FVL Detection Kit v1 USER MANUAL ®
Contents
1. Fig 3b 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 Code MB16v 1f Date April 2011 Fe Edi SetingsiS ViewiV Aralsis A Tools T _HelptH ngmiucsmm smgs 3aseT Module Edit Gene Amplification Test Result Module Mode I Amplification Curve Standard Curve Report Mode ii Melting Curve p 201283 le 120927 156561 134195 r 111829 0 89464 HK 067058 0 44732 022366 000000 022366 HrHEE Fig 4 Amplification Curve of a Bosphore FVL Detection v1 test Analysis of the results should be performed by trained personnel who have received the required training for analyzing 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 The non exponential signals that cut the threshold in the last 5 cycles and with a Rn lower than 0 2 should not be considered as a positive amplification Please consult the manufacturer in case of problems with analyzing the results All analysis is done automatically in routine u2. cooling The main components of PCR are primers dNTPs Taq polymerase enzyme buffer solution and template 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 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 test utilizes the 5 exonuclease activity of Taq Polymerase to cleave a dual labeled fluorescent hydrolysis probe during the extension phase The probe is labeled at the 5 end with a fluorescent reporter 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 even if the reporter is excited by light no reporter fluorescence can be detected During the elongation step Taq Polymerase encounters and cleaves the probe bound to the template As the reporter is relieved from the suppressing effect of the quenc
3. 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 MATERIALS AND DEVICES e Montania 483 Real Time PCR Instrument Anatolia Geneworks or another Real Time PCR system with FAM and HEX 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 0 2 ml Thin Wall PCR tubes or strips e Magnesia 16 Nucleic Acid Extraction System Magnesia Genomic DNA Whole Blood Kit Anatolia Geneworks or other high quality genomic DNA extraction kits systems e Deep freezer 20 C e Desktop centrifuge with rotor for 2 ml microcentrifuge tubes Code MB16v1f Date April 2011 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 5 IMPORTANT NOTES AND SAF
4. more than 3 samples an extra 10 should be added to the total sample number PCR Mix 12 5 ul Detection Mix 1 1 15 ul dH20 6 35 ul Sample DNA Positive Negative Control 5 ul Toplam Hacim 25 0 ul Pipette 20 ul of the master mix into the PCR tubes or strips and add 5 ul of DNA sample 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 4 Programming the Montania 483 Real Time PCR Instrument The thermal protocol for Bosphore FVL Detection 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 95 C 00 30 min Annealing and Synthesis 64 C 00 45 min H 35 cycles 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 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 HEX e Identify unknown samples positive and negative controls e Select the correct therma
5. repeated 11 TROUBLESHOOTING Please contact the manufacturer in case of a problem during a run Signal from FAM HEX Filter in the Negative Control Contamination 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 13 REFERENCES 1 Bertina R M et al Mutation in blood coagulation factor V associated with resistance to activated protein C Nature 1994 369 6475 p 64 7 2 Genetic home reference NIH Resources Factor V Leiden thrombophilia August 2010 3 Herrmann Koesling et al Prevalence of factor V Leiden mutation in various populations Genet Epidemiology 1997 14 4 p 403 411 4 Epidemiology Rees DC Cox M Clegg JB World distribution of Factor V Leiden Lancet 1995 346 1133 4 14 SYMBOLS Use by Lot Batch Catalog number Temperature limitation Caution consult accompanying documents Manufacturer AEDS 8r vD In Vitro Diagnostic Medical Device Code MB16v 1f Date April 2011 15 CONTACT INFORMATION Anatolia Tani 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 Trade
6. ETY 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 209C e PCR and nucleic acid isolation must be performed in different compartments Samples should be stored separately to avoid contact with the kit components e All the biological wastes produced during the nucleic acid isolation step including the blood samples and material contacted with them should be discarded into medical waste and disposed safely 6 PRODUCT USE LIMITATIONS e Allthe 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 MUTATION Factor V Leiden is the name of the single base mutation in the ge
7. FVL Detection Kit v1 USER MANUAL For in vitro Diagnostic Use NAAT LY W o Document Code MB16v2f Approval Date July 2011 Contents 1 Product Description 2 Content 3 Storage 4 Required Materials and Devices 5 Important Notes and Safety Instructions 6 Product Use Limitations 7 Mutation 8 Method 9 Procedure 9 1 DNA Isolation 9 2 Kit Components 9 2 1 PCR Mix 9 2 2 Detection Mix 1 9 2 3 Positive Control 9 3 Preparing the PCR 9 4 Programming the Montania 483 Real Time PCR Instrument 10 Analysis 11 Troubleshooting 12 References 13 Symbols 14 Contact Information Code MB16v1f Date April 2011 10 1 PRODUCT DESCRIPTION Bosphore FVL Detection Kit v1 detects Factor V Leiden mutation namely G1691A a change of glutamic acid to arginine in human biological samples Wild type F5 allele is amplified and fluorescence detection is accomplished using the HEX filter Mutant F5 allele is amplified and fluorescence detection is accomplished using the FAM filter 2 CONTENT Bosphore FVL Detection Kit v1 is composed of Real Time PCR reagents Component REAGENT 100 50 Tests 25 Tests Tests 1 dH O 1000 ul 1000 ul 1000 pl 2 PCR Mix 1375 ul 688 ul 344 ul 3 Detection Mix1 110 ul 55 ul 28 ul 4 Positive Control 44 ul 22 ul 15 ul 3 STORAGE Bosphore FVL Detection Kit v1 PCR reagents should be stored at 20 C Repeated thawing and freezing
