HBV Quantification Kit v1 USER MANUAL ®
Contents
1. 9 4 Kit Components 9 4 1 PCR Mix HotStarTaq DNA Polymerase HotStarTaq DNA Polymerase is a modified form of a recombinant 94 kDa DNA polymerase originally isolated from Thermus aquaticus cloned into E Coli The enzyme is provided in an inactive form It is activated by a 15 minute 95 C incubation step This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step leading to high PCR specificity and accurate quantification PCR Buffer contains Tris Cl KCI NH4 2SOz 8 mM MgCl pH 8 7 20 C dNTP Mix Contains ultrapure quality dATP dGTP dCTP ve dTTP dUTP 9 4 2 Detection Mix 1 Detection Mix 1 contains HBV specific forward and reverse primers and a dual labeled probe 9 4 3 Detection Mix 2 Detection Mix 2 contains internal control specific forward and reverse primers and a dual labeled probe 9 4 4 Internal Control An internal control is included in the kit to control DNA isolation and PCR inhibition The internal control is a synthetic DNA molecule derived from human genome It is added into the serum proteinase K and carrier RNA mixture during DNA isolation to control the isolation efficiency and PCR inhibition The amount of IC that should be added during isolation is 5 ul per serum sample Alternatively the internal control can be added directly into the PCR master mix to control the PCR inhibition exclusively For this purpose 0 1 ul of internal control should be added f2. HBV Quantification Kit v1 USER MANUAL For in vitro Diagnostic Use Document Code MBO1v4f Approval Date April 2011 wo C6 10 11 12 Contents Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Pathogen Method Procedure 9 1 Sample Preparation Storage and Transport 9 2 Interfering Substances 9 3 DNA Isolation 9 4 Kit Components 9 4 1 PCR Mix 9 4 2 Detection Mix 1 9 4 3 Detection Mix 2 9 4 4 Internal Control 9 4 5 Positive Control 9 4 6 Quantitation Standards 9 5 Preparing the PCR 9 6 Programming the Montania 483 Real Time PCR Instrument Analysis Troubleshooting Specifications ll 12 1 Sensitivity 12 1 1 Genotype Detection 12 2 Linear Range 12 3 Cross Reactivity 12 4 Reproducibility and Precision 12 5 Diagnostic Evaluation 12 6 Calibration Against WHO Standard 13 References 14 Symbols 15 Contact Information il 1 PRODUCT DESCRIPTION Bosphore HBV Quantification Kit v1 detects and quantitates Hepatitis B Virus DNA in human serum or plasma encompassing all the major HBV genotypes A H The linear range of quantitation is 1x10 1x10 IU ml and the analytic sensitivity is 10 IU ml A region within the S gene is amplified and fluorescence detection is accomplished using the FAM filter An internal control has been integrated into the kit in order to check PCR inhibition The amplification data o
3. N oe Oo N w D ol D S o wo Fig 7b Linear Range Standard Curve 12 3 Cross Reactivity To eliminate potential cross reactivity both assay design evidence and experimental studies were employed Primer and probe sequences were checked for possible homology to other known pathogen sequences by sequence comparison analysis using database alignment Samples of CMV EBV MTBC Parvovirus B19 BKV with known high positivity were tested and found negative Document Code MB01v4f 12 Date April 2011 12 4 Reproducibility Reproducibility data on C value basis were obtained by the analysis of one of the quantitation standards of the Bosphore HBV Quantification Kit v1 Test was performed in at least 4 replicates by 3 different operators on multiple days using 3 different lots The resulting data is given in Table 1 Table 1 Reproducibility Data HBV Standard Variance Coefficient of 107 1U ml deviation variation Intra assay Variability 0 07 0 005 0 23 N 4 Inter lot Variability 0 34 N 3 0 01 Total Inter assay Variability N 5 Inter operator Variability 0 28 N 3 HBV Quantitation Standards were calibrated against the WHO International Standard for HBV DNA NAT assays NIBSC Code 97 750 by performing two assays using various dilutions and testing them in triplicates 1 IU was found to be equal to 4 5 0 2 copies ml 12 5 Diagnostic Evaluation The diagnostic evaluation was performed by testing 10
4. can be detected During the elongation step of PCR Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is freed from the suppressing effect of the quencher fluorescence signal can be detected The fluorescence generated by the reporter increases as the PCR product is accumulated the point at which the signal rises above background level and becomes distinguishable is called the threshold cycle C There is a linear relationship between the log of the starting amount of a template and its threshold cycle thus starting amount of unknown templates can be determined using standard curves constructed using Crvalues of the known starting amounts of target templates Bosphore HBV Quantification Kit v1 employs multiplex PCR and an internal control is incorporated into the system in order to control the isolation procedure and to check for possible PCR inhibition HBV DNA and an internal control are co amplified in a single reaction using sequence specific primers The fluorescent signal generated by the HBV amplification is detected by a probe labeled at the 3 end with FAM through the FAM channel The fluorescent signal generated by the internal control amplification is detected by a second probe labeled at the 5 end with a different reporter molecule Cy5 through the Cy5 channel 9 PROCEDURE 9 1 Sample Preparation Storage and Transport To isolate serum from the clinical specimen the blood sample shou
