CMV Quantification Kit v1 USER MANUAL
Contents
1. Code MB04v1f 7 Date July 2011 Fig 4 Amplification Curve of a Bosphore CMV 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 TE ERE Seni Wen Ayer Tode epo Oso U BHs193997 Module Edit I ji Test Result Soke Mode T Henphficaton Curve J Standard Curve I Report Mode Logon Fig 5 Standard Curve of a Bosphore CMV 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 s results must be evaluated by an expert clinician taking the patient s clinical findings and the results of other tests into consideration All analysis is done automatically in routine use However when the trained personnel who have received the 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 The table below displays the acceptance criteria for Bosphore CMV v1 Component Parameter Cycle Threshold Cr Standard 1 26 22. 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 For the standards the concentration should also 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 Osa vx gaies or FF Oanei t FAM SYBR T7 vrai 2 HEX VIC JOE TET F Orrel Fig 1 Selecting the Filters in Montania 483 Code MB04v1f 6 Date July 2011 Fig 2 Sample Location and Identification Fig 3 a Selecting the Thermal Protocol LOL RR NS 2023 0 H 193eT isduie Edir enti sven Program Y Gene Amplification Vago Fil Fi a FO Dcum erem Dar Fig 3 b Starting the Experiment 10 ANALYSIS By the end of the thermal protocol the Montania 483 Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Example of an amplification curve is given in Fig 4 a LT L 2 4 6 8 117 2 M 6 V 2 2 6 BH BA FS 9 0 Q 4 6 8 D
3. In this case PCR should be repeated In samples that contain a high bacterial 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 Code MBO4v1f 4 Date July 2011 CMV FAM Internal Control Cy5 Interpretation Sample positive Sample negative Sample positive Repeat the test 9 4 5 Positive Control The positive control contains CMV 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 Quantitation of the viral load is performed using four quantitated DNA controls ranging from 1 x 10 copies ml to 1 x 10 copies ml 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 master mixes for more than 5 samples an extra 10 should be added to the total sample n
4. Standard 2 2942 Standard 3 32 542 Standard 4 35 542 Positive Control 33 4 Correlation Coefficient gt 0 950 PCR Efficiency gt 60 Code MB04v1f 8 Date July 2011 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 having a viral load below the detection limit of the assay For these undetectable samples the Cy5 data of the internal control should also be checked to avoid false negative results Fig 6 File F EdR E Settings S View v Osea oO Anaysi A Tools T Helb Ess1 23207 Module Edit TestResult Amplification Curve Standard Curve Report Mode Case ID inpatient IO Outpatient ID Test Resuk Bed ID Sample Date Doctor Detect Date Dilice Sample Type Diagnosis Experimenter Assessor Remask Type Testitem Property CT Label Name Sex Age Sample CMV 2263 1 762 07 0 s 2
5. copies ml Intra assay Variability N 8 Inter lot Variability N 3 Inter operator Variability N 3 Total Inter assay Variability N 6 Table 1 Reproducibility Data Standard deviation Variance Coefficient of variation 96 Table 2 Precision Data CMV Measured Standard Coefficient Threshold Standard 1 6x10 Quantity Deviation Deviation copies ml variation Ct 96 Intra assay Variability 1801 79927 N 8 Inter lot Variability 17723 75 539 17 N 3 Inter operator 1601417 2829 60 Variability N 3 Total Inter assay 16456 94 2142 77 Variability N 6 12 5 Diagnostic Evaluation The diagnostic evaluation was performed by testing 25 CMV negative serum samples which have been previously analyzed using Fluorion CMV QNP 3 0 Real Time PCR system lontek All of the negative samples were also negative and all of the positive samples were also positive with Bosphore CMV Quantification Kit v1 9 CMV positive serum samples and 1 negative serum sample which have been previously analyzed using COBAS CMV DNA Amplicor were tested with Bosphore CMV Quantification Kit v1 All the positive samples were found to be positive and the negative sample was found negative 13 REFERENCES 1 Mach M Stamminger T Jahn G Human Cytomegalovirus Recent Aspects from Molecular Biology J gen Virol 1989 70 3117 3146 2 Stegmann BJ Carey JC TORCH Infections Toxop
