HCV Real TM Quant 100 PCR NEW
Contents
1. Chick on 5 cycle below to modiy it This cycle repeats 42 tmels Chek an one ol the siege below to noodiy or press gt or to add and remove steps for thir cycle BO dag for 40 sect W Press the button OK 4 Inthe window New Run Wizard click Calibrate Gain Optimisation for Rotor Gene 6000 In the new window Channel Setting select channels Joe Yellow and Fam Green Indicate Min Reading 5 Max Reading 10 and select function Perform calibration Optimization before 1 Acquisition Click the Close button Auto Gain Calibration Setup E alf x Calibration os different gain levels until it finds one at which the fluorescence levels are acceptable The range of fluorescence you are looking for depends on the chemistry you are performing 9 Auto Giain Calibration will read the fluaresence on the inserted sample at Set temperature to degrees Calibrate Al Calibrate Acquiring Perform Calibration At BD D egrees AE Beginning Of Aun Channel Settings 88 Edit Remove Remove ll Sat Hamal Close Help 5 Select Next and click Start run Program the position of the tubes in the carousel of the Rotor Gene 3000 6000 Q and enter the concentrations of the Quantitative Standards reported on the HCV TM RG Quant Data Card in order to generate Standard curves Data Analysis IC amplification analysis Cycling A Fam Green Pre2. um 9 um 3 In the above table the data show that almost all the results obtained from the clinical and control samples with HCV Real TM Quant test differ by less than 0 5 Logio from the values obtained with Reference manufacturer s kit The figure 2 illustrates the graphic correlation between the HCV RNA viral load measured by two tests The correlation factor is 0 975 p lt 0 01 29 Figure 2 Graphic comparison among the results obtained with HCV Real TM Quant Sacace kit and reference manufacturer s kit 6 0 5 0 D4 4 0 j Joy 0160 3 0 3 5 4 5 6 5 em O1 log10 Sacace 30 17 TROUBLESHOOTING l o Weak Ct 34 signal of the IC Fam channel retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended RNA extraction method and follow the manufacturer s instructions gt Re centrifuge all the tubes before pipetting the extracted RNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence cha
3. DefaultUser Dec 18 2004 01 18 PM Default User Dec 18 2004 10 38 AM HBVquan 171204 1 Done DefaultUser Dec 17 2004 11 31 AM HCVquan 161204 1 Done DefaultUser Dec 16 2004 12 53 PM Done DefaultUser Dec 15 2004 02 05 PM Done DefaultUser Dec 14 2004 05 01 PM Done Default User Nov 18 2004 01 01 PM K Done DefaultUser Jul 19 2004 02 23 PM 95 62 65 cmw taq f 22 05 Done Default User May 23 2004 04 24 PM eem 161204 1 User Default User Jeroan standard Started Dec 16 2004 12 53PM Finished Dec 16 2004 02 39 PM est A Temperat wet Notes UM HCVquan V17 HCVquan 0 Temperat Melt raw CVa HCVquan K3 HCVquan B1 J HCVquan B2 r 1 700 01 02 03 0 4 0 5 06 07 08 9 8 10 Degrees C With this euge you can mre a eT curve vy TET emm Sacace HCV Real TM Quant April 16 2011 10 Smart Cycler User Tees Setup Tools Help 4 Run Status Run T 27 2 Run Name Resutts Table Type Seg StdiRes Btd Res HCVquan 1612041 ass Settings Protocols f Temperature FAM 2 orn User Default User Started Dec 16 2004 12 53 PM Finished Dec 16 2004 02 39 PM f Standartcy3 JA HCVquan 25081 426 78 677271 0519 50 Status Done Jaz HcVquan AT6 UNKN 06 1 421696572021 ND HCVquan HCV1000 UNKN
4. l Abbreviations UNKN Samples STD Standards PKO1 PKO2 HCV Positive Controls OKO Negative Control Sample K1 K2 K3 HCV Quantitative Standards B1 B2 B3 IC Quantitative Standards Sacace HCV Real TM Quant April 16 2011 11 RotorGene 3000 6000 Q Corbett Reasearch Qiagen 1 Select New Run and Dual Labeled Probe Click New New Run x Quick Start Advanced i Perform Last Run d SYBR Green Fi 3 Labeled Probe Quenched FRET E P DMA Concentration Measurement E Hybridization Melt m d Rotor Gene Demo Kit Open amp Template In Another Folder Cancel Help Show this Screen when Rotor Gene Opens 2 Program Rotor Gene 3000 6000 Q as follows lt Select Rotor Type 36 Well Rotor and No Domed 0 2 ml Tubes Nepos Rum Wizard Welcome to the Rotor Gene Run Wizard Rolo Type Tawel Rotor Genebisc 7299 8 gt Reaction Volume ul 25 gt Temperature Profile Hold 50 C 30 min Denature 95 C 15 min Cycling 95 C 20 sec 60 C 40 sec Cycle Repeats 42 times Fluorescence is measured at 60 C on FAM Green and JOE Yellow channels Sacace HCV Real TM Quant April 16 2011 12 VR Edit Profile e New pen Save 35 Heb mea m Se LE diedas 0 The run wil take aperasmately 131 mintelz to complete The graph below represents the run to be performed
5. Click the menu Sample Information S lt Sample Data S Insert Sample name Property U for unknown samples and negative PCR control S for Standards and insert standard concentration reported in Data Card Click or click File gt Run Programs R to Run the PCR program Sacace HCV Real TM Quant April 16 2011 23 Results Analysis FAM In the Quantitative Analysis tab select from the menu Dyes D gt FAM F Select standard Concentration boxes left mouse click CTRL for Internal Control see picture below Group Sample 1 Neg extr 1 POS1 1 1 1 12 1 2 1 051 HCV 5 1 QS2 HCV 5 46e 003 1 QS3 HCV 1 1 8 1 88e 005 1 asc 1 QS3IC 8 20e 002 1 NTC Concentration Then do in this order 1 Under Analysis Method select Fit Points 2 Under Analysis Step Click Zero Adjust select Auto and then click OK Then click Baseline 3 Select 2 points and set the base line drag and drop the red baseline as low as possible but above the noise of each sample usually 6 8 4 Under Analysis Step click Analysis Calculated copies reaction of Internal Control and Ct Results should appear in the Ct and the Calculated Concentration column respectively Analysis Method Analysis Step Points Selected Baseline and Threshold Display Mode EJ2nd Driv Max Zero Adjust Baseline Logarithmic ElBaseline Full Scale Fit Points ElAnalyss Show Points Threshold Smooth low Results Analysis J
