GB - Elucigene
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1. 18 65 Deg C 2 Poly_Fill_Vol 6500 6500 38000 steps 3 Current Stability 5 0 int 0 2000 uAmps 4 PreRun_Voltage 15 0 0 15 kvolts 5 Pre_Run_Time 180 1 1000 sec 6 Injection_Voltage 3 0 1 15 kvolts 7 Injection_Time 12 0 1 600 sec 8 Voltage Number Of Steps 20 1 100 nk 9 Voltage_Step_Interval 15 1 60 sec 10 Data_Delay_Time 60 1 3600sec 11 Run_Voltage 15 0 0 15 kvolts 12 Run_Time 1200 300 14000 sec Increase injection time to 36 seconds for analysis of DNA extracted from bloodspots Note Required run time will vary dependent on the ambient temperature of the location in which the Genetic Analyzer has been installed For more information on creating run modules please refer to the Applied Biosystems 3130 Genetic Analyzer User Manual Create the CF EU2 protocol in the Protocol Manager ensure the following are selected e Type Regular Run Module CF EU2 see run module above e Dye Set G5 To run the samples create a sample sheet using the Plate Manager ensure the correct protocol for CF EU2v1 has been selected for the instrument protocol see above Note For more information on instrument setup operation and troubleshooting please refer to the Applied Biosystems 3130 Genetic Analyzer User Manual CF2EUBYEN 002 Sep 2014 Page 12 of 24 Elucigene CF EU2v1 Instructions for Use ABI 3500 Instruments A CF EU2 Instrument Protocol needs to be created which can then be used for each C2. 377 383 35 G85E 384 390 36 R1066C 391 397 37 1898 1G gt A 398 5 404 5 38 W846X 406 41 1 39 2184delA 413 418 40 D1152H 423 429 41 CFTRdel2 3 433 439 42 P67L 439 5 445 5 43 2143delT 446 450 5 44 E60X 453 5 460 5 45 3659delC 461 465 5 46 3272 26A gt G 470 476 47 621 1G gt T 485 5 491 5 CF2EUBYEN 002 Sep 2014 Page 16 of 24 Elucigene CF EU2v1 Instructions for Use Product bp Size Range Maker Markar 3130 POP7 data 48 A455E 496 503 49 R1162X 506 512 50 R1158X 516 5 524 5 5T IVS8 5T 110 130 77 IVS8 7T 140 160 9T IVS8 9T 175 195 S549R T gt G Peak 20 is only present in the A mix 5T Allele sizes Product bp Size Marker Range 3130 POP7 data 5T 9 117 25 118 75 5T 10 119 25 120 75 5T 11 121 25 122 75 5T 12 123 25 124 75 5T 13 125 25 126 75 NOTE Marker sizes may vary due to the instrument and polymer used Examples of Interpretation 1507del Due to the location of the 1507del deletion in the CFTR gene it is possible to detect the presence of this marker through a 3bp shift in size of the F508del peak as well as an 1507del mutant specific peak Where a single 1507del mutant allele is present the 1507del mutant peak will be observed as a blue peak at approximately 159bp A wild type peak for F508 will be observed as being present but at approximately half the peak height normally associated with a homozygous
3. Lot or batch number VD In Vitro Diagnostic Medical Device CF2EUBYEN 002 Sep 2014 Page 6 of 24 Elucigene CF EU2v1 Instructions for Use Materials Provided Storage of the reagents should be in an area free from contaminating DNA or PCR product Store all components below 20 C in the dark The fluorescent dyes used in this product are photosensitive minimise exposure to light Discard 3 months after opening All reagents are supplied ready for use Sufficient materials for 50 10 tests are provided 2 x 120ul 50ul vials of CF EU2v1 A primer mix containing primers to amplify the following mutant alleles R347H R347P 2789 5G gt A 3120 1G gt A 711 1G gt T R334W 1507del F508del 3849 10kbC gt T 1677delTA 1078delT V520F L206W W1282X R560T 2347delG Q890X R553X G551D S549N M1101K G542X 3905insT Y1092X C gt A S1251N 444delA 18114 1 6kbA gt G 1717 1G gt A R117H R117C N1303K Y122X 394delTT G85E R1066C 1898 1G gt A W846X 2184delA D1152H CFTRdele2 3 P67L 2143delT E60X 3659delC 3272 26A gt G 621 1G gt T A455E R1162X and R1158X This mix also contains wild type primers for the detection of normal F508del allele primers for detection of the polythymidine variants IVS8 5T IVS8 7T IVS8 9T and primers for identification of 2 hypervariable short tandem repeat STR markers 404473 450043 2 x 120ul 50ul vials of CF EU2v1 B primer mix containing wild type primers to amplify the normal alleles of
