HCV Quantification Kit v1 USER MANUAL ®
Contents
1. Module Edit Gene Amplification Module Mode Amplification Curve Report Mode Melting Curve 1 34265 1 20839 1 07412 0 93986 0 80559 0 67133 si 0 53706 0 40280 0 26853 0 13427 0 00000 0 13427 Fig 4 Amplification Curve of a Bosphore HCV v1 test Code MBO2v3f Date April 2011 The standard curve is plotted using the data obtained from the defined standards with the axes Ct Threshold Cycle and Log Starting Ouantity Example of a standard curve is given in Fig 5 File F Edit E Settings 5 View V Analysis A Tools T Help H sao Os 123 e 7 LogiC0 Fig 5 Standard Curve of a Bosphore HCV v1 test Analysis of the results should be performed by trained personnel who have received the reguired training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking 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 reguired training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect low positive sa2. Panel Member HCV FAM O i a 2a e zao tt vo i vI fe 12 2 Linear Range The linear range of Bosphore HCV Quantification Kit v1 was determined to be from 1x10 IU ml to at least 1x1071U ml In order to assess the linear range a dilution series which has been calibrated against the WHO International Standard for HCV RNA NAT assays NIBSC Code 06 100 was analyzed by testing each dilution in 2 replicates Fig 7a and 7b The standard curve correlation coefficient was found to be 0 99802 Code MBO2v3f 11 Date April 2011 File F Edit E Settings S View W Analysis 4 Tools T Help H SHO cOx 8s 123 0 7 Module Edit T e Amplificatior Y Test Resulti Amplification Curve 1 36491 1 22842 1 09193 0 95544 0 81895 0 68246 ed 0 54596 0 40947 0 27298 0 13649 0 00000 0 13649 File F Edit E Settings 5 View V Analysis A Tools T Help H s ba Ox s 136 Test Result Logico Fig 7b Linear Range Standard Curve 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 pos
3. HCV Ouantification Kit v1 USER MANUAL For in vitro Diagnostic Use Pilih 4 Nas LLY UY 0 Document Code MBO2v3f Approval Date April 2011 IVD CE 1434 Contents Product Description Content Storage Reguired 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 Internal Control 9 4 6 Positive Control 9 4 7 Quantitation Standards 9 5 Preparing the RT PCR 9 6 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting Specifications 12 1 Sensitivity Code MBO2v3f Date April 2011 10 11 11 12 1 1 Genotype Detection 12 2 Linear Range 12 3 Cross Reactivity 12 4 Reproducibility and Precision 12 5 Diagnostic Evaluation 12 6 Calibration Against WHO Standard 13 References 14 Symbols 15 Contact Information Code MBO2v3f Date April 2011 11 11 12 12 13 13 13 14 14 ili 1 PRODUCT DESCRIPTION Bosphore HCV Quantification Kit v1 detects and quantitates Hepatitis C Virus RNA in human serum and plasma encompassing all the 6 major HCV genotypes The linear range of quantitation is from 1x10 IU ml to at least 1x107IU ml and the analytic sensitivity is 25 IU ml
4. 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 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 RNA Quantification Kit v1 is composed of Real Time RT PCR reagents and quantitation serum standards which have been calibrated against WHO International Standard NIBSC Code 06 100 dH20 PCR Mix RT Mix Detection Mix1 Detection Mix2 Internal Control Positive Control 1 Standard 1 1 x 10 IU ml Standard 2 1 x 10 IU ml Standard 3 1 x 10 IU ml Standard 4 2 x 10 IU ml 2 3 4 5 6 7 8 9 3 STORAGE Bosphore HCV Quantification Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing gt 3x 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 until the expiry dates on the labels if the
5. 8s 123 e7 Po Mode Edit ene Amplification M Niyi Y S e T T S T T Report Mode Well Channel Type Testltem Property CT Test Result Label Name Sex Age CaselD BediD In patientID OutpatientID Sample Type Sample Date Diagnosis Doctor Detect Date Office Experimenter Assessor Remark Ato 1 Sample HCY 40 36 1 414E 01 3 Sample IC 33 07 No Result B3 1 Sample HCY 39 23 3 308E 01 3 Sample IC 32 88 No Result Ba 1 Sample HCY 39 10 3 662E401 3 Sample IC 3311 No Result B5 1 Sample HCV No Ct 0 000E 00 3 Sample Ic 32 72 No Result 86 1 Sample HCY 38 49 5 798E401 3 Sample IC 32 83 No Result B7 1 Sample HCY 40 59 1 186E401 3 Sample IC 33 09 No Result B8 1 Sample HCY 37 62 1 122E402 3 Sample IC 3282 NoResult ci 1 Sample HCV 39 26 3 255E 01 K Sample IC 3312 NoResult c2 1 Sample HCY 39 16 3 490E 01 3 Sample IC 32 80 No Result C3 1 Sample HCV 39 26 3 255E 01 3 Sample Ic 32 78 No Result c4 1 Sample HCY No Ct 0 000E 00 3 Sample IC 33 35 No Result c5 1 Sample HCY 37 19 1 553E402 3 Sample IC 33 16 No Result c6 1 Sample HCV No Ct 0 000E 00 3 Sample IC 3313 No Result 7 1 Sample HCY 41 09 8 123E400 a Sample Ic 33 04 No Result c8 1 Sample HCV No Ct 0 000E 00 3 Sample IC 33 00 No Result c9 1 Sample HCV 39 17 3 459E401 3 Sample IC 3340 NoResult D10 1 Negative HCY No Ct 0 000E 00 3 Negative Ic 3257 NoResult D11 1 Negative HCV No Ct 0 000E 00 3 Negative Ic 3269 NoResult Fig 6 A Report Mode Screen Showing the Resul