6. 6 14 1 6606 06 2 29 86 1 5696 05 3 1 309E 04 08 2 364E 03 5 2 604E 02 0 000 00 33 35 35 90 39 13 No Ct Fig 6 The Report Mode Screen Showing the Results The following table shows the possible results and their interpretation Signal detected in FAM filter pair The sample contains CMV DNA the result is positive No need to check the internal control since the sample is positive high positive samples may suppress the signal from the internal control No signal in FAM The CMV DNA in the Signal from Cy5 filter pair rules signal in Cy5 sample is not out the possibility of PCR detectable inhibition No signal in FAM The diagnosis is No signal in Cy5 points out to and Cy5 inconclusive PCR inhibition 11 Troubleshooting See 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Late or no signal from the FAM filter Wrong thermal protocol is chosen chosen Make sure that the correct thermal protocol is Late or weak signal from the standards No signal from the internal control Code MB04v1f Date July 2011 Deterioration of the standards or the core kit components Don t use expired standards or kit components Follow the instructions for the storage of kit components Section 2 Storage Deterioration of Follow the instructions for the storage of kit the int
7. CMV Quantification Kit v1 USER MANUAL For in vitro Diagnostic Use Code MB04v1f Date July 2011 C Ca Contents 1 Product Description 2 Content 3 Storage 4 Required Materials and Devices 5 Important Notes and Safety Instructions 6 Product Use Limitations 7 Pathogen 8 Method 9 Procedure 9 1 Sample Preparation Storage and Transport 9 2 Interfering Substances 9 3 DNA Isolation 9 4 Kit Components 9 44 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 10 Analysis 11 Troubleshooting Code MB04v1f Date July 2011 10 12 Specifications 12 1 Sensitivity 12 2 Linear Range 12 3 Cross Reactivity 12 4 Reproducibility and Precision 12 5 Diagnostic Evaluation 13 References 14 Symbols 15 Contact Information Code MB04v1f Date July 2011 10 10 11 11 12 12 13 13 iii 1 PRODUCT DESCRIPTION Bosphore CMV Quantification Kit v1 detects and quantitates Cytomegalovirus DNA in human serum encompassing all major CMV genotypes The linear range of quantitation is 1 6x107 1 6x10 copies ml and the analytic sensitivity is 1 2x10 copies ml A region within the DNA Polymerase gene is amplified and the detection is accomplished using the FAM filter To check PCR inhibition an internal control is incorporated into th
8. an Herpes Virus 5 HHV5 HCMV is among the TORCH infections Toxoplasmosis Others Rubella Cytomegalovirus Herpes that are known to cause congenital anomalies 1 2 Code MB04v1f 2 Date July 2011 Epidemiology Infection is common with seroprevalence rates increasing steadily from 65 96 among 40 to 49 year olds to 9196 in those aged 80 years or older 1 3 Congenital CMV infection being the most common congenital viral infection is a significant health problem in developed countries It is responsible for the neurodevelopmental pathological changes involving hearing loss CMV infection is seen in 196 of newborn infants and CMV causes serious life threatening problems in 1096 of these infants The morbidity rates of CMV are 90 95 96 whilst the mortality rates are 4 37 3 4 Modes of Transmission CMV infection can be transmitted via body fluids such as urine saliva tear blood sexual contact plesantal contact breast milk and via solid organ transplantations or blood transfusions Transmission of this virus could easily be prevented because the ubiquitous way of transmission is contact with the body fluids and if hands are washed with soap after the contact the risk of transmission can be prevented The most common way of HCMV transmission is from mother to child in uterus or via perinatal phase during birth 5 6 8 METHOD Bosphore CMV Quantification Kit v1 is based on the real time PCR principle The pathogen
9. e The linear range of Bosphore CMV Quantification Kit v1 was determined to be 1 6x107 1 6x10 copies ml In order to assess the linear range a dilution series of a quantitated DNA Control was analyzed by testing each dilution in 3 replicates Fig 7a The standard curve coefficient was found to be 0 99938 Fig 7b 181288 YI 1631 lug ix 12874 108748 204 6 9 w oU WW U 2 2 4 5 3 9 X 3 9 0 LH b 6 9 Fig 7a Linear Range Amplification Curve Code MB04v1f 10 Date July 2011 12 3 cT D A NM E MEN MN CTS MM HN CNN Ec Cross Reactivity log 0 Fig 7b Linear Range Standard Curve To eliminate potential cross reactivity assay design evidence was used Primer and probe sequences were checked for possible homology to other known pathogen sequences by sequence comparison analysis using database alignment HBV EBV MTBC Parvovirus B19 HSV and BKV samples with known high positivity were tested and found negative in cross reactivity experiments 12 4 Reproducibility and Precision Precision and reproducibility data on Cr value basis and on quantitation basis were obtained by the analysis of the 1 6x1x10 copies ml quantitation control Test was performed in at least 4 replicates by 3 different operators on multiple days using 3 different lots The resulting data is given in Table 1 and 2 Code MB04v1f Date July 2011 CMV 1 6x10