4. Rosenstein BJ Cutting GR The diagnosis of cystic fibrosis a consensus statement Cystic Fibrosis Foundation Consensus Panel J Pediatr 1998 132 589 95 2 De Braekeleer M Allard C Leblanc JP Simard F Aubin G Genotype phenotype correlation in cystic fibrosis patients compound heterozygous for the A455E mutation Hum Genet 1997 101 208 11 3 Drumm ML Konstan MW Schluchter MD Handler A Pace R Zou F Zariwala M Fargo D Xu A Dunn JM Darrah RJ Dorfman R Sandford AJ Corey M Zielenski J Durie P Goddard K Yankaskas JR Wright FA Knowles MR Genetic modifiers of lung disease in cystic fibrosis N Engl J Med 2005 353 1443 53 4 Goss CH Newsom SA Schildcrout JS Sheppard L Kaufman JD Effect of ambient air pollution on pulmonary exacerbations and lung function in cystic fibrosis Am J Respir Crit Care Med 2004 169 81 6 21 5 Kerem B Rommens JM Buchanan JA Markiewicz D Cox TK Chakravarti A Buchwald M and Tsui LC Identification of the Cystic Fibrosis Gene Genetic Analysis Science 1989 245 4922 1073 8 6 Cystic Fibrosis Mutation Database www genet sickkids on ca cftr app 7 Joshua D Groman et al Variation in a Repeat Sequence Determines Whether a Common Variant of the Cystic Fibrosis Transmembrane Conductance Regulator Gene Is Pathogenic or Benign Am J Hum Genet 2004 74 1 176 179 8 Newton CR et al Analysis of any point mutation in DNA The Amplification Refractory Mutation System ARMS Nucleic Ac
5. containing the size standard formamide mix from step 1 already dispensed into the plate Denature the PCR product dispensed into the PCR plate on a thermal cycler using the following parameters 94 C for 3 minutes linked to 4 C for 30 seconds Centrifuge the plate briefly to collect the contents at the bottom of the vials and to remove any bubbles in the wells and immediately load onto the Genetic Analyzer Note Sample injection settings can be modified to suit the amount of amplicon produced during the PCR which can vary due to amount of input genomic DNA added Less amplicon can be applied to the column for analysis by reducing either time or voltage of injection Conversely more amplicon can be applied to the column for analysis by increasing either time or voltage of injection Previously amplified samples can be re injected multiple times for re analysis see Elucigene CF EU2v1 Troubleshooting Guide for more information CF2EUBYEN 002 Sep 2014 Page 11 of 24 Elucigene CF EU2v1 Instructions for Use ABI 3130 Instruments A CF EU2 run module and protocol need to be created which can then be used for each CF EU2 run Create the CF EU2 run module in the Module Manager of the 3130 data collection software Ensure the following are selected e Type Regular Template FragmentAnalysis36_POP7 Enter the settings detailed in the table below 36cm Capillary Module Parameter Name Value Range 1 Oven Temperature 60 int
6. minimise the risk of contamination steps 3 5 must be carried out in an area free from PCR product preferably in a laminar flow cabinet 1 Program the thermal cycler for a single step cycle to activate the HotStart Taq at 94 C for 20 minutes linked to an amplification cycling program of 1 minute at 94 C denaturation 2 minutes at 58 C annealing and 1 minute at 72 C extension for 30 cycles This should be linked to a 20 minutes time delay file at 72 C extension on the final cycle A negative control must be included in each PCR run Thaw the Primer Mixes and the PCR Master Mix and centrifuge briefly to collect the contents at the bottom of the vials Mix gently by vortexing and briefly centrifuge the vials again Prepare sufficient Reaction Mix for the number of samples and controls to be tested Table 1 Note The PCR Master Mix is viscous and care should be taken to ensure that the correct volumes are handled It is recommended that the PCR Master Mix be added to the previously dispensed primer mixes to ensure all liquid is dispensed from the pipette into the Primer Mix Note Do not use different mixes or components from different lots of CF EU2Vv1 kit Enzyme Activation Cycling Final Extension Ambient Temperature 30 Cycles CF2EUBYEN 002 Sep 2014 Page 10 of 24 Elucigene CF EU2v1 Instructions for Use Table 1 Reaction Mix Formulation Number of Samples to be Tested 1 10 25 50