8. her fluorescence signal can be detected Code MB16v1f Date April 2011 As the PCR product accumulates the fluorescence generated by the reporter increases The point at which the signal rises above background level and becomes detectable is called the threshold cycle C Bosphore FVL Detection Kit v1 employs multiplex PCR F5 DNA whether wild type or mutant is amplified in a single reaction using sequence specific primers against mutant and wild type alleles The fluorescent signal generated by the mutant type F5 gene amplification is detected bya probe labeled at the 5 end with FAM through the FAM channel In contrast the fluorescent signal generated by the wild type allele amplification is detected by a second probe labeled at the 5 end with a different reporter molecule HEX through the HEX channel 9 PROCEDURE 9 1 DNA Isolation We recommend that the Magnesia 16 Nucleic Acid Extraction System Magnesia Genomic DNA Whole Blood Kit Anatolia Geneworks isolation system is used with Bosphore FVL Detection 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 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 act
9. ivated 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 SO4 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 F5 gene specific forward and reverse primers 10 uM each and two dual labeled probes against wild type and mutant F5 allele 1 5 uM each 9 2 3 Positive Control The positive control contains heterozygous F5 gene containing one copy of the wild type and mutant F5 allele each It should be included in the PCR to efficiently test whether the samples being analyzed are positive or negative 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 Code MB16v1f Date April 2011 9 3 Preparing the PCR Positive control 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
10. l protocol Code MB16v1f Date April 2011 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 2 HEX are selected Fig 1 Samples positive and negative controls are identified in the Module Edit menu Fig 2 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 Dium 1j H8219223e0 F Charred FAM SYBR T7 Durr HEX VC JOE TET E Owrel2 O15 li A Fig 1 Filter Selection in Montania 483 Fae F ESUE SetigiS View Amaya Tools HelpiM DnazugodUxHSi 922e097 ime Faa Pima Fme 2 Pina Sache ix wr newt Pow wwt newt Sangh Sancte go f g 2 8 8 amp amp Burk Burk Sa Bank Birk Birk Blank Bark Birk Bik Burk Burk Fig 2 Sample Location and Identification Code MB16v1f Date April 2011 zuo D Bs 12307 Module Eat T ene penton T Fig 3a Selecting the Thermal Protocol f e Eee Seong View AnaiyasiA Tooti Hepi suo g 4 123 e7 Gene Amplification I Test Resun o I x Tapen Fen SUA ii FCR Donn Sel OGRA
11. marks Anatolia Geneworks Montania Magnesia and Bosphore are registered trademarks of Anatolia Tani ve Biyoteknoloji Inc Code MB16v1f Date April 2011 10
12. ne referred to as F5 for coagulation Factor V protein molecule This mutation causes a missense substitution of glutamic acid for an arginine at amino acid 506 or 534 depending on the accepted start position Since this is the cleavage site for activated protein C APC to inactivate Factor V this substitution prevents 2 Code MB16v1f Date April 2011 efficient inactivation of factor V resistance to activated protein C resulting in an increased tendency to form abnormal blood clots that causes hypercoagulability disorder Thrombophilia 1 2 Factor V Leiden which is the common inherited form of thrombophilia occurs in 396 596 of the general population and in 2096 4096 of patients with venous thromboembolic disease The risk of blood clot mainly depends on if a person inherits one or two copies of the factor V Leiden mutation Individuals inheriting one copy of the mutation Heterozygous for the factor V Leiden defect have a 5 10 fold increased risk of thrombosis while people inheriting two copies of the mutation homozygous for the factor V Leiden defect one from each parent may have up to 50 100 fold increased risk of developing this type of blood clot than wild type WT healthy individuals 3 4 8 METHOD Bosphore FVL Detection 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
13. se 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 slight amplifications In this case attention should be paid to keep the threshold line above the background Amplification should be observed from both FAM and HEX filters of the heterozygous positive control during the test The amplification of both filters from the tested samples should be compared with that of the positive control 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 qualitative results of the test are displayed on the Report Mode screen The samples that cross the threshold in Channel 1 FAM and Channel 2 HEX are displayed as positive for mutant and wild type alleles respectively The following table shows the possible results and their interpretation Code MB16v 1f Date April 2011 Signal in both FAM and HEX Heterozygous mutant The sample has both wild type and mutant F5 alleles No signal in FAM signal in HEX Homozygous wild type The sample has only wild type FV DNA Signal detected only in the FAM filter Homozygous mutant The FV DNA in the sample is composed of mutant alleles No signal in FAM and HEX The test should be
Download Pdf Manuals
Related Search