HBV Real TM Quant L - bio
Contents
1. BIOTECHNOLOGIES HBV Real TM Quant Dx Handbook Real Time PCR Kit for the Quantitative detection of Hepatitis B Virus in human plasma 1023 For Quantitative inVitro Diagnostic Use V5 96 3FRT Y 96 For use withRotor Gene 6000 Q Qiagen For use with SaCycler 96 Sacace ud Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy LOT Dr E 5 KEY TO SYMBOLS USED List Number Lot Number For inVitro Diagnostic Use Temperature limitations Manufacturer Consult instructions for use Expiration Date Caution O ONTROL IN lt lt m JJ ONTROL 1 ONTROL 2 ONTROL T i gt O gt N Contains sufficient for n tests Version Internal control High Positive Control Low Positive Control Negative Control Standard 1 Standard 2 TABLE OF CONTENTS NAME 4 INTRODUCTION seh ates naam 4 INTENDED etnias nhan ass inaa ra Ene ete dab a 4 PRINCIPLE ASSAY e i eKA 5 MATERIALS PROVIDED 6 MATERIALS REQUIRED BUT NOT PROVIDED bra UNUS 6 WARNINGS AND PRECAUTIONS IS YR EGER ORO Ond il2. Y amm 7 Standard 2 ov ES S222 2222 2 8 K C Y 3 K HBVReeT C Y 2 10 C HevRet C v ie Clicking Apply will bring to the Start Program window The thermalcycling program will be the following 1 950 C 15 00 60 0 00 20 2 950 C 00 05 x5 72 0 00 15 60 0 00 30 Gx 3 950 C 00 05 x40 720 C 00 15 Click Open block insert PCR tubes into the instrument click Close block and then click __ to start the PCR amplification program After the run is complete click OK to interpret the results Select analysis type Multiplex detection Threshold Ct method and Quantitative from results table Analysis type Muliplex detection Resuts Additional standards Method Threshold Ct X Quantitative Click on parameter of data analysis icon 4 V Normalization data Adjust the threshold line using mouse drag and drop Suggested values for Threshold line are 40 for HEX channel 25 for FAM channel slightly adapt threshold level according to your result curves Viral load results will appear in the Results column for HEX channel Final results are already expressed in IU ml select Normalization data and click Apply To import standard curve from other run click on Additional standards tab and select the run file contain
3. 7 STORAGE AND SHIPPING INSTRUCTIONS 8 5 OR 8 QUALITY CONTROL 8 SAMPLE COLLECTION STORAGE AND TRANSPORT OA Rr Un REM pecia is 8 INTERFERING SUBSTANCES oerte dii puni eR ui add a s uade tana aS AU ra 9 REAGENTS PREPARATION asd iret FREE Ke RR DR RU En RA a E tU 9 DIN ALISO que 9 INTERNAL CONT ROMs tie ex basa e Deb undi tes Pisas etx me uM Rura Pado RUD wae 10 ASSAY CALIBRATION mE S 10 5 rs 11 QUALITY CONTROL PROCEDURE eei 12 RESULTS INTERPRETATION URP pad USE Mare bin M RAE E UE d raga Pip RR CUR MR 12 PERFORMANCE CHARACTERISTICS 13 ANALYTICAL SENSITIVITY EUR 13 LINEAR RANGE 13 SPECIFICITY E 14 GENOTYPE DETECTION ebd nt e 14 CROSS BESCTIVIT GRAN SE RUE Cd FUGA lE FON nO RUE UN RUN UL UNE POUR 14
4. Sacace HBV Real TM Quant Dx Control CONTROL INT 4vials with lyophilized reagent HBV IC L CONTROL 1 e 4vials with lyophilized reagent HBV Pos1 L C HBV Real TM Quant Dx High Positive Control CONTROL 2 e A4vials with lyophilized reagent HBV Pos2 L C Sacace HBV Real TM Quant Dx Low Positive Control CONTROL 4 0 vials 4 0 ml per vial with Negative Control Sacace HBV Real TM Quant Dx Calibrator Kit CAL 1 e 4vials with lyophilized reagent HBV Quantitative Standard 1 CAL 2 e 4vials with lyophilized reagent HBV Quantitative Standard 2 Standards and controls concentrations are specific for every lot must be used during the sample preparation procedure see DNA isolation MATERIALS REQUIRED BUT NOT PROVIDED DNA isolation kit see DNA isolation Desktop microcentrifuge for eppendorf type tubes Vortex mixer Disposable gloves powderless Biohazard waste container Refrigerator Freezer Real Time Thermal cycler Biological safety cabinet approved for working with infectious materials Pipettes adjustable Sterile pipette tips with filters Tube racks WARNINGS AND PRECAUTIONS 10 11 14 15 16 17 18 In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only Read the instructions in this package insert carefully before processing samples St
5. Intra assay variability SD Ct HBV log SD Ct IC log 0 006 0 004 CV Ct HBV CV Ct IC 1 31 1 05 Inter assay variability SD Ct HBV log SD Ct IC log 0 006 0 004 CV Ct HBV CV Ct IC 1 32 1 05 CROSS CONTAMINATION The cross contamination of the HBV Real TM Quant Dx kit was evaluated for the manual sample preparation using RotorGene Qinstrument To demonstrate absence of cross contamination a panel of 20 samples consisting of alternate samples of negative plasma spiked with high concentration of HBV and negative plasma samples was tested All the positive samples were correctly detected and gave a fluorescence positive reaction with good and clear sigmoid shaped fluorescence curves and a Cycle threshold Ct less than 35 both in HBV channel and in Internal Control channel All the negative samples gave no fluorescence signal in the HBV channel and the Internal Control channel showing no cross contamination at all 15 PROTOCOL FOR ROTORGENE 6000 Q 1 Switch on instrument by pressing the main switch start Rotor Gene software 2 Start a New Run Empty Run Step with Maintenance Open Template In Another Folder Show This Screen When Software Opens Figure 1 Start New Run 3 Select the appropriate rotor check the Locking Ring Attached box and click Next a Welcome to the Adva