7. p Arai17His Y122X c 366T gt A p Tvri22Xx 621 1G gt T C 489 1G gt T 711 1G gt T c 579 1G gt T _L206W c 617T gt G p Leu206Tro 1078delT c 948del c 948delT p Phe316Leufsx12 R334W c 1000C gt T p Ara334Tro R347P c 1040G gt C p Ara347Pro R347H c 1040G gt A p Ara347His A455E c 1364C gt A p Ala455Glu 1507del c 1519 1521del c 1519 1521delATC p lle507del F508del c 1521 1523del c 1521_ 1523delCTT p Phe508del 1677delTA c 1545 1546del c 1545 1546delTA o Tvr515X V520F c 1558G gt T p Val 520Phe 1717 1G gt A 1585 1G gt A G542x c 1624G gt T p Glv542x S549R T gt G c 1647T gt G p Ser549Ara S549N c 1646G gt A p Ser549Asn G551D c 1652G gt A p Glv551Asp R553X c 1657C gt T p Ara553X R560T c 1679G gt C p Arg560Thr 1811 1 6kbA gt G c 1680 886A gt G 1898 1G gt A c 1766 1G gt A 2143delT c 2012del c 2012delT p Leu671X 2184delA sd c 2052del c 2052delA CCC plys684Asnfsx38 2347delG 22 15del c 2215delG J pVal739TvrfsXi6 W846X c 2538G gt A p Tro846X 2789 5G gt A 2657 5G gt A Q890X c 2668C gt T p GIn890X 3120 1G gt A C 2988 1G gt A CF2EUBYEN 002 Sep 2014 Page 2 of 24 Elucigene CF EU2v1 Instructions for Use 3272 26A gt G c 3140 26A gt G R1066C c 3196C gt T p Ara1066Cvs Y1092X C gt A C 3276C gt A p Tvr1092X M1101K c 3302TSA o Meti10iLvs D1152H 6 3454G gt C p Aso1152His R1158X c 3472C gt T p Ara1158X R1162X c 3484C gt T p Arq1162X 3659delC c 3528del c 3528delC p Lvs11
8. size in the wild type B mix These have been tabulated and are available in a separate document from the Elucigene website www elucigene com products Examples of insertions and deletions detected by the kit and impact they have on the B mix profile are shown below F508del 1677delTA Two peaks ibp apart at position 12 V520F WT will be observed in the B mix results from an individual heterozygous for the F508del 1677delTA mutations asv ll en a a e a a a a E o anaa a aa oO aaa an bea J Ua GO Ae m mam Geer E Ge oY Ge Pee ee oe a ee ee am IETT Sama WT pem ariin nl i y TEL LA CF2EUBYEN 002 Sep 2014 Page 18 of 24 Elucigene CF EU2v1 Instructions for Use V520F A wild type peak of reduced height at position 10 1677delTA WT will be observed in the B mix results from an individual heterozygous for the V520F mutation as this prevents the 1677delTA WT primer working Peaks 10 amp 12 will drop out in the results from an individual homozygous for the V520F mutation VSQ0F WT pes MRAM Hil Fvlituid til tf iii iiaii Wl L aea amn m a A a a a a aT a aaga a pa maa aa e a a a ET TE in A mwa OI te a ew ee UM Me st COR ee ee Me ie se ae 394delTT Two peaks 2bp apart at positions 35 G85E WT 42 P67L WT amp 44 E60X WT will be observed in the B mix results from an individual heterozygous for the 394delTT mutation Peak 34 will drop out and peaks 35 42 amp 44 will be the expect
9. 77SerfsxX15 3849 10kbC gt T 3718 2477C gt T 1251N c 3752G gt A p Ser1251Asn 3905insT c 3773dup c 3773dupT o Leu1258PhefsX7 W1282X C 3846G gt A p Tro1282X N1303K c 3909C gt G p Asn1303Lvys with reference to Mutation Nomenclature in Practice Findings and Recommendations from the Cystic Fibrosis External Quality Assessment Scheme Berwouts S Morris M Girodon G Schwarz M Stuhrmann M and Dequeker E Human Mutation Vol 00 No 0 1 7 2011 CF EU2v1 can distinguish between individuals who are heterozygous and homozygous for the all the above mutations and variants with the exception of S549R T gt G see cross reactivity section of this document Summary and Explanation Cystic Fibrosis CF is the most common life limiting autosomal recessive disorder in the Caucasian population The disease incidence is 1 3200 live births among Caucasians 1 In the Caucasian population the heterozygote frequency is approximately 1 25 Cystic fibrosis affects the epithelia in several organs resulting in a complex multisystem disease that includes the exocrine pancreas intestine respiratory tract male genital tract hepatobiliary system and exocrine sweat glands Disease expression varies by severity of CFTR mutations 2 genetic modifiers 3 and environmental factors 4 The range extends from early childhood death as a result of progressive obstructive lung disease with bronchiectasis to pancreatic insufficiency with graduall