6. Smart Cycler 8 x User Logs Setup Tools Help 1 Create Run Check Status Stop Run 4 Site Protocol Sample ID Eee Status FAM Cy3 Cy3Ct Run Name Std Res 2 JA1 HCVquan W17 UNKN OK POS 4 POS 1950 NEG p HCVquan 476 UNKN OK POS 2681 Pos 2021 HCVquan 00 1000 UNKN OK POS 2710 POS 3270 yp TE ONES Temperature HCVquan 6 1000 UNKN OK Pos 2702 POS 3239 NEG Started Dec 16 2004 12 53 pM FA HCVquan HCV500 UNKN OK POS 2694 POS 3531 NEG OT B HCVquan HCV500 UNKN OK POS 26 88 POS 3537 0 ere minit Standart Cy3 HCVquan PKO1 UNKN OK POS 2713 POS 3267 NEG 4 las HCVquan PKO2 UNKN OK POS 26 76 POS 2572 NEG Pia Done 49 HCVquan OKO UNKN OK Pos 2729 NEG 0 00 NEG Notes jA10 HCvauan 1 UNKN OK NEO 0 00 POS 1867 NEG jaM HcVquan UNNN 20K Neo 000 Pos 2562 NEG jA12 HCVquan 3 UNKN OK NEG 0 00 POS 3425 NEG jA13 HCVquan 4 UNKN OK Pos 22268 NEG X 000 NEG di A14 HCVquan 5 POS 0 00 NEG HCVquan 6 EEE NEG POS Site ID Protocol HCVquan V17 Hein A uan 8 er jStandatCy3 Number of Sites 16 SE in the new TEES Tm experiment ent with me standard curves curves click OK C smart Cycer IE Ereto la x User Logs Setup Tools LLL ain LT Rum Check Static ton Ear Ca Select a Run
7. OK 0 05 555 3270 ND Noise CVquan HCV1000 UNKN OK 2 129 718 3239 ND HCVquan HCV500 UNKN OK 22478 3626 94 18716 35 31 ND 6 HCVquan HCV500 UNKN OK 23429 6626 88 7 987 13537 NO JA HCVquan PKO1 UNKN OK 3 80792 13267 ND Jas HCVquan PK02 UNKN OK 25428 7626 76 10856 606025 72 ND A9 HcVquan OKO UNKN Ok 17713802728 ND 000 ND CVquan K1 UNKN OK ND 0 00 1116980311867 ND CVquan K2 UNKN OK ND 0 00 11608 05725 62 ND CVquan K3 UNKN OK ND 0 00 37714 3428 ND HCVquan B1 UNKN 6 421168 122 08 0 00 ND Dye Set FCTC25 HCVquan B2 OK 148938 7125 81 0 00 0 pa OK 49429 foo Protocols 2 1 Lot Number HCVquan tja HCVquan HCVquan o9 uv 70 5 Pom e Number of Sites 22 8 HCVquan econ 0 00 00 0 00 20 0 00 40 0 01 00 0 01 20 00140 HCVquan s 81204 1 m Ja 5 HCVquan Sunt 3 gp r En NN In any case if the calibrators were inserted with the clinical samples in the same experiment or after the importation of standard curves from another experiment in the table of results in the column FAM Std Res for IC HCV and in the column Cy3 Std Res for cDNA HCV should appear the calculated values oc nim as or 5s 22108 OK 19856 x p a0 pe Hevaan s Bm ox Yoon om em aaps OK Wo000 226 o00 OJ OK X0000 25 OK a
8. Applied Biosystems is trademarks of Applera Corporation SmartCycler is a registered trademark of Cepheid LightCycler is a registered trademark of Roche LineGene K is a registered trademark of Bioer Eco PCR Real Time System is a registered trademark of Illumina Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com SISTEMA SISTEMA DI GESTIONE CERTIFICATO 9001 2008 DI GESTIONE CERTIFICATO 13485 2004 PCR The Polymerase Chain Reaction PCR process is covered by patents owned by Hoffmann La Roche and applicable in certain countries Sacace does not encourage or support the unauthorized or unlicensed use of the PCR process Use of this kit is recommended for persons that either have a license to perform PCR or are not required to obtain a license 32
9. FAM channel for Internal Control amp Smart Cycler User Logs Setup Tools Help 1 A i Maintenance Create Run Check Status Stop Run M Views SitelD Protocol Sample ID Run Name Results Table HCVquan HCVquan 141204 1 f Analysis Settings Sf Standard FAM User Default User Standard cya 8 3 Started Dec 14 2004 05 01 PM c 8 Finished Dec 14 2004 06 46 PM 5 5 Status Done elt 2 FAM Notes 2 Views Resutts Table 2 Analysis Settings Dye Set FCTC25 j 8 A z 2 g Protocols Protocol Lot Number 5 HCVquan Number of Sites 16 j Cycles a HC perm EC EET Select ens View Another Run Delete Run s Update Analysis me E Sacace HCV Real TM Quant April 16 2011 9 Results Analysis Choose in the menu Analysis settings the value 20 for the channels Fam and Cy3 In the table of results Results Table appear the values of Ct Threshold cycle for Fam and Cy3 channels The calculation of HCV RNA concentration in the clinical specimens sample and standards can be performed in the same experiment but with the SmartCycler software it is possible to calculate the samples concentration by importing the experiment with Standard Curve in the experiment with clinical samples You can import curves from another experiment clicking on Import Std Curve
10. Prepare quickly the Reaction mix 10 Specimens may be infectious Use Universal Precautions when performing the assay 11 Specimens and controls should be prepared in a laminar flow hood 12 Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross contamination of specimens or controls 13 Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Follow by wiping down the surface with 70 ethanol 14 Avoid contact of specimens and reagents with the skin eyes and mucous membranes If these solutions come into contact rinse immediately with water and seek medical advice immediately 15 Material Safety Data Sheets MSDS are available on request 16 Use of this product should be limited to personnel trained in the techniques of amplification 17 Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification Area Do not return samples equipment and reagents in the area where you performed previous step Personnel should be using proper anti contamination safeguards when moving between areas 8 STORAGE INSTRUCTIONS Part N 1 Controls must be stored at 2 8 C Part N 2 HCV Real TM Quant must be stored at 20 C The kit can be shipped at 2 8 C for 3 4 days but should be stored at 2 8 C and 20 C immediate
11. 2021 222324 25 26 27 28 29 30 31 32 3334 35 36 37 38 39 40 41 42 12345 6 7 8 91011121314151617 18192021 222324 25 26 27 28 283031 32 3334 35 36 37 38 39 40 41 42 Cycles Cycles Fig 1 FAM 530 channel Internal Control Fig 2 Joe 560 channel HCV Standard Curve t 28 amp 26 Error 0 0104 g Efficiency 1 964 LES 5 22 3 4 5 Log Concentration Fig 3 Standard Curve At first usage the operator must perform a color compensation experiment asking Sacace for the detailed instructions Sacace HCV Real TM Quant April 16 2011 22 LineGene K Bioer Open LineGeneK Software Click File gt New In the Setup Programs tab insert User Name and Test Name Click Dyes button and select FAM JOE Insert the correct liquid quantity 25 ul User nane Testnane Liquid Quant 25 Click the button 9 7 3 times and program the 3 thermalcycling steps At the end it must look like this ed Cycles 1 Cydes 1 Cycles 42 95 0 95 00 On the second phase 60 0 C of step 3 select Single on the Sample Mode column to collect fluorescence data as shown in this picture Target 95 0 00 00 20 4 0 None Y i None Single Click the menu System Parameters P gt Gain Setup G Select Auto Gain box and click OK see picture below for Gain Setup details File E Sample Information S riter Groups G Dyes D T Raw Data R 0L Fao Grad Temp Calculation C 25 25 2 25