6. Sample Temperature C JE Hot Lid Temperature C 0 Running Status Running Cycle times 0 Temperature 0 Holding time 00 00 Running time 00 00 00 onum EQ Progamme J PEGE Control Mode E cw prg With Hot Lid HCV pra Block Control Kiin 20 Experiment Prog Sas Masaiistii New Belgelerim Bigisapanm sei Dosya adr HCY A Ba lantlanm Dosya t r program files I Salt okunur a Fig 3a Selecting the Thermal Protocol Fie F Edt E Settings 5 View Analysis A Tools T Help H sp a ESTES EE EE E i Module Edit Gene Amplification Amplification Program i il emperature Curve Real Time Curve Temperature Monitor C Program Files SLAN Real Time PCR Detection System PROGRAM HCV 5Smodified pra 7 Block Temperature C 0 FF Sample Temperature C Hot Lid Temperature C 0 Running Status Running Cycle times 0 Temperature O Holding time 00 00 Running time 00 00 00 r Control Mode With Hot Lid Block Control Experiment Program Segment 1x1 Segment 2x1 Segment 3x 50 Segment 4x1 Fig 3b Starting the Experiment 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 File F Edit E Settings S View W Analysis A Tools T Help H B A mE 8s 123 e
7. 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 20 C 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 plasma samples including the standards 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 PRODUCT USE LIMITATIONS 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 strande
8. 2010 All of the negative samples were found negative and all of the positive samples were found positive with Bosphore HCV Quantification Kit v1 17 HCV positive and 2 negative serum samples which have been previously analyzed using Roche COBAS Amplicor HCV RNA Monitor v2 0 Bayer Versant HCV RNA v3 0 Abbott HCV RNA m2000 and Roche HCV RNA Taqman were tested with Bosphore HCVQuantification Kit v1 All the positive samples were found to be positive and all the negative samples were found to be negative 12 6 Calibration Against WHO Standard Quantitation Standards were calibrated against the WHO International Standard for HBV DNA NAT assays NIBSC Code 06 100 1 IU was found to be equal to 3 0 2 copies ml 13 REFERENCES 1 By K E Nelson C Williams and N Graham Infectious Disease Epidemiology Theory and Practice July 15 2000 p 923 926 Code MBO2v3f 13 Date April 2011 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 14 SYMBOLS z Use by LOT Lot Batch Catalog number x Temperature limitation A N Caution consult accompanying documents onal Manufacturer IVD 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 90 216 330 00 42 E mail info an
9. atoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji A S Code MBO2v3f 14 Date April 2011
10. 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 specific regions of the template in order to start the synthesis dNTPs are the building blocks 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
11. 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 hybridization 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 as the PCR product is accumulated the point at which the signal rises above background level and becomes distinguishable is called the threshold cycle C There is a linear relationship between the log of the starting amount of a template and its threshold cycle thus starting amount of Code MBO2v3f 3 Date April 2011 unknown templates can be determined using standard curves constructed using Cr values of the known starting amounts of target templates Bosphore HCV Ouantification Kit v1 employs multiplex PCR and an internal control is incorpo
12. d 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 Repeat the test 9 4 6 Positive Control The positive control contains HCV RNA It can be included in the PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria table Section 10 Analysis Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction Code MBO2v3f 5 Date April 2011 9 4 7 Quantitation Standards The guantitation serum standards are calibrated by WHO International Standard NIBSC Code 06 100 9 5 Preparing the RT PCR All four external quantitation standards should be added into the PCR reaction together with the samples and the negative control PCR grade water 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 10 should be added to the total sample number When the Internal Control is added in the extraction step PCR Mix RT mix Detection Mix 1 Detection Mix 2 dH20 Sample RNA Standard Negative Positive Control Total Volume When the Internal Control is a