10. Elucigene CF EU2v1 Instructions for Use Elucigene Diagnostics Elucigene CF EU2v1 Instructions for Use Cat Code CF2EUB2 50 tests CF2EUBX 10 tests For In Vitro Diagnostic Use ual Manufactured by Elucigene Diagnostics Greenheys House Pencroft Way Manchester Science Park Manchester M15 6JJ For Sales Customer Service and Technical Support T 44 0 161 669 8122 F 44 0 161 669 8129 E enquiries elucigene com E techsupport elucigene com Elucigene Diagnostics is the trading name of Delta Diagnostics UK Limited a company registered in England and Wales registration number 8696299 CF2EUBYEN 002 Sep 2014 Page 1 of 24 Elucigene CF EU2v1 Instructions for Use Elucigene CF EU2v1 Intended Use For the simultaneous in vitro qualitative detection of the following human Cystic Fibrosis Transmembrane conductance Regulator CFTR gene mutations in DNA extracted from whole blood EDTA preserved and dried bloodspot samples Traditional HGVS Nomenclature cDNA name Protein name CFTRdele2 3 c 54 5940_273 1025del c 54 5940_273 10250del21080 E60X c 178G gt T p Glu60X P67L c 200C gt T o Pro67Leu G85E c 254G gt A p Gly85Glu 394delTT c 262 263del c 262 263delTT p Leu88llefsX22 444delA c 313del c 313delA p lle105SerfsX2 R117C c 349C gt T p Ara117Cvs R117H c 350G gt A
11. F EU2v1 run Create the CF EU2 Instrument Protocol through the 3500 Instrument Protocols library Ensure the following are selected Run Module FragmentAnalysis50_POP7 Enter the settings detailed in the image below Eda brtrument Protocot CEU Setup an Instrument Protocol A Apphcanon Type Fragment bd apay Length 50 Oye Set GS Inctreenient Crates ol Propertie Bun Module FragenentAmalysnS POPI Protocol Hame CEU Descnpiar Oven Temperature t at Ram Voltage Volts 19 5 Prefas Voltage Volts 15 byection Voltage AValtsh 3 Run Tene liec L200 PreRun Time tech E0 jection Time bech B Data Delay sec b Advanced Options Increase injection time to 36 seconds for analysis of DNA extracted from bloodspots To run the samples create a sample plate by clicking on Create Plate from Template in the Dashboard ensure the correct Instrument Protocol for CF EU2v1 has been assigned see above Sample sheet set up for GeneMarker The GeneMarker software allows the direct comparison between the A and B data from the same individual To facilitate this it is important that the naming of the raw data output file fsa file is consistent across all samples and mixes The sample sheet should contain the unique sample name for each sample being tested suffixed by either _A or _B depending which mix is being tested If the Plate ID is to be included in the fsa name then a fixed format should be used each
12. Primer Mix ul 4 5 45 112 5 225 PCR Master Mix ul 7 5 75 187 5 375 Total ul 12 120 300 600 Pipette 10ul of each reaction mix into the bottom of the appropriately labelled 0 2ml PCR vials or plates Using separate pipette tips add 2 5ul of test DNA sample to each of the vials and cap Do not add DNA to the vial for the negative control substitute with 2 5 of sterile deionised water Centrifuge the PCR vials briefly to collect the contents at the bottom of the vials Place the vials firmly in the thermal cycler block Initiate the 94 C single step cycle followed by the amplification cycling program On completion of the amplification cycling program the samples may be stored at room temperature overnight or at 2 8 C for up to 7 days in the dark before analysis by capillary electrophoresis Capillary Electrophoresis It is recommended that each user ensure that the chosen capillary electrophoresis equipment is used according to the manufacturer s instructions and is compatible with this test In this context the key parameters are the polymer and the capillary array Optimal results can be obtained using the following capillary electrophoresis conditions 1 Combine 6 8ul of GS600v2 LIZ size standard with 250ul Hi Di Formamide and mix thoroughly sufficient mix for 16 wells Dispense 15ul of the mix into each well of a 96 well PCR plate Add 3ul of PCR product from each of the A and B mix amplifications to separate wells