12. 6 BIOTECHNOLOGIES HCV Real TM Quant Handbook Real Time Kit for the Quantitative detection of Hepatitis C Virus in human plasma for use with RotorGene 3000 6000 Q Corbett Research Qiagen SmartCycler Cepheid 1Q 1Cycler and 1Q5 Biorad MX3000P and MX3005P Stratagene Applied Biosystems 7300 7500 StepOne Real Time PCR Systems Applera LightCycler 2 069 Roche LineGene K Bioer Eco Real Time PCR System Illumina V1 100 2FRT 16 04 2011 NV 0 Sacace HCV Real TM Quant April 16 2011 TABLE OF CONTENTS v Ge E RADI Roo ER gt 07 N mM 15 16 17 18 19 EI M PUY OCU CHOM M E B c R Principle 0I XS9dV oeesses vetas E FEE E UP bx cR TQUE ME MEX DERI EMI MK EECU DENEN Materials Provided e Materials Required but Not Provided eere eee e rese ue S ev roe ERE ERR E E EN PRA ER SEP FEES UE TERI es Warnings and Precautions c sievev vastos duo upEvan UE RREFENUERULUM FI icQuIM EVI S NOT PUn MES CAVE FER UNDE MEE Virg 1010 TR wICIDILAT E E EU Et Specimen collection Storage and Transport ccccccccccccccccccccccccccc
13. 6 53 7 27 10 IEEE Analytical sensitivity The kit HCV Real TM Quant allows to detect HCV RNA in 100 of the tests with a sensitivity not less than 20 U ml value obtained using the Magno Virus extraction kit Sacace REF K 2 16 and RotorGene 6000 The detection was carried out on the control standard and its dilutions by negative plasma Human immunodeficiency virus 1 Sa Linearity The linearity of the HCV Real TM Quant assay was tested with the HCV RNA Standard and it s dilution using HCV negative human plasma Each dilution was analysed three times and the mean HCV RNA titer of each sample was determined HCV Real TM Quant is linear from 2 5 x 10 to 5 x 10 IU mL Test results greater than 50 000 000 IU mL are above the upper limit of quantitation of the test and should be reported as greater than 50 000 000 IU mL If quantitation results are desired for such samples the specimen should be diluted 1 10 with negative serum and retested Test results less than 250 U mL are below the lower limit of quantitation of the test and should be reported as less than 250 IU mL 28 Table 1 Comparison among the results of HCV RNA viral load detection using HCV Real TM Quant Sacace kit and reference manufacturer s kit Sample ae xl cdi pie wm sm e sw 99 ww ww 9 sm omm w ome o 99 mw wem 7 sem mee os mee e 3 sew nem aor ome o 99
14. C ResWs Area to analyze Amplification plots C Plate sample values C Standard curve C Initial template quantity C Dual color scatter plot C Text report Last cycle ls Fluorescence dA Threshold fluorescence v JOE 500 0002 Reset FAM 1000 00 Reset NVA Reset Fluorescence dR Select amplification plots to display B2 JOE Unknown 22 18 B2 FAM Unknown 5 B3 JOE Unknown 25 38 B3 FAM Unknown 24 07 B4 JOE Unknown 28 71 B4 FAM Unknown 25 25 B5 JOE Unknown 34 32 B5 FAM Unknown 8 BB JOE Unknown 32 58 BE FAM Unknown 25 05 Cycles B JOE Unknown 35 32 B FAM Unknown 8 Select All Disable Dyes Dyes shown Well types shown JOE FAM Unknown Standard Results Door Closed Lamp Off F Sacace HCV Real TM Quant April 16 2011 18 Applied Biosystems 7300 7500 StepOne Real Time PCR Systems Applera 1 Select in the main menu the option New Experiment Advanced Setup insert experiment details Select your Instrument Type 7500 7500 fast StepOne StepOne Plus Quantitation Standard curve Taqman Reagents Standard ramp speed DO NOT select Fast RampSpeed Which instrument are you using to run the experiment Which reagents do you want to use to detect the target sequence Set up run and analyze an experiment using a 4 or 5 color 96 well system The PCR reacticns cottain primers des gned to amalify the
15. Cal E 2D ul suggest to select 20 ul and use 30 ul polycarbonate capillaries 2 Program the thermalcycling as follows ied Real TM Quant one None Stage Profile NENNEN yu Hi 39c 309b cycles None 3 95 C 15 00 T 95 C 0 15 Progra None 60 C 0 40 a a a Program 42 E Quantification Program Temperature Targets Acquisition mode Single Target C Hold hh mm ss Ramp Rate C s Sec J Step Size C Step Delay Acquisition Mode cycles 5 00 g Overview 100 O eee d 0 00 00 0 30 02 0 45 09 0 57 27 1 10 05 1 22 23 1 34 46 Time h mm ss 3 Click and insert sample names then select Absolute Quantification from Analysis Type and select 530 560 in Selected Channels section Sample data 1 Analysis Type Reset Samples Impe P Absolute Quantification Color Compensation Genotyping 4 X Nucleic Acid Quantification Qualitative Detection Relative Quantification Dual Color Relative Quantification Monocolor Tm Calling Selected Channels 530 560 61 o 64 4 Insert target Name Sample Type Standard for standards Unknown for all other samples and insert sample concentration values as reported in the Data Card Pos Sample Name Channel Target Name
16. HCV Reporter HEX and the other one for Internal Control Reporter Fam select Quencher Non fluorescent for both targets Click Assay Mame Color Reporter Quencher Internal Control C FAM NonFluorescent HBV 6 HEX NanFlucrescent 4 Click and enter name for all samples controls and standards Click 5 Associate samples and standards with the two previously designed assays following this procedure select the well select Unknown for samples and Standard for standards and to assign the corresponding assay click the white circle of the HCV and Internal control assays the circle will become colored for each well both Assays must be assigned 6 Click m and in the Set Up Standards box insert each value reported in the DataCard click Enter after inserting each value both for HCV assay and Internal control assay then associate concentration values to the corresponding well In the end it should look like above Set Up Standards 1 2 3 Sampei ENENEEMNEEEEENNM Sample3 vas zu 9086 2 ee Apply Standards 68350 Sampe4 SampeS ample ple 8220 gos go 0 Ww w an D 760 B wy e uj s 68350 s8220 s 760 w 0 ermal Profile s 7 Click on mE and program the instrument as follows reaction volume is 20 ul1 ILL HCV Real TM Quant Reverse Polymerase PCR Cycling Transcription Activation 3 95 C 0 15 42 60 C 1 00 Add 10 ul of Reaction Mix into eac