13. d RNA virus that is classified into six main genotypes 1 6 with more than one hundred different subtypes 1 Epidemiology It is estimated that HCV has a worldwide prevalence of 3 affecting around 180 million people with between 3 to 4 million new infections each year The vast majority of infected people 70 90 develop chronic infection Code MBO2v3f 2 Date April 2011 Though chronic infection may be asymptomatic it is a leading cause of chronic liver diseases including cirrhosis in between 20 to 50 of patients Treatment may be effective in 10 50 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 routes 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 Quantification Kit v1 is based on the Real Time RT PCR method HCV genetic material is amplified
14. dded in the PCR step PCR Mix RT mix Detection Mix 1 Detection Mix 2 Internal Control Sample RNA Standard Negative Positive Control Total Volume 20 ul 0 4 ul 2 28 ul 1 2 ul 0 12 ul 16 ul 40 ul 20 ul 0 4 ul 2 28 ul 1 2 ul 0 4 ul 16 ul 40 28 ul Pipette 24 ul of the master mix into the PCR tubes or strips and add 16 ul of RNA sample standard 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 Quantification Kit v1 is composed of two steps firstly a reverse transcription step and secondly Real Time PCR steps an initial denaturation for activation the HotStarTag 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 Reverse Transcription 50 C Initial denaturation 95 C Denaturation 97 C Annealing 59 C Data Collection Synthesis 72 C Hold 228 Code MBO2v3f Date April 2011 30 00 min 14 30 min 00 30 min 50 cycles 01 20 min 0 15 min 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 and Cy5 e Identify unknown samples standards positive and negative controls assign quant
15. er in the Negative Control 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 Code MBO2v3f Date April 2011 10 be manually adjusted signals Avoid the background and the signal from negative control 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 guantitated as an exact value The analytical sensitivity or detection limit for NAT assays is expressed by the 95 positive cut off value The analytical detection limit for Bosphore HCV v1 was found to be 2 5x10 IU ml p 0 05 The sensitivity was determined using serial dilutions of RNA calibrated with the WHO International Standard for 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 1 1 Genotype Detection and Quantitation Efficiency 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
16. itative values to the standards 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 and 3 Cy5 are selected Fig 1 Standards samples and negative controls are identified in the Module Edit menu Fig 2 Standards should only be defined for the FAM channel and their concentration viral load should be entered To select the thermal protocol Gene Amplification menu is used The Open button in the Experiment 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 FiF EdR E Settings wV AnalysislA TookiT HebiH Dekha zij E212 8e W Dharrel 1 FAM SYBA IC Channel 2 HEX VIC JOE TET 7 Orrel CY5 Fig 1 Filter Selection in Montania 483 File F Edt E Settings 5 Vie Analysis ools T osbogoOxesia2ezsie Fig 2 Sample Location and Identification Code MBO2v3f 7 Date April 2011 File F Edit E Settings S Vieww Analysis A Tools T Help H Dae Olio x es i1zziel2 Module Edit Gene Amplification Amplification Program Temperature Monitor JE Block Temperature C 0 JE
17. lts bilirubin or lipids 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 Quantification Kit v1 The RNA isolation should be performed according to the manufacturers instructions The starting volume is 400 ul the elution volume is 60 ul and the amount of internal control that should be used during isolation for each system is 5 ul The external quantitation standards are provided as serum so that they undergo the same steps as the patient samples starting from RNA isolation 9 4 Kit Components 9 4 1 PCR Mix PCR mix contains HotStarTag DNA Polymerase Probe RT PCR Buffer and ROX passive reference dye HotStarTag DNA Polymerase HotStarTag DNA Polymerase is a modified form of Tag DNA Polymerase and is provided in an inactive state and has no enzymatic activity at ambient temperature The enzyme remains completely 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
18. mples In this case attention should be paid to keep the threshold line above the background and to keep the correlation coefficient at the maximum possible value and within its acceptance criteria The table below displays the acceptance criteria for Bosphore HCV v 1 Component Parameter 3 Standard 4 3 3 Correlation Coefficient gt 0 950 PCR efficiency is calculated by the following formula 10 seP 1 x100 Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if an impairment in the product s performance is observed See the last page for contact information The guantitative results of the test are displayed on the Report Mode screen A spread sheet containing the calculated starting guantities of the unknown samples in each tube is shown The samples that cross the threshold in Channel 1 FAM are displayed with a calculated starting guantity 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 Code MBO2v3f 9 Date April 2011 assay For these undetectable samples the Cy5 data of the internal control should also be checked to avoid false negative results Fig 6 File F Edit E Settings S View YW Analysis A Tools T Help H Melting Curve eho Ox