5. establish an appropriate link between the system components first the thermal cycler and the optical module and then the PC and the software should be started Before starting a Real Time PCR reaction using the Bosphore Kits the following steps should be completed e Choose the filter pairs to be used FAM and Cy5 e Identify unknown samples standards positive and negative controls assign quantitative values to the standards e Select the correct thermal protocol These steps are described below From the main menu of the Montania 483 Real Time PCR Instrument File and then New is selected Create a new Experiment is selected In the Select Channel window channels 1 FAM and 3 Cy5 are selected Fig 1 Standards samples and negative controls are identified in the Module Edit menu Fig 2 Standards should only be defined for the FAM channel and their concentration viral load should be entered To select the thermal protocol Gene Amplification menu is used The Open button in the Experiment Program is clicked and the appropriate thermal protocol is selected Fig 3a The thermal cycles of the selected protocol is displayed The experiment starts by clicking the Start button Fig 3b Osta Ox FESE EN JR Fig 1 Filter Selection in Montania 483 Document Code MB01v4f 7 Date April 2011 PF CRM SNOEN Von Aneel ToT HebOd sao 70x Bs 12838 of Module
6. 0 HBV negative and 7 HBV positive serum Samples which have been previously analyzed using Roche Diagnostics Elecsys 2010 All of the negative samples were found negative and all of the positive samples were found positive with Bosphore HBV Quantification Kit v1 15 HBV positive serum samples which have been previously analyzed using HBV DNA Amplicor Monitor Test v2 0 Quantitative COBAS HBV DNA Amplicor Monitor Quantitative and Diasorin ETI MAK2 HBsAg Plus Assay were tested with Bosphore HBV Quantification Kit v1 All the samples were found to be positive including the sample that could only be detected as positive by Diasorin ETI MAK2 HBsAg Plus Assay but was noted as below detection limit using HBV DNA Amplicor Monitor Test v2 0 Quantitative and COBAS HBV DNA Amplicor Monitor Quantitative 12 6 Calibration Against WHO Standard Quantitation Standards were calibrated against the WHO International Standard for HBV DNA NAT assays NIBSC Code 97 750 1 IU was found to be equal to 4 5 0 2 copies ml Document Code MB0O1v4f 13 Date April 2011 13 REFERENCES 1 K E Nelson C Williams and N Graham Infectious Disease Epidemiology Theory and Practice July 15 2000 p 907 921 Barbara Rehermann and Michelina Nascimbeni Immunology of Hepatitis B virus and Hepatitis C virus Infection NATURE REVIEWS Volume 5 March 2005 p 215 229 3 Hepatitis B Fact Sheet No 204 2008 World Health Organization Jinlin Hou Zhihua Liu
7. Edit C GeneAmpiiication tetReuk SSS A CHI 2 gt 2a 2o 2o Ge Fig 2 Sample Location and Identification Fig 3a Selecting the Thermal Protocol Fig 3b Starting the Experiment Document Code MB01v4f Date April 2011 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 FiF EROE Settings S View Analysis A Took T Hebi uo 0 B2 123 9 7 Module Edit Test Result 143190 1 28871 1 14552 1 00233 0 85914 0 71595 0 57276 0 42957 0 28638 0 14319 0 00000 Eisamen ee 0 14319 Fig 4 Amplification Curve of a Bosphore HBV v1 test The standard curve is plotted using the data obtained from the defined standards with the axes Ct Threshold Cycle and Log Starting Quantity Example of a standard curve is given in Fig 5 FF ERIE Settinge S Yie Anaiysis A Toob T Heb t CA ES o Bz 1239 7 Module Edit 2 S 8 UENS EE 3 5 6 7 4 Logi00 Fig 5 Standard Curve of a Bosphore HBV v1 test Analysis of the results should be performed by trained personnel who have received the required training for analysing Real Time PCR data We recommend that the test results must be evaluated by an expert clinician taking the patient s clin
8. S AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Document Code MB0O1v4f 1 Date April 2011 Real Time PCR System ABl Stratagene Mx3005P Mx3000P Agilent LineGeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen 0 2 ml Thin Wall PCR tubes or strips Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks or other high quality viral DNA extraction kits and systems Deep freezer 20 C Desktop centrifuge with rotor for 2 ml microcentrifuge tubes Calibrated adjustable micropipettes DNAse RNAse pyrogen free micropipette tips with filters DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes Disposable laboratory gloves 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important The product should be delivered on dry ice Check for presence of dry ice upon arrival Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used Before starting a test procedure all components should be thoroughly thawed After thawing all components should be centrifuged briefly soin down for 3 5 seconds and mi