6. all of the specimens and controls from that run must be processed beginning from the sample preparation step RESULTS INTERPRETATION The Internal Control IC is detected on FAM Green channel and HBV DNA on Joe Yellow HEX channel Each DNA amplification is associated with generation of a fluorescence signal measurable in FAM Green channel for IC or in Joe Yellow HEXchannel for HBV DNA resulting in a sigmoid growth curve log scale The data analysis is performed according to manufacturer s instructions Operation manual Rotor Gene 6000 Q Qiagen and Instrument Manual SaCycler 96 Sacace using the respective software and considering the recommendations of the handbook Check the obtained results to ensure that the run is valid and to interpret results HBV DNAis determined basing on CT values and a standard curve resulting from analysis of quantitation standards HBV DNAconcentration is expressed in IU ml If the result is more than 100 000 000 IU ml then it will be displayed as the result more than 100 000 000 IU HBV ml If the result is more than the linear measurement range the sample can be analyzed after 10x dilution and the obtained result should be multiplied by 10 If the result is less than 7 IU ml in case of extraction from 1 ml then it will be displayed as the result less than 7 IU HBV ml 12 PERFORMANCE CHARACTERISTICS The HBV Real TM Quant Dx Kit was evaluated according to the common tech
7. 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 70 Do not freeze whole blood Specimens anti coagulated with heparin are unsuitable for this test Thaw frozen specimens at room temperature before using gt 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 Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Serum can be used also as starting material on some occasions In this cases the analytical sensitivity of the kit HBV Real TM Quant Dx is the same but the clinical sensitivity may be significantly decreased because of the precipitation of viral particles during the clot retraction phase of serum preparation INTERFERING SUBSTANCES Elevated levels of bilirubin 215 mg dl and lipids 2800 mg dl do not influence the system Heparin 210 IU ml affects the PCR Samples that have been collected in tubes containing heparin as ananticoagulant should not to be used Also samples of heparinized patients mustnot be used REAGENTS PREPARATION Before starting any HBV Real TM Quant Dx protocol
8. illness that begins with general ill health loss of appetite nausea vomiting body aches mild fever and dark urine and then progresses to development of jaundice It has been noted that itchy skin has been an indication as a possible symptom of all hepatitis virus types The illness lasts for a few weeks and then gradually improves in most affected people A few people may have more severe liver disease fulminant hepatic failure and may die as a result The infection may be entirely asymptomatic and may go unrecognized Chronic infection with hepatitis B virus either may be asymptomatic or may be associated with a chronic inflammation of the liver chronic hepatitis leading to cirrhosis over a period of several years This type of infection dramatically increases the incidence of hepatocellular carcinoma liver cancer Chronic carriers are encouraged to avoid consuming alcohol as it increases their risk for cirrhosis and liver cancer Approximately 300 millionindividuals are chronically infected with hepatitis B virus in theworld Enzyme linked immunosorbent assay ELISA is still a main detection method for HBV infection but ELISAresult can neither efficiently reflect serum viral load or hepatitis activity nor monitor the efficacy of antiviral treatments Currently polymerase chain reaction PCR assay has been widely used for monitoring HBV load HBV DNA monitoring has become an important tool to identify individuals with high viral replic