17. Sample Type Concentration 8 Sample 8 530 IC Unknown 560 HCV Unknown 3 Sample 3 530 JIC Unknown 560 HCV Unknown 10 Cneg EXTR 530 JIC Unknown 560 HCV Unknown 11 POS 1 530 IC Unknown 560 HCY Unknown 12 POS 2 530 JIC Unknown 560 HCV Unknown 13 Neg PCR 530 JIC Unknown 560 HCV Unknown 14 Q51 HCV 530 IC Unknown 560 HCY Standard 8 72E5 15 Q52 HCV 530 IC Unknown 560 HCV Standard 5 46E3 16 Q53 HCY 530 IC Unknown 560 HCV Standard 3 10E1 17 Q81 IC 530 t Standard 1 88E5 560 HCV Unknown 18 QS2IC 530 IC Standard 1 88E4 560 HCV Unknown 19 QS31C 530 IC Standard 8 20E2 560 HCV Unknown Click totus to start PCR reaction 6 After Run is completed click Analysis button and select Absolute quantification Select Method Auto Standard Curve In Run Ct values and calculated results will appear for the selected channel under the CP and Concentration column in the Absolute Quantification table Ese ee Quantification HCV Quant FAM JOE Channel 560 Color Compensation On Program Samples Results n vi Sample 1 2 nple 2 32 92 2 40E 1 8 nple 3 1 29E0 Sample 4 31 98 1 Sample 5 31 17 9 27E1 6 Sample 6 31 72 6 06E1 Sample 7 31 59 6 67E1 8 Sample 8 31 8 5 53E1 v 7 Calculate results manually using the usual formula as indicated in the User Manual of the kit Amplification Curves Amplification Curves 8 z z 12345 6 7 8 9 1011121314151617 1819
18. cccccccsccssssssssssssssecs 2 RNA COLO es esiersts Bhuodgcenig c Rear PEeDAIEOUOIL ececaciccioe access neues OR EEE e RU PEU Dong eiF Usa cta D sio Vota rU ERES RIT FOE Protocol and Data Analysis SmariCycler 6 inm e RotorGene M 3000 6000 Corbett Research ccccccccccccccccccccccccccccccccccsccccesccscces 6 jw IWC ycler and 105 BiOrad sicicccccsasensevssadeccscsaetver venues UI URS EEN DIR DEDI NAAR Ua FA EEUU MX3000P and MX3005P Stratagene ccccccccccccccccccccccccccccccsssssssssssssssssssseees e Applied Biosystems 7300 7500 Real Time PCR Systems Applera ccccccscccccccceees e gt yder 2 08 0076 e LimeGene KO 1067 6 Eco Real Time PCR System Tlumima ccccccccccccccsscccccccccccsssccsssccccccsscsssseees Results tes Performance 6 06001 0 exe eeeressenss 0 Explanation of 005 10 tine even eonadsetareudsaacoesserdisecieiess 1 NAME HCV Real TM Quant 2 INTRODUCTION Hepatitis C virus HCV is a single stranded RNA virus that belongs to the family Flaviviridae In 1974 HCV was initially recognized as non A non B hepatitis virus NANBH until 1989 when the etiologic agent was cloned It is estimated by the World Health Organization that worldwide there are 170 million HCV infected persons The prima
19. ch the amplification will be carried out Make sure that the following settings for dynamicwf are selected 1 cycle 95 C 8 1 cycle 95 C 30 s fluorescence measurement on the second step 1 Select in the main menu Define Protocols and click Create a new protocol Set the following parameters 30 0 2 1 9s 2 B 00 20 p w dw pm PCR Melk Data Acquisition 1 30 00 50 0 1 1500 95 0 1 0 20 95 0 gt 2 1 00 60 0 i Real Time 2 Select Edit Plate for iQ5 or View Save Data for iQiCycler to create the plate for samples and controls Enter the concentrations of the Quantitative Standards reported on the HCV TM 1Q Quant Data Card in the Standard Quantity box QS1 HCV QS2 HCV QS3 HCV as Standard in the HEX channel and Unknown in the Fam QS1 IC QS2 IC QS3 IC as Standard in the Fam channel and Unknown in the HEX channel Use icon Unknown for all samples 3 Choose the FAM and HEX channels for all samples 4 For the iQ5 instrument enter the reaction volume Sample volume 25 yl Select caps type Seal Type and tubes type Vessel Type Save plate setup 5 Begin the amplification For the 1Q iCycler instrument activate the key Run with selected plate and choose in the window of the opened menu the reaction volume 25 ul select PCR Quantification Melt Curve and Experimental Plate Click the button Begin Run and save the experiment settings For t
20. entration in the Quantity field as indicated in the Data Card Target HCY RNA v Task Standard v Quantity 1000 0 Target Internal Control Task Unknown v Quantity Sample Q81 HCV vi 6 Click on id oes insert Reaction volume 25 ul Number of Cycles 42 2 HCV Real TM Quant See Pete a 3 95 C 0 15 42 60 C 1 00 Legend Data Collection On Data Collection Off 7 Click to begin PCR reaction 8 After the run is complete select all samples and standards and click Analysis Settings D Real TM Quant April 16 2011 19 NONCAIM UC V Sacace HCV 9 10 11 D 13 900 000 850 000 800 000 750 000 1 700 000 650 000 600 000 550 000 600 000 c 450 000 AR 350 000 300 000 250 000 200 000 150 000 gt 100 000 60 000 o 50 000 NS 400 000 Deselect Use default settings Automatic threshold and automatic baseline suggested values are Cr Settings for HCV Cr Settings for Internal Control CT Settings to Use Use Default Settings Cr Settings to Use Use Default Settings E Automatic Threshold Automatic Threshold Threshold 30 000 Threshold 35 000 E Automatic Baseline Automatic Baseline Baseline Start Cycle 3 5 End Cycle 4 Baseline Start Cycle 3 End Cycle 15 7 for StepOne instrument recommended Threshold values are Joe HCV 1 000 Fam IC 2 000 NOTE
21. f patients with chronic hepatitis C Virus Infection HIGH VIRAL LOAD O Treatment Started LOW VIRAL LOAD Virological Response me D Nonresponse E Slow Response F Early Response G Rapid Response H Relapse D Sustained Response Log g HCV RNA HCV RNA Testing 4 Quantitative 4 Qualitative or Highly Sensitive Stop Treatment x All Genotypes 69 Genotype 2 or 3 Genotype 1 or 4 6 12 18 24 30 3 42 48 54 60 66 72 A A A Time From Treatment Onset wks 4 PRINCIPLE OF ASSAY Kit HCV Real TM Quant is a Real Time test for the Quantitative detection of Hepatitis C Virus in human plasma HCV RNA is extracted from plasma amplified using real time amplification and detected using fluorescent reporter dye probes specific for HCV or HCV IC Internal Control IC serves as an extraction and an amplification control for each individually processed specimen and to identify possible inhibition IC is detected in a channel other than the HCV RNA Monitoring the fluorescence intensities during Real Time allows the detection and quantification of the accumulating product without having to re open the reaction tube after the real time amplification 5 MATERIALS PROVIDED Part N 1 Controls RNA isolation controls Part N 2 HCV Real TM Quant Real Time kit Part N 2 HCV Real TM Quant RT PCR mix 1 TM Standards and controls concentrations are specific for every lot mu
