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BKV Quantification Kit v1 USER MANUAL

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1. 53123 el Module Edit Gene Amplification if Test Result Module Mode Ampiicabon Curve if Standard curve Report Mode ii Tauna Curve 122989 110690 d 0 98301 t 0 85092 am Lae 073183 5 4 MALE 0 81494 Me 5 SIAE 4 KZ 049198 TAZ a 035867 4 AEN Le Te 024508 DE LY LAT T7 hd 0 12289 WA l 2 00000 ina 212209 2 4 6 e w x nu 6 w a z 2 2 O2 OS x u 36 38 4 u 46 4 m ole Fig 1 An Amplification Curve obtained using Montania 483 Real Time PCR Instrument 0 4 w Norm Fluoro N S ah 5 10 15 20 25 30 35 40 45 Cycle Fig 2 An Amplification Curve obtained using RotorGene Q Real Time PCR Instrument 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 standard curves are given in Fig 3 4 File F Edit SettingsG View V Anahsis A Toels T Help H ego Ms Sale Module Edit Gene Amplification Test Result Module Mode ji Amplification Curve f Standard Curve if Report Mode iL Melting Curve sl Channel a e Skpe 3780 Intercept 4745087 RValue 099985 Test item DRY 5 Testo ET Save Standard A Curve a 2 a 5a a 2 zl 5 a 2 2 a a 2 3 4 5 6 7 Logi Fig 3 Standard Cur
2. The thermal protocol for Bosphore BKV 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 min Annealing and Synthesis 53 C 01 30 min 30 cycles Data Collection Hold 22 C 05 00 Before starting a Real Time PCR reaction using the Bosphore Kits the following steps should be completed e Choose the correct filters to be used FAM and Cy5 e Identify unknown samples standards positive and negative controls assign quantitative values for the standards e Select the correct thermal protocol 10 ANALYSIS By the end of the thermal protocol the Real Time PCR Instrument software automatically calculates the baseline cycles and the threshold Examples of amplification curves are given in Fig 1 2 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 clinical findings and the results of other tests into consideration Code MB14v2f 5 Date Dec 2011 Vin Analysis Too Helpt
3. Follow the instructions for the storage of kit components Section 3 Storage No signal from the internal control Deterioration of the internal control or detection mix 2 Follow the instructions for the storage of kit components See Section 3 Storage PCR inhibition Make sure that you use the recommended DNA isolation method See DNA isolation DNA lost during isolation Make sure that you use the recommended DNA isolation method See DNA isolation Signal from FAM Filter in the Negative Control Contamination Use filter tips Repeat PCR with new kit components The Threshold is Abo ve Low Signals The threshold should be manually adjusted Using the mouse pull the threshold down until it cuts the low signals Avoid the background and the signal from negative control Uneven Traces Baseline cycles should be readjusted Assign new baseline cycles manually and recalculate threshold cycles 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 val
4. PCR to test the efficiency of the PCR exclusively The threshold cycle for the positive control is given in the acceptance criteria table Analysis section Threshold cycles higher than the acceptance criteria may indicate an efficiency loss in the reaction 9 2 6 Quantitation Standards Quantitation of the viral load is performed using four quantitated DNA controls ranging from 1 x 10 copies ml to 2 x 10 copies ml 9 3 Preparing the PCR AII four 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 Code MB14v2f 4 Date Dec 2011 below for preparing the PCR It is for only one reaction multiply these values with the sample number to find the values required for the master mix While preparing master mixes for more than 5 samples an extra 10 should be added to the total sample number PCR Mix 12 5 pl Detection Mix 1 1 9 pl Detection Mix 2 0 5 pl Internal Control 0 1 pl Sample DNA 10 pl Standard Negative Positive Control Total Volume 25 pl Pipette 15 ul of the master mix into the PCR tubes strips or plate wells and add 10 ul of DNA sample standard positive or negative control Close the tube cap seal the plate carefully Make sure that the solution in each tube is at the bottom of the tube well Centrifuge if necessary 9 4 Programming the Real Time PCR Instrument
5. be stored separately to avoid contact with the kit components e Pathogen information should be reviewed to be aware of the health related risks e Biological samples 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 All the pathogenic wastes produced during the nucleic acid isolation step including the serum plasma samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCT USE LIMITATIONS 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 BK virus BKV is a polyomavirus belonging to polyomaviridae family which is composed of an icosahadral viral particle virion containing 5000 base pair double stranded circular DNA molecule and surrounded by a protein capsid It does not possess a lipid envelope BKV infections are asymptomatic as they remain latent in about 90 of the main human population and may lead to infections resulting in severe diseases such as of the immune system There are 4 distinct genotypes of BK classified 1 2 3 Epidemiology Most of the primary polyomavirus infections occur during childhood Serological studies