9. is detected Thecalibration curve slope and intercept are calculated and stored on the instrument The concentrationof HBVDNA in a sample is calculated from the stored calibration curve The quantitative standards CAL1 and CAL2 must be treated in the same way as patients specimens Before the first use of a new lot of HBV Real TM Quant Dx 6 calibrators run must be performed beginning from DNA extraction procedure to generate a calibration curve two calibrators are run in replicates of three Prepare sample preparation tubes for CAL1 3 tubes for CAL2 for each calibration run Add 10 CONTROL INT to each tube e Add CAL1 and CAL2 to the appropriate tubes inthe quantity indicated in the manual of DNA purification kit for example 1000 ul using Magno Virus kit Sacace REF K 2 16 Follow the sample preparation procedure described in the Handbook of used DNA purification kit 10 ASSAY PROTOCOL Note Reaction Mix volume No attempt should be made to weigh out any portion of the freeze dried material prior to reconstitution REAL TIME PCR CALIBRATION PROCEDURE 1 Prepare 6 reactiontubes with lyophilized reagents to perform PCR of extracted calibrators 50 pl 2 Add 50ul of eluted samples obtained from DNA purification of 6 calibrators Follow the procedure for amplification and detection as described in this manual Once an HBV Real TM Quant Dx calibration run is accepted a
10. structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of HBV IC L allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the DNA during extraction procedure thus enabling to calculate precisely the HBV viral load An IC threshold cycle Ct assay validity parameter is established during a calibration run A defined consistent quantity of IC 10 pl is introduced into each specimen calibrator and control at the beginning of sample preparation and measured on the real time pcr instrument to demonstrate proper specimen processing and assay validity The median amplification cycle at which the IC target sequence fluorescent signal is detected during the calibration procedure two calibrators are run in replicates of three establishes an IC Ct validity range to be met by all subsequent processed specimens The established range of samples IC Ct Medium IC Ct of 6 calibrators 2 cycles Specimens whose IC Ct value exceeds the established range must be retested starting from sample preparation ASSAY CALIBRATION A calibration curve is required to quantitate the HBVDNA concentration of specimens and controls Two assay calibrators are run in replicates of three to generate a calibration curve HBV concentration versus the threshold cycle Ct at which a reactive level of fluorescent signal
11. will take approximately 91 minute s to complete The graph below represents the run to be performed Click a cycle below to modify it Insert after 228 Insert before L Remove Remove This cycle repeats 5 time s Click on one of the steps below to modify it or press or to add and remove steps for this cycle 95 deg for 5 secs 72 deg for 15 secs Long Range Touchdown Figure 6 Cycling 1 Amplification Step Sacace HBV Real TM Quant Dx VER 06 06 2013 17 Open SaveAs Help The run will take approximately 91 minute s to complete The graph below represents the run to be performed _ Click on a cycle below to modify it Insert after Insert before Remove 60 deg 95 deg for 5 secs 20 seconds 72 deg for 15 secs Long Range 50 deg for 20 secs Figure 7 Cycling 2 Amplification and Detection Step 6 Click Gain Optimisation in the New Run Wizard menu and set the following parameters m Auto Gain Optimisation Setup x m Optimisation 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 Settemperature 10 60 H degrees Optimise Optimise Acquiring Perform Optimisation Before 1st Acquisition Perform Optimisation At 6
12. 0 Degrees At Beginning Of Run Auto Gain Optimisation will read the fluoresence on the inserted sample at Channel Settings Add Max Gain Edit Green 1 1061 10 Yelow 1 5FI 10Fl 10 Remove Remove All Figure 8 Gain Optimisation 7 Place PCR tubes carefully onto RotorGene 6000 Q rotor close lid and start run Sacace HBV Real TM Quant Dx VER 06 06 2013 18 ICCA Setting Value Green Gain 10 Yellow Gain 10 Auto Gain Optimisation Before First Acquisition Rotor 36 Well Rotor Sample Layout 1 2 Reaction Volume in microliters 50 Once you ve confirmed that your run settings are correct click Start Run to Save Template begin the run Click Save Template 10 save settings for future runs Skip Wizard Beck Figure 9 Starting the run 8 Enter sample positions and names for samples Negative Control and Positive Control for controls If the kit calibration run is performing use name standard for CAL1 and CAL2 and enter the concentrations of the Quantitative Standards reported on HBV Real TM Quant Dx Data Card Rotor Gene Q Series Software VIRTUAL MODE Lyo standard curve lot 10 03 13 Run 2013 03 28 1 File Analysis Run Gain View Window Help Zea gt O F B mo New Open Save tart Help Settings Progress Profile Temp Samples Anal