10. e system The internal control is detected with the Cy5 filter The internal control can be added into the PCR reaction mix Quantitation of the viral load is done using internationally accepted DNA quantitation controls 2 CONTENT Bosphore CMV DNA Quantification Kit v1 is composed of PCR reagents and DNA standards Component Reagent 100 Tests 50 Tests 25 Tests 1 dH20 1000 ul 1000 ul 500 ul 2 PCR Mix 2240 ul 1120 ul 560 ul 3 Detection Mix1 228 ul 114 ul 57 ul 4 Detection Mix2 132 ul 66 ul 33 ul 5 internal Kontrol 15 ul 15 ul 15 ul 6 Pozitif Kontrol 1 70 ul 35 ul 17 5 ul 7 Standard 1 1x 105 copies ml 140 ul 70 ul 35 ul 8 Standard 2 1x 10 copies ml 140 ul 70 ul 35 ul 9 Standard 3 1x 10 copies ml 140 ul 70 ul 35 ul 10 Standard 4 1x 10 copies ml 140 ul 70 ul 35 ul 3 STORAGE Bosphore CMV Quantification Kit v1 PCR reagents should be stored at 20 C the serum standards can be stored at 80 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 15 min The detection mix components should not be exposed to light more than 1 2 min The components maintain their stability until the expiry dates on the labels if they are stored at p
11. ernal components See 2 Storage control or detection mix 2 PCR inhibition Make sure that you use the recommended DNA isolation method See 9 3 DNA isolation DNA lost during Make sure that you use the recommended DNA isolation isolation method See 9 3 DNA isolation Signal from FAM Filter in the Negative Control Contamination Use filter tips Repeat PCR with new kit components The Threshold is Above Low Signals The threshold Using the mouse pull the threshold down until it should be cuts the low signals Avoid the background and manually adjusted the signal from negative control Uneven Traces Baseline cycles Assing new baseline cycles manually and should be recalculate threshold cycles readjusted 12 SPECIFICATIONS 12 1 Sensitivity Analytical sensitivity may be expressed as the limit of detection i e the smallest amount of the target marker that can be precisely detected The detection limit of an individual analytical procedure is the lowest amount of nucleic acid in a sample which can be detected but not necessarily quantitated as an exact value The analytical sensitivity or detection limit for NAT assays is expressed by the 9596 positive cut off value The analytical detection limit for Bosphore CMV v1 was found to be 1 2x10 Copies ml p 0 05 The sensitivity was determined using serial DNA dilutions The results were analyzed by probit method 12 2 Linear Rang
12. is detected using fluorescent dyes that are incorporated into oligonucleotide probes The assay utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent hybridization probe during the extension phase of PCR The probe is labeled at the 5 end with a fluorescent reporter molecule and at the 3 end with another fluorescent molecule that acts as a quencher for the reporter When the two fluorophores are in close proximity and the reporter is excited by light no reporter fluorescence can be detected During the elongation step of PCR Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is released from the suppressing effect of the quencher fluorescence signal can be detected At each cycle of the reaction the product that is formed is sensitively detected by the increase in the fluorescence level 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 Cr There is a linear relationship between the log of the starting amount of a template and its threshold cycle thus starting amount of unknown templates can be determined using standard curves constructed using Crvalues of the known starting amounts of target templates In contrast to conventional PCR Real Time PCR eliminates the need for further analysis methods like gel elect
13. ity prior to use e The kit components should be kept on ice or a cooling block until the reaction is prepared and they should be quickly returned to 209C e PCR and nucleic acid isolation should be performed in different compartments Samples should be stored separately to avoid contact with the kit components The safety instructions below should be followed e Serum samples including the standards should be handled with extreme caution 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 e Pathogenic wastes serum samples and material contaminated with them produced during the nucleic acid isolation step should be discarded into medical waste and disposed safely e Pathogen information should be reviewed for the health risks 6 PRODUCTUSELIMITATIONS 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 Cytomegalovirus belongs to the herpes family and is composed of a linear double stranded DNA genome containing aproximatally 230 000 base pairs It is believed that cytomegalovirus genome contains nearly 165 protein encoding genes This virus is also known as Hum