9. and Fan Gu Epidemiology and Prevention of Hepatitis B Virus Infection Int J Med Sci 2005 2 1 p 50 57 14 SYMBOLS be Use by Lot Batch Catalog number K Temperature limitation A N Caution consult accompanying documents onal Manufacturer IVD In Vitro Diagnostic Medical Device 15 CONTACT INFORMATION Pri VA Ate LY WA Anatolia Tani ve Biyoteknoloji A S Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 0455 Fax 90 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com RegisteredTrademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji A S Document Code MB01v4f 14 Date April 2011
10. aster mixes for more than 5 samples an extra 10 should be added to the total sample number When the Internal Control is added in the extraction step PCR Mix 12 5 ul Detection Mix 1 0 7 ul Detection Mix 2 0 85 ul dH20 0 95 ul Sample DNA Standard 10 0 ul Negative Positive Control Total Volume 25 0 ul When the Internal Control is added in the PCR step PCR Mix 12 5 ul Detection Mix 1 0 75 ul Detection Mix 2 0 85 ul Internal Control 0 1 ul dH20 0 85 ul Sample DNA Standard 10 0 ul Negative Positive Control Total Volume 25 0 ul Pipette 15 ul of the master mix into the PCR tubes or strips and add 10 ul of DNA sample Standard positive or negative control Close the tube cap Make sure that the solution in each tube is at the bottom of the tube Centrifuge if necessary 9 6 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore HBV Quantification Kit v1 is composed of an initial denaturation for activation the HotStarTaq DNA Polymerase a two step amplification cycle and a terminal hold The real time data is collected at the second step of the amplification cycle Initial denaturation 95 C 14 30 min Document Code MB0O1v4f 6 Date April 2011 Denaturation 97 C 00 30 min 50 cycles Annealing and Synthesis 54 C 01 30 min Data Collection Hold 22 C 05 00 min Montania 483 Real Time PCR Instrument is installed and calibrated as it is delivered to the end user In order to
11. f the internal control is detected with the Cy5 filter The internal control can be added either during DNA extraction or PCR step 2 CONTENT Bosphore HBV Quantification Kit v1 is composed of Real Time PCR reagents and quantitation serum standards which have been calibrated against WHO International Standard NIBSC Code 97 750 Component REAGENT 100 50 Tests 25 Tests aT dH20 1000 ul 500 ul PCR Mix 700 ul 350 ul Detection Mix1 39 5 ul 19 5 ul Detection Mix2 47 5 ul 23 75ul Internal Control 280 ul 140 ul Positive Control 1 Standard 1 1 x 10 IU ml Standard 2 1 x 10 IU ml Standard 3 1 x 10 IU ml Standard 4 5 x 107 IU ml iO CON AUN BRWDN O 3 STORAGE Bosphore HBV Quantification Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing gt 3x should be avoided since it may reduce sensitivity If the components are to be used in small amounts they should be frozen in aliquots While preparing the PCR the components should not be exposed to room temperature for more than 10 min and the detection mix components should not be exposed to light more than 1 2 min We recommend preparing the PCR on a cooling block and keeping the detection mixes within a closed container The components maintain their stability until the expiry dates on the labels if they are stored at advised conditions 4 REQUIRED MATERIAL
12. ical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the required training from manufacturer consider it as necessary the system allows pulling down the threshold as much as possible in order to detect low positive samples In this case attention should be paid to keep the threshold line above the background and to keep the correlation coefficient at the maximum possible value and within its acceptance criteria Document Code MB01v4f 9 Date April 2011 The table below displays the acceptance criteria for Bosphore HBV v 1 PCR efficiency is calculated by the following formula 10 5 1 x100 Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if an impairment in the product s performance is observed See the last page for contact information The quantitative results of the test are displayed on the Report Mode screen A spread sheet containing the calculated starting quantities of the unknown samples in each tube is shown The samples that cross the threshold in Channel 1 FAM are displayed with a calculated starting quantity samples that do not cut the threshold are displayed as No Ct These samples are regarded as negative or having a viral load below the detection limit of the assay For these undetectable samples the Cy5 data