22. h tube Add 10 ul of extracted RNA sample or quantitative standards to the appropriate tube with Reaction Mix Number of Cycles 42 8 Click Start Run to begin PCR reaction E 9 After the run is complete click then click o tab to interpret the results 25 10 Click v to Export the samples in the Export Options select Results Table 6 Export Options Experiment File Name HCV Quant exp 2 results run Export Location CN Browse CSV PDF Report Excel Export Options CO Component Data O Plate Layout O Component Melt Data C Thermal Profile O Amplification Data E Results T able CO Melt Data 11 Using the exported file calculate results pasting data from the Sample Name and the Quantity columns in the provided HCV Quant Result Calculation sheet FAM Internal Control Color Slope Y Intercept Efficiency 2 10 103 10 105 10 107 108 Bn 3 376 39 324 97 783 0 999 10 103 10 105 105 Standard Curve Standard Curve 26 15 RESULTS INTERPRETATION The Internal Control IC is detected on the FAM channel and HCV RNA on the Joe HEX Cy3 channel For each control and patient specimen calculate the concentration of HCV RNA using the following formula HCV DNA copies specimen Joe HEX Cy3 channel x coefficient IU HCV mL IC DNA copies specimen FAM channel coefficient is specific for each lot and reported in the HCV TM RG Quant Data Card provided in
23. he 1Q5 instrument activate the button Run and choose in the window of the opened menu Collect Well Factors from Experimental Plate Click the button Begin Run and save the experiment settings 15 Data Analysis The results are interpreted with the software of IQ iCycler or 1Q5 through the presence of crossing of fluorescence curve with the threshold line Internal Control IC is detected on the FAM channel and HCV RNA on the HEX channel Activate the button PCR Quant for the results analysis Results analysis for Fam channel IC Activate the button Log View Put the threshold line with the left button of the mouse at such level where curves of fluorescence are linear see figure Results analysis for HEX channel HCV RNA Activate the button Log View Put the threshold line with the left button of the mouse at such level where the curves of fluorescence are linear see figure ue 1 1 77 i t 15 5 40 16 MX3000P and MX3005P Stratagene 1 Open the program select Quantitative POR pe Standarts and click OK New Experiment Options E xj CA ble 1 Feal ane i Molocuar Haacan bise Dome Plate read T Quantitative Plate Read C Plate Read Allele Discrimination Meo Tumdempenderwemrug f Don t show this again At the top left of the window choose Plate Setup In the window Well type set Unknown for
24. into the tube with DTT 300 ul of RT PCR mix 1 200 ul of RT PCR mix 2 and vortex for at least 5 10 seconds This mix is stable for 1 month at 20 C Add for each sample N in the new sterile tube 12 5 N ul of mix 0 5 N ul of TaqF Polymerase and 0 25 N ul of M MLV For the second run it s recommended to import the standard curve generated in the first run provided that at least one HCV and IC standards is used 4 Add 12 5 ul of Reaction Mix into each tube 5 Add 12 5 ul of extracted RNA sample to the appropriate tube with Reaction Mix and mix by pipetting If the Ribo Sorb isolation kit is used as a RNA extraction kit re centrifuge all the tubes with extracted RNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B don t disturb the pellet sorbent inhibit reaction 6 Prepare for each run 6 standards and 1 negative control e add 12 5 ul of Quantitation Standards HCV QS1 HCV QS2 HCV QS3 HCV into 3 labelled tubes e add 12 5 ul of Quantitation Standards IC QS1 IC QS2 IC QS3 IC into 3 labelled tubes e add 12 5 ul of TE buffer to the tube labelled Negative Control Close the tubes and transfer them into the Real Time PCR instrument 14 PROTOCOL DATA ANALYSIS SmartCycler Cepheid 1 Select in the main menu Define Protocols and press the button New Protocol Give a name to the protocol and set the following parameters 1 Stagel 50 C 1800 sec 2 Stage2 95 C 900 sec 2 Temperatu
25. ion of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DOES UN 11 RNA ISOLATION The following isolation kits are recommended gt Ribo Virus 100 spin column extraction kit Sacace K 2 C 100 Ribo Sorb 100 Sacace Please carry out the RNA extraction according to the manufacturer s instructions Add 5 ul of Internal Control during the RNA isolation procedure directly to the sample lysis mixture see 12 Internal Control 12 INTERNAL CONTROL HCV Rec IC HCV Rec IC is a quantitative Internal Control concentration reported in Data Card and represents recombinant RNA containing structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of quantitative HCV Rec IC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the RNA during extraction procedure thus enabling to calculate precisely the HCV viral load 13 REAGENT PREPARATION Note Reaction Mix volume 25 ul 1 Thaw one set of reagents vortex and centrifuge briefly the tubes 2 Prepare reaction tubes or PCR plate 3 Prepare Reaction Mix add into the tube with DTT 300 ul of RT PCR mix 1 200 ul of RT PCR mix 2 20 ul of Hot Start Taq Polymerase and 10 ul of M MLV Revertase Vortex thoroughly and centrifuge briefly If it is necessary to test less than 25 samples add
26. l 16 2011 17 if Mx3000P Standalone Quantitative PCR New Experiment mxp 5 x File Edit Instrument Tools Options Section View Window Help Plate Setup Thermal Profile Setup Thermal Profile gt Deas Add Normal Segment Plateau with Ramp Data collection marker by dragging Q All points Enders Selection Edit Properties Delete Temperature a Thermal profile comments Segment 1 Segment 2 Segment 3 1 Cycle 1 Cycle 45 Cycles Full Screen Profile Start Hun To edit select plateaus ramps or segment boundaries Door Closed Lamp Off zm Data Analysis 1 Soon after the amplification is over choose the button Analysis at the top left of the window 2 Choose the button Results 3 At the right angle of the window Area to analyze select Amplification plots 4 Set Threshold fluorescence 500 for the Joe channel and 1000 for the Fam channel 5 Inthe window Text report appear for each sample the values of Ct and experimental values of copies cDNA HCV and cDNA IC 6 Take care that the value of RSq correlation coefficient in the window Standard curve is not lower than 0 9 for both channels f Mx3000P Standalone Quantitative PCR Server Documents 3a es Banepa Real Stratagene Quantitative PCR 08 18 2005 17Hr 42Min mxp E la x File Edit Instrument Tools Options Section view Window Help Analysis Selection Setup C