14. lasmosis Other syphilis varicella zoster parvovirus B19 Rubella Cytomegalovirus CMV and Herpes infections Curr Womens Health Rep 2002 2 4 253 8 3 Staras SAS Dollard SC Radford KW Flanders WD Pass RF Cannon MJ Seroprevalence of cytomegalovirus infection in the United States 1988 1994 Clin Infect Dis 2006 43 9 1143 1151 4 Hodinka RL Friedman HM Human Cytomegalovirus in Balows A ed Manuel of Clinical Microbiology 5 th ed Washington American Society for Microbiology 1991 pp 829 36 5 Adler SP Transfusion associated cytomegalovirus infections Review oflnfeetious Diseases 5 1983 977 993 6 Mandell GL Bennett JE Dolin R Principles and practice of infectious diseases 5th ed Philadelphia Churchill Livingstone 2000 1586 96 Code MB04v1f 12 Date July 2011 14 SYMBOLS Use by o Lot Batch Catalog number Temperature limitation Caution consult accompanying documents E gt s Manufacturer IVD In Vitro Diagnostic Medical Device 15 CONTACT INFORMATION AnatoliaTani ve Biyoteknoloji A Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 490 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji Inc Code MB04v1f Date July 2011
15. roper conditions 4 REQUIRED MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and Cy5 filters iCycler iQ5 CFX BioRad LightCycler 1 5 2 0 480 Roche 7500 Real Time PCR System ABI Stratagene Mx3005P Mx3000P Agilent LineGeneK LineGene 9600 Bioer Rotorgene 2000 3000 6000 Q Qiagen e 02ml Thin Wall PCR strips or tubes e Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks e Deep freezer 20 C e Vortex mixer e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes e Adjustable micropipettes e Sterile micropipette tips with filters Code MB04v1f 1 Date July 2011 e Sterile 1 5 or 2 ml microcentrifuge tubes e Disposable gloves 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Caution e The product should be delivered on dry ice Check for presence of dry ice upon arrival and do not use products that have been delivered without it e Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components Attention should be paid also to the following points e Sterile micropipette tips with filters and sterile microcentrifuge tubes should be used e Before starting a test procedure all components should be thoroughly thawed at room temperature After thawing all components should be mixed and centrifuged briefly to ensure homogene
16. rophoresis whereby minimizing the risk of contamination 9 PROCEDURE 9 1 Sample Preparation Storage and Transport To isolate serum from the clinical specimen the blood sample should be collected into sterile vacutainers without any anticoagulant For venipuncture only sterile material should be used Attention EDTA or heparin containing vacutainers should not be used for sample collection Code MB04v1f 3 Date July 2011 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 To avoid 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 must not be used 9 3 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks isolation system or an alternative high quality DNA extraction system is used with Bosphore CMV Quantification Kit v1 The DNA isolation should be performed according to the manufac
17. turers instructions The starting volume is 400 ul the elution volume is 60 9 3 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 QuantiTect Probe PCR Buffer contains Tris Cl KCI NH4 SO 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 CMV 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 PCR inhibition The internal control is a synthetic DNA molecule derived from human genome It is added directly into the PCR master mix to control the PCR inhibition exclusively For this purpose 0 1 ul of internal control should be added for each reaction into the master mix The internal control generally has a Ct of 34 3 Lack of internal control amplification in the FAM negative samples may indicate a problem in PCR or PCR inhibition
18. umber PCR Mix 20 ul Detection Mix 1 2 1 ul Detection Mix 2 1 2 ul Internal Control 0 1 ul dH20 0 6 ul Sample DNA Standard 16 ul Negative Positive Control Total Volume 40 ul Pipette 24 ul of the master mix into the PCR tubes or strips and add 16 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 CMV 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 Denaturation 97 C 00 30 iud a cycles Annealing and Synthesis 53 C 01 30 min Code MB04v1f 5 Date July 2011 Data Collection Hold 22 C 10 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
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