepatitis C in humans It is a small enveloped 9 6kb single str
10. her than the chronic infection of genotype 1 11 12 13 The nucleotide sequences of genotypes differ around 31 3496 from each other the subtypes differ around 20 to 2396 Though the genotypes first appeared endemically in geographically distinct regions currently they are spread all over the world As the Genotypes 1 2 and 3 are widely seen all over the world genotype 4 and 5 are predominant in Africa For instance in the U S approximately 7596 of all cases are caused by genotype 1 1596 by genotype 2 596 by genotype 3 and 196 by genotype 4 Genotype 6 is typical to Southeast Asia genotype 1 is prevalent in Western Europe and U S genotype 3 is very common in the UK 14 15 16 Epidemiology It is estimated that HCV has a worldwide prevalence of 396 affecting around 180 million people with between 3 to 4 million new infections each year The vast majority of infected people 70 9096 develop chronic infection Though chronic infection may be asymptomatic it is a leading cause of chronic liver diseases including cirrhosis in between 20 to 5096 of patients Treatment may be effective in 10 5096 of patients depending on the applied therapy 2 Modes of Transmission Hepatitis C is believed to be spread through contact with infected blood However unlike many other blood borne viruses HCV may be transmitted even through indirect sources like a used razor making HCV more transmissible than other blood borne viruses including HIV Common r
11. il the expiry dates on the labels if they are stored at advised conditions 4 REQUIRED MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM HEX and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABI Stratagene Mx3005P Mx3000P Agilent LineGeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e 02 ml Thin Wall PCR tubes or strips e Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks or other high quality viral RNA extraction kits and systems e Deep freezer 20 C e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes e Calibrated adjustable micropipettes Code MBO8v3f 1 Date July 2011 DNAse RNAse pyrogen free micropipette tips with filters DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes Disposable laboratory gloves 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important The product should be delivered on dry ice Check for presence of dry ice upon arrival Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used Before starting a test procedure all components should be thoroughly thawe
12. letely inactive during the reverse transcription reaction and does not interfere with it This prevents formation of misprimed RT PCR products and primer dimers during reaction setup reverse transcription and the first denaturation step The enzyme is activated after the reverse transcription step by a 15 minute 95 C incubation step The hot start also inactivates the reverse transcription enzymes ensuring temporal separation of reverse transcription and PCR and allowing both steps to be performed sequentially in a single tube Probe RT PCR Buffer It is a unique OneStep RT PCR buffer system and has been specifically adapted for realtime RT PCR using sequence specific probes The buffer contains a balanced combination of KCl and NH4 2SO4 ROX passive reference dye For certain real time cyclers the presence of ROX passive reference dye in real time PCR compensates for non PCR related variations in fluorescence detection Fluorescence from ROX dye does not change during the course of real time PCR but provides a stable baseline to which PCR related fluorescent signals are normalized Thus ROX dye compensates for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position The use of ROX dye is necessary for all instruments from Applied Biosystems and is optional for instruments from Stratagene e g Mx3000P Mx3005P and Mx4000 Montania 483 Rotor Gene cyclers and instrumen
13. od collection To separate the serum the blood container should be centrifuged at 800 1600 x g for 20 minutes The separated serum should be transferred to polypropylene tubes and stored at 20 C or lower until use The samples should be transported in containers with capacity to resist pressure Transportation should be done according to local and national regulations for pathogen material transport 9 2 Interfering Substances To avoid possible influences on PCR e Hemolytic samples Code MBO8v3f 4 Date July 2011 e Samples of heparinized patients e Samples of patients with elevated levels of bile salts bilirubin or lipids must not be used 9 3 RNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks isolation system is used with Bosphore HCV Genotyping Kit v1 The RNA isolation should be performed according to the manufacturers instructions The recommended starting volume is 400 ul and the elution volume is 60 ul The amount of internal control that should be used during isolation for each system is 5 ul 9 4 Kit Components 9 4 1 PCR Mix PCR mix contains HotStarTaq DNA Polymerase Probe RT PCR Buffer and ROX passive reference dye HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form of Taq DNA Polymerase and is provided in an inactive state and has no enzymatic activity at ambient temperature The enzyme remains comp