27. ly on receipt 9 STABILITY HCV Real TM Quant Test is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results 10 SAMPLE COLLECTION STORAGE AND TRANSPORT Note Handle all specimens as if they are potentially infectious agents 1 EDTA tubes may be used with the HCV Real TM Quant Follow sample tube manufacturer s instructions 2 Whole blood collected in EDTA should be separated into plasma and cellular components by centrifugation at 800 1600 x g for 20 min within six hours The isolated plasma has to be transferred into a sterile polypropylene tube Plasma may be stored at 2 8 C for an additional 3 days Alternatively plasma may be stored at 18 C for up to one month or 1 year when stored at 70 C Do not freeze whole blood Specimens anti coagulated with heparin are unsuitable for this test Thaw frozen specimens at room temperature before using Whole blood must be transported at 2 25 C and processed within 6 hours of collection Plasma may be transported at 2 8 C or frozen 7 Transportat
28. nnel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the RNA extraction procedure Weak Ct 40 signal on the Cy3 channel retesting of the sample is required The correlation coefficient R is less than 0 9 retesting of all samples is required The calculated concentrations of HCV Rec Pos and or HCV Rec Pos2 are different from given control concentrations reported in the Data Sheet retesting of all samples is required Any signal on the Joe HEX Cy3 channel with Negative Control of extraction e Contamination during RNA extraction procedure All samples results are invalid gt Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol gt Use only filter tips during the extraction procedure Change tips among tubes Repeat the RNA extraction with the new set of reagents Any signal with Negative PCR Control e Contamination during PCR preparation procedure All samples results are invalid gt Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end Repeat the PCR preparation with the new set of reagents 18 REFERENCES l 3 11 L2 13 14 15 16 World Health Organization Hepatitis C Global prevalence update Wkly Epidemiol Rec 1999 74 425 427 Alter H 1999 Discovery of non A non B hepati
29. oe In the Quantitative Analysis tab select from the menu Dyes D gt Joe F Select standard Concentration boxes left mouse click CTRL for HCV Status C Sample Concentration P Neg extr POS1 m POS1 POS2 m POS2 Ej QS1 HCV 8 72e 005 Bou 5 46e 003 E Q53 HCV 310e 001 QS1 IC 1 88e 005 E QS2 IC 1 88e 004 QS3 IC 8 20e 002 NTC Then do in this order 1 Under Analysis Method select Fit Points 2 Under Analysis Step Click Zero Adjust select Auto and then click OK Then click Baseline 3 Select 2 points and set the base line drag and drop the red baseline as low as possible but above the noise of each sample usually 15 20 4 Under Analysis Step clic Analysis Calculated copies reaction of HCV and Ct Results should appear in the Ct and the Calculated Concentration column respectively Analysis Method Analysis Step Points Selected Baseline and Threshold Display Mode Driv Max Elzero Adjust Baseline Logarithmic ElBaseline ElFull Scale MIFit Points Analysis Show Points Threshold 1 00 Smooth hi 24 Eco Real Time PCR System Illumina Quantification Hydrolysis Probe 1 Open Eco software click experiment and under Experiment Type select l select RNA as starting material for HCV Real TM Quant 2 U o Standard Curve nder Quantification Method select and insert experiment name Click 3 Click Q set up two assays one for
30. patients who have recovered from infection may remain anti HCV positive for many years Conversely during seroconversion antibody tests may be negative The direct molecular biology detection of HCV RNA by reverse transcriptase polymerase chain reaction RT PCR is considered the gold standard for the diagnosis of HCV infection 3 INTENDED USE Kit HCV Real TM Quant is a Real Time Amplification test for the Quantitative detection of Hepatitis C Virus in human plasma and the simultaneous detection of a HCV specific Internal Control IC by dual color detection Quantitative HCV RNA testing provides prognostic information regarding likelihood of treatment response and it plays an important role in monitoring the antiviral response to treatment Sustained virological response is defined as testing negative for HCV RNA 6 months after cessation of therapy Recent studies suggest that the rate of response to therapy is also important For example conversion to an HCV RNA negative test result after 4 weeks of therapy constitutes a rapid virological response and is a strong predictor of treatment success Patients who have not had an early virological response defined as at least a 2 log decline in HCV RNA after 12 weeks of therapy are unlikely to respond with an additional 36 weeks of therapy and should stop therapy The same type of quantitative HCV RNA test should be used throughout a patient s treatment course Fig 2 Monitoring treatment response o
31. please slightly adapt suggested values according to your result curves Apply Analysis Settings Click S Select all samples and standards click View well table and then Click File gt Export select Results only Custornise Export Sample Setup v Results E Raw Data E Multicomponent Data Amplification Data Export Properties 1 Select data to export Click Customize Export and sort samples by reporter as indicated in the above image Then click CS J export results in a file Using the exported file calculate results pasting data from the Sample and the Quantity columns in the provided HCV Quant Result Calculation sheet Amplification Plot Amplification Plot 650 000 600 000 550 000 500 000 1 450 000 400 000 350 000 ARn 300 000 250 000 1 200 000 150 000 100 000 1 50 000 28 x 32 3 35 38 40 2 4 2 4 6 a 10 12 14 16 18 20 2 24 28 28 2 E 3 3a 4n 42 2 24 2 Cycle Cycle FAM Internal Control Joe HC 20 LightCycler 2 0 Roche 1 Select New LightCycler Experiment Click E and insert as follows Sety Default Channel 530 e Max Seek Pos indicates the number of samples used in the experiment e Choose Instrument Type according to your instrument Seek Temperature 0 Max Seek Pos 13 Instrument Type E Ch Choose capillary Size according to your capillaries we