6. suggest that more than 70 of adults possess antibodies against BK virus There are serological evidences suggesting the reactivation of the BK virus during the pregnancy period of 5 10 of the women 4 5 Code MB14v2f 2 Date Dec 2011 Modes of Transmission Although little is known about the route of transmission among humans high infection rate suggests airborne transmission Abundance of BKV in sewages also suggests transmission via contaminated food and water Semen blood products and organ transplantation particularly renal allografts are other potential routes 6 8 METHOD Bosphore BKV Quantification Kit v1 is based on the Real Time PCR method Polymerase chain reaction is a technique that is used for amplification of a DNA region The reaction occurs by the repeating cycles of heating and cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template As a brief explanation primers are small synthetic DNA those anneal to the specific regions of the template in order to start the synthesis dNTPs are the building blocks of the amplified products Taq polymerase amplifies the DNA template Buffer solution provides the pH adjustment required for the reaction and template as referred is the target region for synthesis In 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 an
7. the threshold in FAM filter are displayed with their calculated starting quantities samples that do not cut the threshold are displayed as Negative or 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 of the internal control should also be checked to avoid false negative results The following table shows the possible results and their interpretation Signal detected in FAM The sample contains BKV No need to check the internal filter pair DNA the result is positive control since the sample is positive high positive samples may suppress the signal from the internal control No signal in FAM The BKV DNA in the Signal from Cy5 filter pair rules signal in Cy5 sample is not detectable out the possibility of PCR inhibition No signal in FAM and The diagnosis is No signal in Cy5 points out to Cy5 inconclusive PCR inhibition or to a problem in DNA isolation Troubleshooting See Code MB14v2f Date Dec 2011 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 Make sure that the correct thermal protocol is chosen Late or weak signal fr om the standards Deterioration of the standards or the core kit components Don t use expired standards or kit components
8. 00 pil 2 PCR Mix 1375 ul 688 pl 344 pl 3 Detection Mix1 210 pl 105 pl 53 pl 4 Detection Mix2 55 pl 28 pl 14 pl 5 Internal Control 15 pl 15 pl 15 pl 6 Positive Control 1 45ul 23 ul 15 pl 7 Standard 1 1 x 10 copies ml 88 ul 44 ul 22 ul 8 Standard 2 1 x 10 copies ml 88 pl 44 ul 22 ul 9 Standard 3 1 x 10 copies ml 88 pl 44 ul 22 ul 10 Standard 4 2 x 10 copies ml 88 ul 44 ul 22 ul 3 STORAGE Bosphore BKV Quantification Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing more than 3 times 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 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 Ro
9. BKV Quantification Kit v1 USER MANUAL For in vitro Diagnostic Use Code MB14v2f Date December 2011 10 11 12 13 14 15 Contents Product Description Content Storage Required Materials and Devices Important Notes and Safety Instructions Product Use Limitations Pathogen Method Procedure 9 1 DNA Isolation 9 2 Kit Components 9 2 1 PCR Mix 9 2 2 Detection Mix 1 9 2 3 Detection Mix 2 9 24 Internal Control 9 2 5 Positive Control 9 2 6 Quantitation Standards 9 3 Preparing the PCR 9 4 Programming the Real Time PCR Instrument Analysis Troubleshooting Specifications 12 1 Sensitivity 12 2 Cross Reactivity References Symbols Contact Information Code MB14v2f Date Dec 2011 Code MB14v2f Date Dec 2011 iii 1 PRODUCT DESCRIPTION Bosphore BKV Quantification Kit v1 detects and quantitates four main genotypes of BK Virus DNA in human serum or plasma samples The analytic sensitivity is 800 copies ml A region within the large T antigene encoding 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 of the internal control is detected with the Cy5 filter 2 CONTENT Bosphore BKV DNA Quantification Kit v1 is composed of the following reagents Bilesen icerik 100 Test 50Test 25 Test 1 dH 0 1000 ul 1000 pl 5
10. alysis methods like gel electrophoresis 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 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 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 C values of the known starting amounts of target templates Bosphore BKV Quantification Kit v1 employs multiplex PCR and an internal co
11. hak D Farrah SR Influence of salts on virus adsorption to microporous filters Appl Environ Microbiol 2000 66 2914 2920 6 McQuaig SM Scott TM Harwood VJ Farrah SR Lukasik JO Detection of human derived fecal pollution in environmental waters using a PCR based human polyomavirus assay Appl Environ Microbiol 2006 72 7567 7574 14 SYMBOLS Use by Lot Batch e 4 Catalog number Temperature limitation Caution consult accompanying documents Manufacturer In Vitro Diagnostic Medical Device lt i EDs E 15 CONTACT INFORMATION Egitim Mh Kasap Ismail Sk No 10 23 Kadikoy 34722 ISTANBUL TURKEY Phone 90 216 330 04 55 Fax 90 216 330 00 42 E mail info anatoliageneworks com www anatoliageneworks com Registered Trademarks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji Inc Code MB14v2f 9 Date Dec 2011