13. Thermal cycler to accommodate 96 well microtitre plates or 0 2ml vials with a minimum temperature accuracy of 1 C between 33 C and 100 C and static temperature uniformity of 1 C Note Thermal cycler equipment should be regularly maintained in accordance with the manufacturer s instructions and calibrated to ensure accurate PCR cycling and optimal performance The Elucigene CF EU2v1 assay was developed on Applied Biosystems 9700 thermal cyclers Other makes and models should be fully tested and evaluated for optimal performance by the user before reporting results with CF EU2v1 CF2EUBYEN 002 Sep 2014 Page 7 of 24 Elucigene CF EU2v1 Instructions for Use Capillary Electrophoresis ABI 3130 3500 Genetic Analyzer with GeneMapper fragment analysis software 36cm or 50cm ABI3500 capillary array 96 well optical plates 96 well septas 96 well cassettes Note Capillary electrophoresis equipment should be regularly maintained in accordance with the manufacturer s instructions and calibrated to ensure optimal performance Sample Collection and Storage Whole blood EDTA samples and dried bloodspots have been evaluated to be compatible with this test Sample collection devices have on occasion been reported to be detrimental to the integrity of certain analytes and could interfere with some method technologies 9 It is recommended that each user ensure that the chosen device is used according to the manufacturer s instruc
14. U2v1 kit uses fluorescent ARMS Amplification Refractory Mutation System allele specific amplification technology which detects point mutations insertions or deletions in DNA 8 The principle of ARMS is that oligonucleotides with a 3 mismatched residue will not function as Polymerase Chain Reaction PCR primers under specified conditions Selection of appropriate oligonucleotides allows specific mutant or normal DNA sequences to be amplified and detected Amplified sequences amplicons are separated by capillary electrophoresis using an Applied Biosystems Genetic Analyzer instrument Analysis software enables amplicons to be identified and labelled according to their size and dye colour Elucigene CF EU2v1 is a highly multiplexed assay comprising two A and B PCR reactions Fifty mutant sequences within the CFTR gene are detected in the A mix and are visualised as blue amplicon peaks Corresponding non mutated normal wild type sequences are detected in the B mix and are visualised as green amplicon peaks The A mix also detects normal sequence for the most commonly observed mutation causing cystic fibrosis in Caucasian populations called F508del and visualised as a green amplicon peak Additionally polyT repeat sequences are detected in the A mix and visualised as black amplicon peaks Internal amplification control markers non cystic fibrosis are included in both the A and B mixes to monitor the efficiency of sample amplification and are vi
15. ed height but shifted 2bp smaller than expected size in the results from an individual homozygous for the 394delTT mutation pt a aa ama l Ge Get a al a aed fori et fe TA F508del CFTRdele2 3 Reduced peak heights at positions 34 394delTT 35 G85E WT 41 CFTRdele2 3 WT 42 P67L WT amp 44 E60X WT will be observed in the B mix results from an individual heterozygous for the CFTRdele2 3 mutation Peaks 34 35 41 42 amp 44 will drop out in results from an individual homozygous for the CFTRdele2 3 mutation e 1 gt s r gt gt gt ISaderTT WT peh ME e Ladd lei ba cee Lana a 4H CF2EUBYEN 002 Sep 2014 Page 19 of 24 Elucigene CF EU2v1 Instructions for Use 444delA Two peaks 1bp apart at positions 30 R117H WT 31 R117C WT 33 Y122X WT amp 47 62141 WT will be observed in the B mix results from an individual heterozygous for the 444delA mutation Peak 27 will drop out and peaks 30 31 33 amp 47 will be the expected height but shifted 1bp smaller than expected size in the results from an individual homozygous for the 444delA mutation dtidel WT peak 2347delG Two peaks 1bp apart at positions 39 2184delA WT amp 43 2143delT WT will be observed in the B mix results from an individual heterozygous for the 2347delG mutation Peak 16 will drop out and peaks 39 amp 43 will be the expected height but shifted 1bp smaller than expected size in the results