32. re 95 C 20 sec Cycle 60 C 40 sec Repeat 42 times optics ON 2 Choose Save Protocol 3 Click the Create Run button in the main menu then the button Dye Set and select FCTC25 amp Smart Cycler 18 x User Logs Setup Tools Help Check Status Stop Run sitein Protocol Sample Sample Type Notes FAM Std 3 TXR Std Cy5 Std Run Name 2 E 2 Selections Site Protocol Notes atl AddRemow Sites Dye Set FCTC25 v ch Dye Usage Bkand Name Sub Mir 11 FAM Assay ON 5 Protocols Protocol toNumer 3 Assay ON 5 213 TXR Assay ON 5 Hn N Cy5 Assay O 5 4 Graphs HNA EPEA Standard FAM 2 Select All Sites Standard Cy3 E d Cance Ol FAM E FR SST St SEE HIER Rox emperature m secre err TE ERE EE EE Start an CancetRun Setup Report Run Setup SelectGranhe ConyRun Setup 4 Choose Add Remove Sites and select in the new windows the protocol and the sites for analysis Click OR Choose Start Run and give a name to the experiment 6 Insert in the column Sample Type UNKN for samples Enter the concentrations of the Quantitative Standards reported on the HCV TM SC Quant Data Card in the columns Fam Std and Cy3 Std in order to generate Standard curves 7 Fluorescence is observed in Real Time on the Cy3 channel for HCV RNA and
33. region c100 3 as well as a part of c100 3 named 5 1 1 Third generation anti HCV ELISA was introduced in Europe in 1993 and in the USA in 1996 In addition to the antigens of ELISA 2 third generation anti HCV ELISA uses a NS5 region antigen of the viral genome However synthetic peptide antigens c22 and c 100 replaced recombinant antigens of ELISA 2 Other manufactures for example Abbott Diagnostics used recombinant antigens derived from the same regions of HCV genome Despite the increased sensitivity and specificity with each generation of ELISA false positive antibody results continue to be observed particularly among low risk blood donors Thus supplemental or confirmatory assays were developed in parallel with ELISA The recombinant immunoblot assay RIBA was used extensively to confirm the presence or the absence of antibody to HCV epitopes In RIBA recombinant or peptide HCV antigens are blotted as separate bands onto a nitrocellulose strip flanked by a weak positive Level I and a moderately positive Level II strip control Fig 1 Genome organization of HCV and antigens licensed for diagnostic use Structural proteins gt gt 4 amp Nonstructural proteins EN EN a 5 1 1 NS5 c33c c100 3 c22 3 c200 Since ELISA and RIBA are antibody tests the positivity of either one or both does not necessarily indicate current HCV infection as
34. ribavirin compared with interferon alfa 2b plus ribavirin for initial treatment of chronic hepatitis C a randomized trial Lancet 2001 358 958 965 Weiland O Braconier JH Fryden A Norkrans G Reichard O Uhnoo I Influence of pretreatment factors on outcome of interferon alpha treatment of patients with chronic hepatitis C Scand J Infect Dis 1999 31 115 118 Fried MW Shiffman ML Reddy KR et al Peginterferon alfa 2a plus ribavirin for chronic hepatitis C virus infection N Engl J Med 2002 347 975 982 Jensen DM Morgan TR Marcellin P et al Early identification of HCV genotype 1 patients responding to 24weeks peginterferon alpha 2a 40kd ribavirin therapy Hepatology 2006 43 954 960 Davis GL Wong JB McHutchison JG Manns MP Harvey J Albrecht J Early virologic response to treatment with peginterferonalfa 2b plus ribavirin in patients with chronic hepatitis C Hepatology 2003 38 645 652 Terrault NA Pawlotsky JM McHutchison J et al Clinical utility of viral load measurements in individuals with chronic hepatitis C infection on antiviral therapy J Viral Hepat 2005 12 465 472 31 19 EXPLANATION OF SYMBOLS Catalogue Number RUO For Research Use Only LOT Lot Number b Expiration Date Contains reagents Temperature limitation Cycler and iQ5 are trademarks of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Corbett Research MX3000P and MX3005P are trademarks of Stratagene
35. ry mode of HCV transmission is the exposure to infected human blood via intravenous drug use or unscreened transfusions Nosocomial HCV transmission during dialysis colonoscopy and surgery has also been reported Perinatal and sexual transmission of the virus is inefficient but occurs more frequently if the HCV infected mother or sexual partner is also infected with human immunodeficiency virus type 1 Most people with acute HCV infection are asymptomatic or have mild symptoms fatigue nausea jaundice but they are unable to clear the virus and in approximately 80 of cases this leads to chronic infection In 15 to 20 of patients chronic HCV infection progresses at a variable rate to cirrhosis with a 1 to 4 annual risk of developing hepatocellular carcinoma The discovery of HCV genome in 1989 by Choo et al paved the way for the development of serological and molecular assays for viral hepatitis C In the first generation of an enzyme linked immunosorbent assay ELISA wells of microtitre plates were coated with purified recombinant antigen c100 3 which was derived from the non structural 4 NS4 region of the HCV genome However ELISA 1 was associated with a high percentage 50 to 70 of false positive results among low risk blood donors and in the presence of hyperglobulinemia Thus second generation anti HCV ELISAs were developed ELISA 2 by Ortho Diagnostics contained recombinant antigens from the core c22 3 NS3 region c33c and NS4
36. ss Analysis and then select Quantitation Cycling A Fam Cycling A Green Show Turn off the automatic option Threshold Press the buttons Dynamic Tube Slope Correct In the table of results Quantitation Analysis select More settings and set NTC Threshold to 10 Select Threshold 0 03 In the table of results Quantitation Results appear the values of Ct Threshold cycle which should be lt 30 In the menu window Quantitation Results column Calculation concentration appear values of IC cDNA copy specimen Quantitation Analysis Cycling AFAM Sybr Page t aS c ps ee 9 ABVRBLSRL SRS 5 HCV amplification analysis Cycling A Joe Yellow 1 Press Analysis and then select Quantitation Cycling A Joe Cycling A Yellow Show 2 Turn off the automatic option Threshold 3 Press the buttons Dynamic Tube Slope Correct 4 Inthe table of results Quantitation Analysis select More settings and set NTC Threshold to 6 5 Select Threshold 0 03 6 Inthe table of results Quantitation Analysis appear the values of Ct Threshold cycle 7 Inthe new window Quantitation Results appear values of HCV cDNA copy specimen amp Quantitation Analysis Cycling A JOE Page 1 Sacace HCV Real TM Quant April 16 2011 14 iQ iCycler and iQ5 Biorad Make sure that the 1Q5 instrument is calibrated for working with 50 ul reaction mix Perform the calibration with the same tubes in whi