12. imer dimers during reaction setup and the first denaturation step leading to high PCR specificity and accurate quantification PCR Buffer contains Tris Cl KCI NH4 2SO4 8 mM MgCl pH 8 7 20 C dNTP Mix Contains ultrapure quality dATP dGTP dCTP ve dTTP dUTP 9 2 2 Detection Mix 1 Detection Mix 1 contains BKV specific forward and reverse primers and a dual labeled probe 9 2 3 Detection Mix 2 Detection Mix 2 contains internal control specific forward and reverse primers and a dual labeled probe 9 2 4 Internal Control An internal control is included in the kit to control PCR inhibition and possible problems The internal control is a synthetic DNA molecule It is added directly into the PCR master mix to control the PCR inhibition exclusively For this purpose 0 1 pl of internal control should be added for each reaction into the master mix Lack of internal control amplification in the FAM negative samples may indicate a problem in PCR or PCR inhibition In this case PCR should be repeated In samples that contain a high viral load internal control may be suppressed and no increase of the signal is detected Please use the table below for the interpretation of internal control data BKV FAM Internal Control Cy5 Interpretation Sample positive Sample negative Sample positive Repeat the test 9 2 5 Positive Control The positive control contains BKV DNA It can be included in the
13. ntrol is incorporated into the system in order to control the isolation procedure and to check for possible PCR inhibition BKV DNA and an internal control are co amplified in a single reaction using sequence specific primers The fluorescent signal generated by the BKV 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 Code MB14v2f 3 Date Dec 2011 9 PROCEDURE 9 1 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks isolation system other high quality viral DNA extraction kits and systems are used with Bosphore BKV Quantification Kit v1 The DNA isolation should be performed according to the manufacturers instructions The recommended starting volume for Magnesia Viral Nucleic Acid Extraction Kit is 400 ul and the elution volume is 60 ul 9 2 Kit Components 9 2 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 pr
14. torgene 2000 3000 6000 Q Qiagen e 0 2 ml Thin Wall PCR tubes strips or PCR plates e Magnesia 16 Nucleic Acid Extraction System Magnesia Viral Nucleic Acid Extraction Kit Anatolia Geneworks or other high quality viral DNA extraction kits and systems e Deep freezer 20 C e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes e Calibrated adjustable micropipettes e DNAse RNAse pyrogen free micropipette tips with filters e DNAse RNAse pyrogen free 1 5 or 2 ml microcentrifuge tubes e Disposable laboratory gloves Code MB14v2f 1 Date Dec 2011 5 IMPORTANT NOTES AND SAFETY INSTRUCTIONS Important e The product should be delivered on dry ice Check for presence of dry ice upon arrival e Check for the expiry dates on the box and tube labels upon arrival Do not use expired products or components e Calibrated or verified micropipettes DNAse RNAse pyrogen free micropipette tips with filters and DNAse RNAse pyrogen free microcentrifuge tubes should be used e Before starting a test procedure all components should be thoroughly thawed After thawing all components should be centrifuged briefly spin down for 3 5 seconds and mixed well to ensure homogeneity 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 20 C e PCR and nucleic acid isolation must be performed in different compartments Samples should
15. ue The analytical detection limit for Bosphore BKV v1 was found to be 800 Copies ml The sensitivity was determined using serial DNA dilutions The dilutions were tested in different runs in replicates The results were analyzed by probit method 12 2 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 JCV HBV HSV EBV MTBC and Parvovirus B19 with known high positivity were tested and found negative 13 REFERENCES 1 Baron Samuel 1996 Medical Microbiology 4th ed The University of Texas Medical Branch at Galveston ISBN 0 9631172 1 1 Code MB14v2f 8 Date Dec 2011 2 Rajpoot DK Gomez A Tsang W Shanberg A Ureteric and urethral stenosis a complication of BK virus infection in a pediatric renal transplant patient Pediatr Transplant 2007 11 4 433 5 3 Fioriti D Degener AM Mischitelli M Videtta M Arancio A Sica S Sora F Pietropaolo V BKV infection and hemorrhagic cystitis after allogeneic bone marrow transplant Int J Immunopathol Pharmacol 2005 18 2 309 16 4 Padgett BL Walker DL Prevalence of antibodies in human sera against JC virus an isolate from a case of progressive multifocal leukoencephalopathy J Infect Dis 1973 127 467 470 5 Lukasik J Scott TM Andrys
16. ve of a Bosphore BKV v1 test performed using Montania 483 Real Time PCR Instrument Code MB14v2f 6 Date Dec 2011 40 38 34 32 30 Concentration Fig 4 Standard Curve of a Bosphore BKV v1 test performed using RotorGene Q Real Time PCR Instrument AII 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 may allow adjusting the threshold as much as possible for example 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 BKV v1 Component Parameter Cycle Threshold Cr Standard 1 28 2 Standard 2 31 2 Standard 3 34 2 Standard 4 36 2 Positive Control 33 4 Correlation Coefficient gt 0 960 PCR Efficiency gt 60 Test results should not be reported unless the assay results meet the criteria stated above Please contact the manufacturer if an impairment is observed in the product s performance See the last page for contact information The quantitative results of the test are displayed on a spread sheet containing the calculated starting quantities of the unknown samples in each tube Samples that cross

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