13. 68 188 Import cr Cancel Sacace HBV Real TM Quant Dx 06 06 2013 19 Data Analysis IC amplification analysis Cycling A Green Fam 1 Press Analysis and then select Quantitation Cycling A Green Cycling A Fam Show 2 Std Curves Other A Green rage T A Yellow Page Hide Auto shrink window Turn off the automatic option Threshold Activate the Dynamic tube and Slope Correct buttons in the main window menu Quantitation analysis 2 Quantitation Analysis Cycling A Green Page 1 celts c 4 Dynamic Slope Correct 3 Ignore First Outlier Removal Save Defaults E04 02 w 3 2 In the table of results QuantitationAnalysis select More settings and set NTC Threshold to 10 Select Threshold 0 03 In the table of results QuantitationResults appear the values of Ct Threshold cycle which should be lt 30 see Internal Control of the IC Fam Green channel HBV amplification analysis Cycling A Yellow Joe 1 Press Analysis and then select Quantitation gt Cycling A Yellow Cycling A Joe Show 2 Std Curves Other Cycling A Green Page T V Cycling Yellow Page 1 Auto shri
14. 8 64 PMID 17206754 Shibayama T Masuda G Ajisawa A Hiruma K Tsuda F Nishizawa T Takahashi M Okamoto H May 2005 Characterization of seven genotypes A to E G and H of hepatitis B virus recovered from Japanese patients infected with human immunodeficiency virus type 1 Journal of Medical Virology 76 1 24 32 doi 10 1002 jmv 20319 PMID 15779062 Bonino F Chiaberge E Maran E Piantino P 1987 Serological markers of HBV infectivity Ann Ist Super Sanita 24 2 217 23 PMID 3331068 Zoulim F November 2006 New nucleic acid diagnostic tests in viral hepatitis Semin Liver Dis 26 4 309 317 doi 10 1055 s 2006 951602 PMID 17051445 Rotor Gene Technology is a registered trademark of Qiagen SaCycler is a registered trademark of Sacace Biotechnologies SacaceBiotechnologiesSrl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 info sacace com web www sacace com 24
15. ROBUSTNESS css cp 14 PRECISION T HR 15 CROSS CONTAMINATION Ss saab ribera P pU Ret REA E IHRE ROREM DIU MG bello td PCR 15 PROTOCOL FOR ROTORGENE BDOO GI RR d cu cuu Re ao e av nv e a 16 PROTOCOL FOR SaCycler 96 Real Time PCR system 21 TROUBLESHOOTING Oe UH Ip ei tov 23 Real TM Quant INTRODUCTION Hepatitis B virus HBV is a member of the Hepadnavirus family The virus particle virion consists of an outer lipid envelope and an icosahedral nucleocapsid core composed of protein These virions are 42 nM in diameter and are sometimes referred to as Dane particles The nucleocapsid encloses the viral DNA and a DNA polymerase that has reverse transcriptase activity The outer envelope contains embedded proteins that are involved in viral binding of and entry into susceptible cells The virus is one of the smallest enveloped animal viruses but pleomorphic forms exist including filamentous and spherical bodies lacking a core These particles are not infectious and are composed of the lipid and protein that forms part of the surface of the virion which is called the surface antigen HBsAg and is produced in excess during the life cycle of the virus Acute infection with hepatitis virus is associated with acute viral hepatitis an
16. ation to monitor patients on therapy and to predict whether antiviral therapy is successful For example with the introduction of new antiviral agents like lamivudine close monitoring of patients has become increasingly important due to the occurrence of antiviral drug resistant virus strains or the presence of flares after withdrawal of antiviral therapy INTENDED USE Kit HBV Real TM Quant Dx is a Real Time Amplification test for the Quantitative detection of Hepatitis B Virus in human plasma and the simultaneous detection of a HBV specific Internal Control IC by dual color detection The HBV Real TM Quant Dx Real Time HBV assay is not for screening blood plasma serum or tissue donors for HBV or to be used as a diagnostic test to confirm the presence of HBV infection PRINCIPLE OF ASSAY Kit HBV Real TM Quant Dx is a Real Time test for the Quantitative detection of Hepatitis B Virus in human plasma HBVDNA is extracted from plasma amplified using real time amplification and detected using fluorescent reporter dye probes specific for HBV or HBV IC During each round of thermal cycling amplification products dissociate to single strands at hightemperature allowing primer annealing and extension as the temperature is lowered Exponentialamplification of the product is achieved through repeated cycling between high and lowtemperatures resulting in a billion fold or greater amplification of target sequences Amplifi