14. of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In addition to these components in RT PCR reverse transcriptase is added to the reaction and cDNA synthesis from the RNA template is acquired In Real Time PCR technique in contrast to conventional PCR PCR product can be monitored during the reaction Therefore Real Time PCR obviates the need for further analysis methods like gel electrophoresis whereby minimizing the risk of contamination Dual labeled probes employed in the reaction in addition to the conventional PCR reagents enable detection of the amplified target with increased sensitivity The assay utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent hydrolysis probe during the extension phase of PCR The probe is labeled at the 5 end with a fluorescent reporter molecule and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity and the reporter is excited by light no reporter fluorescence can be detected During the elongation step of PCR Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is freed from the suppressing effect of the quencher fluorescence signal can be detected The fluorescence generated by the reporter increases
15. on the HotStarTaq DNA Polymerase a two step amplification cycle and a terminal hold The real time data is collected at the second step of the amplification cycle The thermal protocol required to run with the Detection Mix 1 2 and 3 is in the table below Reverse Transcription 50 C 30 00 min Initial denaturation 95 C 14 30 min Denaturation 97 C 00 30 min Annealing 54 C 01 20 min Data Collection 50 cycles Synthesis 72 C 0 15 min Hold 22 C 05 00 The thermal protocol required to run with the Detection Mix 1 2 and 3 is in the table below Reverse Transcription 50 C 30 00 min Initial denaturation 95 C 14 30 min Denaturation 97 C 00 30 min Annealing 58 C 01 10 min 50 cycles Data Collection Hold 22 C 05 00 Before starting a Real Time PCR reaction using the Bosphore Kits the following steps should be completed e Choose the filter pairs to be used FAM HEX and Cy5 e Identify unknown samples positive and negative controls e Select the correct thermal protocol These steps are described below From the main menu of the Montania 483 Real Time PCR Instrument File and then New is selected Create a new Experiment is selected In the Select Channel window channels 1 FAM 2 HEX and 3 Cy5 are selected Fig 1 Samples positive and negative controls are identified in the Module Edit menu Fig 2 To select the thermal protocol Gene Amplification menu is used The Open button in the E
16. outes of transmission include transfusion of blood products intravenous and percutaneous drug and needle use work accidents among healthcare workers and any other blood to blood contacts such as sexual practices and from mother to newborn maternal infant transmission Statistical studies have revealed no risk factors for HCV transmission in the activities of daily living sneezing coughing hugging etc 2 3 8 METHOD Bosphore HCV Genotyping Kit v1 is based on the Real Time RT PCR method HCV genetic material is amplified by reverse transcription technique since it is composed of RNA RT PCR which is also referred as RNA PCR is a two step reaction First complementary DNA is synthesized from RNA by reverse transcription and then complementary DNA is amplified by standard PCR The primer binds to the target RNA region in RT PCR and RNA DNA double strand is synthesized by reverse transcriptase enzyme using the RNA template for complementary DNA Afterwards standard PCR continues Polymerase chain reaction is a technique that is used for amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template As a brief explanation primers are small synthetic DNA those anneal to the Code MBO8v3f 3 Date July 2011 specific regions of the template in order to start the synthesis dNTPs are the building blocks
17. results of the unknown samples in each tube is shown in the Test Result column The samples that cross the threshold in Channel 1 FAM Channel 2 HEX and Channel 3 Cy5 are displayed with their positive negative results samples that do not cut the threshold are displayed as No Ct These samples are regarded as negative or having a viral load below the detection limit of the assay For these undetectable samples the Cy5 data of the internal control should also be checked to avoid false negative results The table below shows the possible results and their interpretations Channel Detection Mix No FAM HEX Cy5 Result 1 HCV 4 1 HCV 1b 1 Test must be repeated 2 t HCV 1a 2 HCV 2 2 Test must be repeated 3 none HCV 3 3 none Test must be repeated 4 none HCV 1 1c d eff g h lj k 4 none Test must be repeated 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal from the FAM or HEX filter Wrong thermal Make sure that the correct thermal protocol is chosen protocol is chosen Late or no signal from the Positive Control Deterioration of the Do not use positive controls or other kit components after positive control or their expiration date Follow the instructions for the storage other kit components of kit components See Section 3 Storage No signal from the in