37. st be used during the sample preparation procedure see RNA isolation must be used during the sample preparation procedure add 100 ul of C Negative Control to labeled Cneg add 90 ul of C Negative Control and 10 ul of HCV Rec Pos controls to the tubes labeled Cposl and Cpos2 6 MATERIALS REQUIRED BUT NOT PROVIDED RNA isolation kit see 11 RNA isolation Desktop microcentrifuge for eppendorf type tubes Vortex mixer Disposable gloves powderless Biohazard waste container Refrigerator Freezer Real Time Thermal cycler Workstation Pipettes adjustable Sterile pipette tips with filters Tube racks Sacace HCV Real TM Quant April 16 201 1 6 7 WARNINGS AND PRECAUTIONS 1 Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward 2 Use routine laboratory precautions Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not pipette by mouth 3 Do not use a kit after its expiration date 4 Do not mix reagents from different kits 5 Dispose all specimens and unused reagents in accordance with local regulations 6 Heparin has been shown to inhibit reaction The use of heparinized specimens is not recommended 7 Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test 8 Once the reagents have been thawed vortex and centrifuge briefly the tubes 9
38. targat sequence and a TaqMan probe t What type of experiment do you want to set up Which ramp speed do you want to use in the instrument run cus For optimal results with the standard ramp speed Applied Biosystems recommerds using standart Use standards to determine the absolute quantity oftargel nucleic acid sequence in samples Note when using StepOne software be sure to click New Experiment Advanced Setup 5 StepOne Software v2 1 File Edit Instrument Analysis Wdvenced Satun New Experiment Advanced setup Design Wizard From Template 2 Click EM add two targets one for HCV Reporter Joe and the other one for Internal Control Reporter Fam select Quencher None for both targets 3 Click Add new samples multiple times and enter name for all samples controls and standards 4 Click Assign target and Samples tab associate samples m h and standards ES with the two targets and select None as passive reference dye as shown in the following pictures 3 1 2 Assign target s to the selected wells Q 1 ic 52 IC 53 Ic Task Assign Target Neg Control Sample 1 Sample 2 nev RNA OT a Select the dye to 5 Double click on each standard well a new small window will appear and enter both for HCV and Internal control the corresponding conc
39. the kit Results may by calculated also using HCV Quant Result Calculation sheet provided with the kit To obtain the results in copies mL multiply the IU HCV ml value by 4 IU RNA HCV mL x 4 copies RNA HCV mL 21 16 PERFORMANCE CHARACTERISTICS The sensitivity the specificity the performance and the linearity of the kit HCV Real TM Quant were determined with the following samples 1 The HCV RNA standard with the concentration of 2x10 U ml and its dilution 2 The samples of blood plasma obtained from 215 patients with IgG antibodies to HCV The results were compared to those ones obtained by the reference manufacturer s kit 3 80 samples of blood plasma obtained from donors Analytical specificity The analytical specificity of the primers and the probes was validated with 80 negative samples They did not generate any signal with the specific HCV primers and probes The specificity of the kit HCV Real TM Quant was 100 The potential cross reactivity of the kit HCV Real TM Quant was tested also against the group control listed in the following table It was not observed any cross reactivity with these pathogens Table 1 Testing the specificity of the kit with other pathogens Adenovirus type o Adenovirus type3 Herpes simplex virus 1 Herpes simplex virus 2 Human herpes virus 6 Human herpes virus 8 HPV groups 6 y u 1 3 4 5 8 37 38 65 20 24 49 50 15 HPV group a 6 11 2
40. the samples and Standard to identify calibrators In the window Collect fluorescence data select for all samples the channels Fam and Joe Enter the concentrations of the Quantitative Standards reported on the HCV TM MX Quant Data Card in the Standard Quantity box yf Mx3000P Standalone Quantitative PCR New Experiment mxp E 2 18 x File Edit Instrument Tools Options Section View Window Help De B amp B oo 8 RE Thermal Profile Setup bm 9 Import Defaults bg Buick Setup Welltype Standard gt Collect fluorescence data Iv JOE HEX Rox FAM Reference dye lt none gt All wells Standard quantity lt all gt 2 O0e 000 Auto Increment 10x m Standard units copies Identify replicates Replicate symbol lt none gt x Auto Increment Clear Selected Wells Plate setup comments Full Screen Plate Next gt FAM Pe Assign Well Type and Dyes to selected wells The Ctrl key selects multiple wells Door Closed LampOff Sm 6 At the top left of the window select button Thermal Profile Setup 7 Set the following parameters of amplification 1 50 C 30 min 2 95 C 15 min 3 05 C 20 sec 60 C 60 sec 45 Cycles 8 Fluorescence is measured at 60 C To do this set on the Thermal Profile graph the Endpoints marker 9 Click Run button enter a name for the experiment and save it Sacace HCV Real TM Quant Apri
41. tis and identification of its etiology Am J Med 107 Supp1 6B 16S 20S Bendinelli M M Pistello F Maggi and M Vatteroni 2000 Blood borne hepatitis viruses hepatitis B C D and G viruses and TT virus p 306 337 In S Specter R L Hodinka and S A Young ed Clinical virology manual 3 ed ASM Press Washington D C Choo Q L G Kuo A J Weiner L R Overby D W Bradley and M Houghton 1989 Isolation of a cDNA clone derived from a blood borne non A non B viral hepatitis genome Science 244 359 362 Lauer G M and B D Walker 2001 Hepatitis C virus infection N Engl J Med 345 41 52 De Medina M Schiff ER Hepatitis C diagnostic assays Sem Liver Dis 1995 1 33 40 Kao J H M Y Lai Y T Hwang P M Yang P J Chen J C Sheu T H Wang H C Hsu and D S Chen 1996 Chronic hepatitisC without anti hepatitis C antibodies by second generation assay a clinicopathologic study and demonstration of the usefulness of a third generation assay Dig Dis Sci 41 161 165 Schiff ER de Medina M Kahn RS New perspectives in the diagnosis of hepatitis C Sem Liver Dis 1999 19 3 15 Lok ASF Gunaratnam NT Diagnosis of hepatitis C Hepatology 1997 26 Suppl 1 48 565 Pawlotsky J M Bouvier Alias M Hezode C Darthuy F Remire J Dhumeaux D Standardization of hepatitis C virus RNA quantification Hepatology 2000 32 654 659 Manns MP McHutchison JG Gordon SC et al Peginterferon alfa 2b plus
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