17. cationof both targets HBV and IC takes place simultaneously in the same reaction 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 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 HBVDNA HBV IC L is a lyophilized Internal Control and represents recombinant DNA containing structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of HBVIC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the DNA during extraction procedure thus enabling to calculate precisely the HBV viral load The assay is standardized against the 3rd WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques NIBSC code 10 264 and results are reported in International Units mL IU mL The target sequence for the HBV Real TM Quant Dx assay is in the 5 gene coding HBsAg This region is specific for HBV and is highly conserved MATERIALS PROVIDED Sacace HBV Real TM Quant Dx Amplification Reagent Kit RT PCR REAGENT PACK e 96vials 0 2 ml with lyophilized amplification reagents
18. contamination of specimens or controls Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 596 sodium hypochlorite or other suitable disinfectant Follow by wiping down the surface with 7096 ethanol 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 This product does not contain potentially infectious components Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of amplification 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 STORAGE AND SHIPPING INSTRUCTIONS All components of the HBV Real TM Quant Dx PCR kit must be stored at 2 8 C when not in use All components of the HBV Real TM Quant Dx PCR kit are stable until the expiration date on the label The shelf life of reagents before and after the first use is the same unless otherwise stated Ship HBV Real TM Quant Dx PCR kit at 2 8 C STABILITY HBV Real TM Quant Dx Test is stable up to the expiration date indicated on the kit label The prod
19. from 46 to 52 ul 20 HBV DNA negative plasma pools spiked with HBV DNA to a final concentration of 3 times 95 per cent cut off value were added with small variations from 46 to 52 ul to the vials containing lyophilized reagents All tested samples were found positive for HBV DNA 14 PRECISION The precision of the HBV Real TM Quant Dx kit was evaluated for the manual sample preparation using the SaCycler 96 instruments The precision data of the HBV Real TM Quant Dx kit allow the determination of the total variance of the assay The total varianceconsists of the intra assay variability variability of multiple results of samples ofthe same concentration within run the inter assay variability variability of multiple results of the assay generated on different instruments and different days and the inter lot variability variability of multiple results of the assay using various lots The data obtained were used to determine the standard deviation and the coefficient of variation for the specific pathogen and the internal control All tested samples in all combination analyzed intra assay inter assay inter lot variability as indicated in the below tables showed Standard Deviation in log and CV within the acceptance interval 0 5 log of set point The total variability calculated was Mean SD Ct HBV log Mean SD Ct IC log 0 006 0 004 Mean CV 96 Ct HBV Mean CV Ct IC 1 31 1 05
20. ification Program click to create a new program 7 Select Preliminary heating and choose the template below Click Apply then program the instrument as follows Amplification 8 Click and save the amplification program file in a convenient folder Then click OK in the previous window 9 Click add test button T amp then select HBV Real TM Quant DX from dropdown menu Test indicate the number of samples and click first Add then OK Sacace HBV Real TM Quant Dx VER 06 06 2013 21 10 Use the default Autofill positioning of PCR tubes into the plate or the Free Filling to chose manually 11 12 13 14 15 16 17 18 19 Concentration the position of the tubes In the Concentration column click the green circle gt and insert the HBV standard values reported in the DataCard see example below Protocol number 50 Operator name P Tube Concentration 3 a r 1 Sample 1 HBV Reat T 4 2 1 3 000 000 3 Standard_1 HBVReeT 4 Standard_1 O h 5 Standard 2 g HBVRe amp T 6 Standard 2 8
21. ing standards Then come back to Results tab to see viral load results NOTE for IVD version of SaCycler since the HBV Real TM protocol is already inserted in the PC supplied with the instrument ignore points 4 8 and just follow instructions from point 9 22 TROUBLESHOOTING 1 Weak Ct gt 30 or exceeding the established range signal see Internal Control of the IC Fam Green channel retesting of the sample is required The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow the manufacturer s instructions e The reagents storage conditions didn t comply with the instructions Check the storage conditions The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol The IC was not added to the sample during the pipetting of reagents Make attention during the DNA extraction procedure The correlation coefficient is less than 0 9 retesting of all samples is required The calculated concentrations of CONTROL 1 and or CONTROL 1are different from given control concentrations reported in the Data Sheet retesting of all samples is required 4 Any signal on the Joe HEX Yellow channel with Negative Control of extraction e Contamination during DNA extraction procedure All samples results are invalid Decontaminate all surfaces and instrumen