18. ternal control Deterioration of the Follow the instructions for the storage of kit components See internal control or Section 3 Storage detection mix PCR inhibition Make sure that you use the recommended RNA isolation method See 9 3 RNA isolation RNA lost during Make sure that you use the recommended RNA isolation isolation method See 9 3 RNA isolation Signal from FAM or HEX Filter in the Negative Control Contamination Use filter tips Repeat PCR with new kit components The Threshold is Above Low Signals The threshold should Using the mouse pull the threshold down until it cuts the low be manually adjusted signals Avoid the background and the signal from negative control Code MBO8v3f 10 Date July 2011 12 SPECIFICATIONS 12 1 Sensitivity Analytical sensitivity may be expressed as the limit of detection i e the smallest amount of the target marker that can be precisely detected The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value The analytical sensitivity or detection limit for NAT assays is expressed by the 9596 positive cut off value The analytical detection limit for Bosphore HCV Genotyping Kit v1 was found to be 1x10 IU ml p 0 05 The sensitivity was determined using serial dilutions of RNA calibrated with the WHO International Standard for
19. th Hot Lid Block Control Experiment rogram Giese Module Edit Trade Mode DIT I Report Mode anes nass I nass 054612 045427 F 035342 027253 D moss 0008 009085 FEE Analysis of the results should be performed by trained personnel who have received the required training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking Fig 4 Amplification Curve of a Bosphore HCV Genotyping Kit v1 the patient s clinical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the required training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect low positive samples In this case attention should be paid to keep the threshold line above the background Code MBO8v3f Date July 2011 Test results should not be reported unless there is amplification of the internal control in negative samples Please contact the manufacturer if an impairment in the product s performance is observed See the last page for contact information Qualitative results of the test are displayed on the Report Mode screen A spread sheet containing the
20. ts from Bio Rad MJ Research Cepheid Eppendorf and Roche do not require ROX dye The presence of ROX dye in the master mix does not interfere with real time PCR on any instrument since the dye is not involved in the reaction and has an emission spectrum completely different from fluorescent dyes commonly used for probes 9 4 2 RT mix RT Mix contains a unique Omniscript and Sensiscript blend Both enzymes exhibit a high affinity for RNA facilitating transcription through secondary structures that may inhibit other reverse transcriptases Omniscript is designed for reverse transcription of RNA amounts greater than 50 ng and Sensiscript is optimized for use with very small amounts of RNA lt 50 ng This enzyme combination provides highly efficient and sensitive reverse transcription over a wide range of RNA template amounts 9 4 3 Detection Mix 1 Detection Mix 1 contains forward and reverse primers and dual labeled probes specific for HCV genotypes 1b and 4 and for internal control Code MBO8v3f 5 Date July 2011 9 4 4 Detection Mix 2 Detection Mix 2 contains forward and reverse primers and dual labeled probes specific for HCV genotypes 1a and 2 and for internal control 9 4 5 Detection Mix 3 Detection Mix 3 contains forward and reverse primers and dual labeled probes specific for HCV genotype 3 and for internal control 9 4 6 Detection Mix 4 Detection Mix 4 contains forward and reverse primers and dual labeled probes specific for HC
21. xperiment Program is clicked and the appropriate thermal protocol is selected Fig 3a The thermal cycles of the selected protocol is displayed The experiment starts by clicking the Start button Fig 3b Code MBO8v3f 7 Date July 2011 simi x SS aiza or Fig 1 Filter Selection in Montania 483 Fie EdE SetinosS View Analysis TooisT Helo I5 2a o Test Result 1 a a 10 m a2 B j Hoy Hoy Hoy paa Sarpe Sangle Podivo HV Negative ay m ri gs a Pasivo Fig 2 Sample Location and Identification Fig 3a Selecting the Thermal Protocol Code MBO8v3f Date July 2011 le Edi E Setin uci Analysis A ToolsT Help H 1232097 Module Edit Gene Amplification I Test Result Amplification Program Curve i Real Tme Curve ve PROCRAMHCV SSnodied pr E soc sor p re poc ii Segment 1s 1 cope og Segment 2x1 Segment 3250 Segment tu 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically Fig 3b Starting the Experiment calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 Hep EAE anms Ww Andes TOT Hel Domo Barat Control Mode Wi
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