13. ld be collected into sterile vacutainers without any anticoagulant For venipuncture only sterile material should be used The serum should be separated from blood within 6 hours after blood collection To separate the serum the blood container should be centrifuged at 800 1600 x g for 20 minutes The separated serum should be transferred to polypropylene tubes and stored at 20 C or lower until use The samples should be transported in containers with capacity to resist pressure Transportation should be done according to local and national regulations for pathogen material transport 9 2 Interfering Substances The following factors may have possible influences on PCR e Hemolytic samples e Samples of heparinised patients e Samples of patients with elevated levels of bile salts bilirubin or lipids Document Code MB0O1v4f 4 Date April 2011 9 3 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks isolation system is used with Bosphore HBV Quantification Kit v1 The DNA isolation should be performed according to the manufacturers instructions The starting volume is 400 ul the elution volume is 60 ul and the amount of internal control that should be used during isolation for each system is 5 ul The external quantitation standards are provided as serum so that they undergo the same steps as the patient samples starting from DNA isolation
14. m the FAM filter Wrong thermal protocol is chosen Make sure that the correct thermal protocol is chosen Late or weak signal from the standards Deterioration of the standards or Don t use expired standards or kit components Follow the the core kit components instructions for the storage of kit components Section 3 Storage Document Code MB0O1v4f 10 Date April 2011 No signal from the internal control Deterioration of the internal Follow the instructions for the storage of kit components See control or detection mix 2 Section 3 Storage See 9 3 DNA isolation See 9 3 DNA isolation Signal from FAM Filter in the Negative Control Use filter tips Repeat PCR with new kit components The Threshold is Above Low Signals The threshold should be manually Using the mouse pull the threshold down until it cuts the low adjusted signals Avoid the background and the signal from negative control 12 SPECIFICATIONS 12 1 Sensitivity Analytical sensitivity may be expressed as the limit of detection i e the smallest amount of the target marker that can be precisely detected The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value The analytical sensitivity or detection limit for NAT assays is expressed by the 95 positive cut off value The analytical detection limit for Bosphore HBV v1 wa
15. of the internal control should also be checked to avoid false negative results Fig 6 Pie F ELE Settinge S VievAV Analyss A Tools T Hebi D0 Saa oO 8a 123 Module Edit AN Gene Amplification Test Result I Module Mode Amplification Curve l Standard Curve Report Mode Wel Channel Type Testitom Property CT Test Rew Label Name Sex Age Case iD BediD Inpatient 1O Ourpatiort iD Sample Type Sample Date Diagnosis Doctor Detect Date Oifice Expaimerter Assessor Remak J Semple H 947 00 0I gt amp HBV WGA 4 124E 0 bangle 283E N N eeFNUEM2eo Vegveee 2eee Oe SES ee 882293 2888288888 Fig 6 A Report Mode Screen Showing the Results The following table shows the possible results and their interpretation Signal detected in FAM The sample contains No need to check the internal control since the sample filter pair HBV DNA the result is positive high positive samples may suppress the is positive signal from the internal control No signal in FAM signal The HBV DNA in the Signal from Cy5 filter pair rules out the possibility of in Cy5 sample is not PCR inhibition detectable No signal in FAM and The diagnosis is No signal in Cy5 points out to PCR inhibition or to a Cy5 inconclusive problem in DNA isolation See 11 Troubleshooting 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal fro
16. or each reaction into the master mix Lack of internal control amplification in the FAM negative samples may indicate a problem in isolation or PCR inhibition In this case isolation and PCR should be repeated In samples that contain a high viral load internal control can be suppressed and no increase of the signal is detected Please use the table below for the interpretation of internal control data HBV FAM Internal Control Cy5 p Sample negative p Sample positive po Repeat the test Document Code MBO1v4f 5 Date April 2011 9 4 5 Positive Control The positive control contains HBV DNA It can be included in the PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria table Section 10 Analysis Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction 9 4 6 Quantitation Standards The quantitation serum standards are calibrated by WHO International Standard NIBSC Code 97 750 9 5 Preparing the PCR All four external quantitation standards should be added into the PCR reaction together with the samples and the negative control PCR grade water Make sure that all the kit components are thawed before use Refer to the table below for preparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing m