22. n the following table It was not observed any cross reactivity with these pathogens Table 1 Testing the specificity of the kit with other pathogens Control group Results HBV Joe Yellow Results IC Fam Green channel channel Cytomegalovirus CMV Epstein Barr virus EBV Hepatitis C virus HCV Hepatitis D virus HDV Hepatitis A virus HAV Parvovirus B19 Herpes Zoster Virus VZV Human immunodeficiency virus 1 Herpes simplex virus 1 Herpes simplex virus 2 Human herpes virus 6 Human herpes virus 8 West Nile Virus WNV Tick borne encephalitis virus TBEV Adenovirus type 2 Adenovirus type 4 Adenovirus type 7 Escherichia coli E coli Staphylococcus aureus Streptococcus pyogenes f f f f I Streptococcus agalactiae ROBUSTNESS The robustness of the assay is a measure of its capacity to remain unaffected by small and accidental variations in method parameters and provides an indication of its reliability during normal usage As HBV Real TM Quant Dx kit contains RT PCR REAGENTS PACK 96 vials with ready to use lyophilized reagents the only probable variation during normal usage of the kit could be the different volume of added eluted samples obtained from DNA purification step The robustness of HBV Real TM Quant Dx kit was evaluated adding different volume of eluted sample
23. nced Run Wizard Rotor Type 72 Rotor Figure 2 Rotor and Locking Ring 4 Input operator s name notes and reaction volume 50 Subsequently press Next Ths screen dapheys mixcafaneous optans for the rus Complete te feld Nut when you are mady t move fe next page Operae Sacaco nom HEV Aes TM OF Figure 3 Reaction Volume Sacace Real TM Quant Dx 06 06 2013 16 5 Click the Edit Profile and program the temperature profile New Run Wizard Temperature Profile his box displays help on elements in the wizard For help on an item hover your mouse over the item for help You ALANNAH ca iso its available settings Channel Setup Create New Green 470nm 510nm 10 Edi Yellow 530pm 555nm 10 us Orange X 585nm 610nm 5 Edit Gai Red 625nm 660nm 5 Crimson 680nm 710 Remove Reset Defaults Gain Optimisation 2 Skip Wizard lt lt Back Next gt gt Figure 4 Edit Profile 8 New Open SaveAs Help The run will take approximately 91 minute s to complete The graph below represents the run to be performed Click on a cycle below to modify it Hold Temperature 95 deg Hold Time mns 0 secs Figure 5 Hold Enzyme activation step ok Open SaveAs Help The run
24. nd stored it may be used until the lot in use expires During this time all subsequent samples may be tested without further calibration by importing the experiment with Calibration Curve in the experiment with clinical samples using the same HBV Real TM Quant Dx lot number REAL TIME PCR SAMPLE PROCEDURE 1 Prepare requested quantity of reactiontubes with lyophilized reagents to perform PCR of extracted samples and controls 2 Add 50ul of eluted samples obtained from DNA purification step Close the tubes and transfer them into the Real Time PCR instrument Create a temperature profile on your Real time instrument as follows Stage Temp C Time Fluorescence detection Cycle repeats Hold 95 15 min 1 95 5s 60 20s 5 72 15s 95 55 FAM Green Cycling2 60 308 JOE Green HEX 40 72 155 20 using Rotor Gene 6000 Qinstrument 11 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition One replicate each of the Negative Control CONTROL the HBV High Control CONTROL 1 and the HBV Low Control CONTROL 2 must be included in each test run If the controls are out of their expected range see Data Sheet
25. nicalspecifications CTS for in vitro diagnostic medical devices 2009 108 EC and European Pharmacopoeia 7 0 p 2 6 21 Nucleic acid amplification techniques 07 2010 20621 ANALYTICAL SENSITIVITY The analytical sensitivity or Limit of Detection LOD is defined as the smallest amount of HBV DNA that can be precisely detectedwith a probability of 95 or greater The LOD in consideration of DNA purification from the sample is determined using HBV positive clinical specimens in combination with a particular extraction method HBV DNA was extracted with Magno Virus purification kit Sacace REF K 2 16 according to the manufacturer s instructions The LOD of the HBV Real TM Quant Dx assay is 7 IU mL with the 1 0 mL sample preparation procedure The LOD was determined by testing dilutions of the 3rd WHO International Standard for Hepatitis B Virus for Nucleic Acid Amplification Techniques NIBSC code 10 264 prepared in HBV negative human plasma The resultswere determined by probit analysis LINEAR RANGE Linear range or Linearity of an assay is its ability to obtain test results that are directly proportional to the concentration of the nucleic acid In other words it is the interval between the upper and the lower concentration where test results are directly proportional to the concentrations The upper limit of quantitation for the HBV Real TM Quant Dx assay is 100 million IU mL and the l