19. performed sequentially in a single tube Code MBO2v3f 4 Date April 2011 Probe RT PCR Buffer It is a unigue OneStep RT PCR buffer system and has been specifically adapted for realtime RT PCR using seguence specific probes The buffer contains a balanced combination of KCI and NH4 2S04 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 instruments from Bio Rad MJ Research Cepheid Eppendorf and Roche do not reguire 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 RTmix RT Mix contains a unigue Omniscript and Sensiscript blend Both enzymes exhibit a high affinity for RNA facilitating t
20. ranscription 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 HCV specific forward and reverse primers and a dual labeled probe 9 4 4 Detection Mix 2 Detection Mix 2 contains internal control specific forward and reverse primers and a dual labeled probe 9 4 5 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 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 4 ul of internal control should be added for each reaction into the master mix 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 loa
21. rated 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 seguence 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 The serum should be separated from blood within 6 hours after blood 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 The following factors may have possible influences on PCR e Hemolytic samples e Samples of heparinized patients e Samples of patients with elevated levels of bile sa
22. sible 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 12 4 Reproducibility and Precision Reproducibility data on Cr value basis were obtained by the analysis of one of the quantitation standards of the Bosphore HCV Quantification Kit v1 Test was performed in at least 4 replicates by 3 different operators on multiple days using 3 different lots The resulting data is given in Table 1 and Table2 Code MBO2v3f 12 Date April 2011 Table 1 Reproducibility Data HCV Standard Variance Coefficient of 107 IU ml deviation variation Intra assay Variability 0 001 N 4 Inter lot 0 08 N 3 Inter operator Variability 0 08 N 3 Total Inter assay Variability 0 N 5 Table 2 Precision Data 0 Variability 0 0 03 29 26 25 HCV Measured Standard Coefficientof Threshold Standard 10 IU ml Quantity Deviation variation Cycle Deviation MO IU ml MO MO Ct Ct Intra assay 10852 5 267 87 2 46 Variability l l N 4 Inter lot Variability 11072 17 SEE iii N 3 Inter operator 9290 58 1354 50 Variability N 3 Total Inter assay 10047 15 1596 95 Variability N 5 12 5 Diagnostic Evaluation The diagnostic evaluation was performed by testing 100 HCV negative and 5 HCV positive serum samples which have been previously analyzed using Roche Diagnostics Elecsys
23. ts The following table shows the possible results and their interpretation Signal detected in FAM The sample contains HCV No need to check the internal filter pair RNA the result is positive control since the sample is positive high positive samples may suppress the signal from the internal control No signal in FAM The HCV RNA in the Signal from Cy5 filter pair rules signal in Cy5 sample is not detectable out the possibility of PCR inhibition No signal in FAM and The diagnosis i No signal in Cy5 points out to Cy5 inconclusive PCR inhibition or to a problem in RNA isolation See 11 Troubleshooting 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal from the FAM filter Wrong thermal Make sure that the correct thermal protocol is chosen protocol is chosen Late or weak signal from the standards Deterioration of the Don t use expired standards or kit components Follow the standards or the core instructions for the storage of kit components Section 3 kit components Storage No signal from the internal control Deterioration of the Follow the instructions for the storage of kit components See internal control or Section 3 Storage detection mix 2 Make sure that you use the recommended RNA isolation method See 9 3 RNA isolation Make sure that you use the recommended RNA isolation isolation method See 9 3 RNA isolation Signal from FAM Filt
24. y 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 and Cy5 filters iCycler i05 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABl Stratagene Mx3005P Mx3000P Agilent LineGenekK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e 0 2 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 MBO2v3f 1 Date April 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 thawed After thawing all components should
Download Pdf Manuals
Related Search