17. r amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template As a brief explanation primers are small synthetic DNA those anneal to the specific regions of the template in order to start the synthesis dNTPs are the building blocks of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In Real Time PCR technique in contrast to conventional PCR PCR product can be monitored during the reaction Therefore Real Time PCR obviates the need for further analysis methods like gel electrophoresis Document Code MBO1v4f 3 Date April 2011 whereby minimizing the risk of contamination Dual labeled probes employed in the reaction in addition to the conventional PCR reagents enable detection of the amplified target with increased sensitivity The assay utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent probe during the extension phase of PCR The probe is labeled at the 5 end with a fluorescent reporter molecule and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity and the reporter is excited by light no reporter fluorescence
18. s found to be 1x10 IU ml p 0 05 The sensitivity was determined using serial dilutions of DNA calibrated with the WHO International Standard for HBV DNA NAT assays NIBSC Code 97 750 The dilutions were tested in different runs in replicates The results were analyzed by probit method 12 1 1 Genotype Detection and Quantitation Efficiency Efficiency of detecting and quantitating different genotypes were ensured both by sequence comparison analysis and a Real Time PCR assay using HBV DNA Genotype Performance PHD 350 Seracare The following genotypes were tested and found positive Panel Member HBV FAM a ee ee ae ee ee Document Code MB0O1v4f 11 Date April 2011 12 2 Linear Range The linear range of Bosphore HBV Quantification Kit v1 was determined to be 1x10 1x10 IU ml In order to assess the linear range a dilution series of a member of HBV DNA Genotype Performance PHD 350 Seracare which has been calibrated against the WHO International Standard for HBV DNA NAT assays NIBSC Code 97 750 was analyzed by testing each dilution in 4 replicates Fig 7a and 7b The standard curve correlation coefficient was found to be 0 99968 1200 097222 1 asg Q69144 OR O4i667 027773 QU 2 4 6 8 0 2 4 6 8 DB 2 4 oe 3 d R A S amp S BZ H LR U4 E 8 BD Fig 7a Linear Range Amplification Curve 8 6 i CT amp RRS FR SS RR FS HRS Be S 2 NR Sm 23 8 ee ee oe d w S nn
19. ss of HBV that takes place in liver is unique among the animal DNA viruses in which reverse transcription is involved HBV may destroy the liver and cause diseases such as cirrhosis and hepatocellular carcinoma There are 8 distinctly classified genotypes of hepatitis B virus and further recognized subgenotypes 1 2 Epidemiology Hepatitis B virus HBV infection is a worldwide health problem with the highest burden of disease in Asia Pacific Islands and Sub Saharan Africa There are 2 billion people infected worldwide one third of world s population and 400 million suffering from chronic HBV infection 90 of infants and up to 50 of young children infected with hepatitis B will develop chronic infections HBV infections result in roughly 1 million deaths per year including the deaths caused by HBV and its complications HBV related liver diseases 3 4 Modes of Transmission Transmission of hepatitis B virus follows the same modes as HIV but unlike HIV HBV is 50 100 times more infectious and survives in the open air for at least 7 days Common modes of transmission are perinatal from mothers to infants primarily at birth early childhood infections inapparent infection through close contact with infected household unsafe injection practices blood transfusions and sexual contact 4 8 METHOD Bosphore HBV Quantification Kit v1 is based on the Real Time PCR method Polymerase chain reaction is a technique that is used fo
20. xed well to ensure homogeneity prior to use The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 20 C PCR and nucleic acid isolation must be performed in different compartments Samples should be stored separately to avoid contact with the kit components Pathogen information should be reviewed to be aware of the health related risks Serum samples including the standards should be handled with extreme caution suitable class microbiological safety cabinet should be used Physical contact with pathogens should be avoided by wearing lab coats and gloves no allowance for eating or drinking within the workspace prevention of unauthorized individuals access to the working area All the pathogenic wastes produced during the nucleic acid isolation step including the serum Samples and material contacted with them should be discarded into medical waste and disposed safely Document Code MB01v4f 2 Date April 2011 6 PRODUCT USE LIMITATIONS e All the components may exclusively be used for in vitro diagnostics e This product should be used in accordance with this user manual e This product is to be used by personnel specially trained to perform in vitro diagnostic procedures 7 PATHOGEN Causative Agents Hepatitis B virus HBV is one of the smallest enveloped double stranded DNA viruses and a member of the Hepadnaviridae family The replication proce
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