26. nk window Turn off the automatic option Threshold Press the buttons Dynamic Tube Slope Correct Quantitation Analysis Cycling AYellow Page 1 fool 5 Dynamic Tube amp Ignore First f Outlier Removal Save Defaults j 3 4 p id In the table of results QuantitationAnalysis select More settings and set NTC Threshold to 10 Select Threshold 0 03 In the table of results QuantitationAnalysis appear the values of Ct Threshold cycle In the new window QuantitationResults appear values of HBV DNA IU ml 20 PROTOCOL FOR SaCycler 96 Real Time PCR system Recommended settings 1 Double click on the RealTime_PCR software icon 2 Select Device handling 3 Select the plugged device and click Connect Create Edit Test 4 Select from Menu Test gt Create Edit test Click Create new test button and enter name HBV Real TM Quant click OK 5 Select Analysis Type Multiplex detection and Method Threshold Standard Quantity 2 and copy 3 total 6 standards 2 positive controls 1 negative control FAM IC and R6G s 50 ul of Mixture volume 5 volume 50 mal 6 Fluorofors Bars Positive K 2 Em Mutiplex detection gE fma RG 9 Rx 9 O5 9055 Method Threshold 0 Negative K 1 B s 6 Under Ampl
27. nts with clinical samples to have quantitative results To do this click Import Curve select From Other Run option choose the Calibration file to be imported and Import Joe Yellow Channel 3 08 5 KE E 7 P a 202 2 f Im p BarkOn Z Namedon Allon AIC 0 Named on EditSamples 5 10 15 20 25 30 35 40 Import Standard Eye Import Standerd Curve po oireenon Adjust Scale Auto Scale Default Scale Log Scale C irent fun inven Faw Data From Oher Run Threshold my 9 Results Cycling AGreen Page 1 eju A E enal Eliminate Cycles fj No Nema Tyee e Given Cane Cop Calc Conc Copie Ver Ec 1 5000 _ Unknown TESI 183 2 O1 GENB 2500 Urknown 1885 188 AoT ea Tasia 3 01 GENE 1250 Urknawn 18 39 183 4 101 0 25 Urknown 1815 101 E 5 O1 GENE 312 Unknown 1888 EE _ 5000 Unknown SD jai From Extemal Source Post Standard Curve Format PE 7 107 0 250 Unknown 1658 185 O7 GENA Urknown 188 Reset 8 07 E26 Unknown 1843 184 EEE n Imported Settings 185 From External Source 1 6 0 Standerd Curve Format 3 neg ext Unknown 1
28. ower limit of quantitation is equal to the Limit of Detection 7 IU mL with the 1 0 mL sample preparation procedure The linear range of the HBV Real TM Quant Dx kit was determined by analyzing a dilution series from 8 00 log IU ml to 1 00 log IU ml of an HBV synthetic quantitative standard calibrated against the 3rd WHO International HBV DNA Standard The results representative of the HBV Real TM Quant Dx assay linearity areshown in Figure 1 y 0 92x 0 185 2 E 2 5 5 x A 5 E 24 gt en 2 00 3 00 4 00 5 00 6 00 Target Conc log IU ml 13 SPECIFICITY The analytical specificity of the primers and the probes was validated with 100 negative plasma samples obtained from the European Directorate for the Quality of Medicine amp HealthCare EDQM They did not generate any signal with the specific HBV primers and probes The specificity of the kit HBV Real TM Quant Dx was 100 GENOTYPE DETECTION The ability to detect different genotypes of the HBV virus depend on the specificity of primers and probes The detectability of all relevant subtypes and genotypes has thus been ensured Genotypes tested A B C D H All tested HBV genotypes gave positive results with HBV Real TM Quant Dx kit CROSS REACTIVITY The potential cross reactivity of the kit HBV Real TM Quant Dx was tested also against the group control listed i
29. prepare the following reagents 1 Choose the requested quantity of lyophilized controls and calibrators and centrifuge briefly 2 Add Negative Control CONTROL as for table below Lyophilized reagent CONTROL ul CAL 1 1100 CAL 2 1100 CONTROL 1 1100 CONTROL 2 1100 CONTROL INT 300 3 Close the tubes and incubate all tubes for 2 min at room temperature Vortex peridiocally Centrifuge the tubes for 5 sec 5 Dissolved reagents must be stored at 2 8 and always protected from light up to 30days do not freeze ISOLATION The following isolation recommended Magno Virus Sacace REF K 2 16 sample volume 1000 ul gt SaMag 24 Sacace REF SM102 automated nucleic acid purification system SaMag Viral Nucleic Acid Extraction Kit sample volume 400 ul Please carry out the DNA extraction according to the manufacturers instructions Add of CONTROLINT during the DNA isolation procedure directly to the sample lysis mixture in all samples controls calibrators see Internal Control High and Low Positive Controls CONTROL 1 and CONTROL 2 as well as Neg Control CONTROL must be always used with patients specimens during DNA isolation procedure These controls must be treated in the same way as patients specimens following the Handbook of used DNA purification kit INTERNAL CONTROL HBV IC Lrepresents recombinant DNA containing
30. rict compliance with the user manual is required for optimal PCR results Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Use routine laboratory precautions Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not pipette by mouth Do not use a kit after its expiration date Do not mix reagents from different kits To reduce the risk of nucleic acid contamination due to aerosols formed during pipetting pipettes with aerosol barrier tips must be used Change aerosol barrier pipette tips between all liquid transfers During preparation of samples compliance with good laboratory practices is essential to minimize the risk of cross contamination between samples as well as the inadvertent introduction of nucleases into samples during and after the extraction procedure Proper aseptic technique should always be used when working with RNA or DNA Use only powder free gloves Dispose all specimens and unused reagents in accordance with local regulations Heparin has been shown to inhibit reaction The use of heparinized specimens is not recommended Specimens may be infectious Use Universal Precautions when performing the assay 12 13 Specimens and controls should be prepared in a laminar flow hood Handle all materials containing specimens or controls according to Good Laboratory Practices in order to prevent cross
31. ts with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes Repeat the DNA extraction with the new set of reagents 5 Any signal with Negative PCR Control Contamination during PCR preparation procedure All samples results are invalid 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 23 REFERENCES 1 2 Zuckerman AJ 1996 Hepatitis Viruses In Baron S et al Baron s Medical Microbiology 4th ed University of Texas Medical Branch ISBN 0 9631172 1 1 Locarnini S 2004 Molecular virology of hepatitis B virus Semin Liver Dis 24 Suppl 1 3 10 doi 10 1055 s 2004 828672 PMID 15192795 Terrault N Roche B Samuel D July 2005 Management of the hepatitis B virus in the liver transplantation setting a European and an American perspective Liver Transpl 11 7 716 32 doi 10 1002 It 20492 PMID 15973718 Harrison T 2009 Desk Encyclopedia of General Virology Boston Academic Press p 455 ISBN 0 12 375146 2 Howard C R 1986 The Biology of Hepadnaviruses Journal of General Virology 67 7 1215 1235 doi 10 1099 0022 1317 67 7 1215 PMID 3014045 Beck J Nassal M January 2007 Hepatitis B virus replication World J Gastroenterol 13 1 4
32. uct 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 Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results When a positive or negative control value is out of the expected range it may indicate deterioration of the reagents Associated test results are invalid and samples must be retested Assay recalibrationmay be necessary QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality SAMPLE COLLECTION STORAGE AND TRANSPORT Note Handle all specimens as if they are potentially infectious agents Current studies refer to EDTA or citrate plasma as the most suitable sample materials for HBV detection Therefore we recommend the use of these materials with the HBV Real TM Quant Dx The internal validation of the HBV Real TM Quant Dx assay has been performed using human EDTA plasma samples Other sample materials are not validated HBV Real TM Quant Dx assay is only for use with human plasma specimens 1 EDTA tubes may be used with the HBV Real TM Quant Dx Follow sample tube manufacturer s instructions 2 Whole blood collected in EDTA should be separated into plasma and cellular components by
33. ysis Reports Arrange Channels 7 Cycling AGreen 2 Cycling AYellow Page 1 i 1 lt lt Quantitation Analysis Cycling AGreen Z8 Quantitation Analysis Cycling A Yello c 22 lt Ignore First Outlier Removal Save Defaults gt ignore First Outlier Removal led Save Defaults 06 o 3 a e m 2 3 4 5 6 7 8 9 Norm Fluoro Be Norm Fluoro eee E S re FRR RMD 14 15 ee ey 16 i Quant Results Cycling AYellow Page 8 i lt j al No Name Type ct Given Cone Cop 1 CALTHBV Standard 1076 350000000 IL E MEE CALI Stenderd 1070 350000000 Re2 0 99943 CALI HBV Standard 1034 350000000 3 360 CAL2 Standard 2071 3 500 00 8532 789 CAL2 HBV Standard 21 04 350000 CAL Stenderd 20 89 3 40 15 20 25 30 35 4 510 15 20 25 3 35 2 Auto Scale Default Scale Log Scale si Auto Scale Default Scale Log Scale E w Concentration ls Rotor Gene Q Series Software 2 0 2 VIRTUAL MODE 9 User must import the calibration experiment with Standard Curves to subsequent experime
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