16. en site an individual is described as being heterozygous for this site Once data collection has finished CF EU2v1 PCR fragments should be sized against the GS600v2 LIZ size standard using fragment analysis software The Elucigene CF EU2v1 Guide to Analysis Software document provides further detailed guidelines for software settings analysis and interpretation Guide to Analysis Software procedures are available for GeneMapper and GeneMarker software from the Elucigene website www elucigene com products CF2EUBYEN 002 Sep 2014 Page 14 of 24 Elucigene CF EU2v1 Instructions for Use The results from the A mutant mix determines if an individual carries a mutation and is shown by the presence of a blue peak The mutant mix also contains primers for the normal F508 allele therefore this mix can determine if an individual is normal for F508 green peak only homozygous for the mutant F508del blue peak only or heterozygous for F508del blue and green peak If any other mutation is observed the results from the B wild type mix can be analysed to determine homozygous or heterozygous status The presence of a green peak for the particular allele in the wild type mix indicates the individual is heterozygous and the absence of the green peak indicates the individual is homozygous for the particular mutation Hypervariable STR markers red are included in both mixes This enables the comparison between the sample amplified with the mutant
17. following effects on the Elucigene CF EU2v1 kit results 1 The G551D mutant primer in the A mix will also detect the S549RT gt G mutation The analysis software will label this as peak 20 There will not be a corresponding wild type peak in the B mix 2 The 2184delA mutant primer in the A mix will cross react with 2183AA gt G mutant DNA sequence and result in a mutant peak at the 2184delA position 3 The 1078delT mutant primer in the A mix will cross react with F316L mutant DNA sequence and result in a mutant peak at the 1078delT position 4 The R347P mutant primer in the A mix will cross react with R347H mutant DNA sequence and result in a mutant peak at the R347P position of reduced height compared to a true R347P peak 5 The presence of the F508C 1655T gt G polymorphism will result in the reduction of the height of the 1507del peak in the CF EU2v1 B mix 6 The presence of the R117H will result in the reduction of the height of the R117C peak in the CF EU2v1 B mix In a sample homozygous for R117H the R117C peak will be absent 7 The presence of the G85E will result in the reduction of the height of the 394delTT peak in the CF EU2v1 B mix In a sample homozygous for G85E the 394delTT peak will be absent 8 A very small artefact peak in the 1507del position can sometimes be observed in the results from an F508del heterozygote and in particular an F508del homozygote sample 9 The following mutations which have not been checked
18. for possible cross reactivity due to unavailability of relevant samples may interfere with test function R117P R117L 1717 2A gt G 621 2T gt C 621 2T gt G R553G R553Q R347L I506T I506S I506V and the rare combination of 1507del with the polymorphism 1651A G Performance Characteristics One hundred and ten DNA samples extracted from liquid whole blood EDTA were tested blind using Elucigene CF EU2v1 Five samples failed after PCR which correlated to low DNA concentration less than 1 5ng ul Of those that gave interpretable results 96 were normal 5 were heterozygous for F508del 1 was heterozygous for 1717 1G gt A 1 was heterozygous for G551D 1 was heterozygous for 621 1G gt T 1 was heterozygous for G542X and 1 was compound heterozygous for G542X F508del Ninety one DNA samples extracted from dried bloodspots were tested blind using Elucigene CF EU2v1 Five samples failed to amplify Of those samples that did amplify 20 failed to meet analysis criteria for interpretation following a 12 second injection onto a 3500 Genetic Analyzer all failed samples correlated to low concentration DNA less than 1 5ng ul The 20 failed samples were re injected for 36 seconds on the 3500 Genetic Analyzer and analysed 7 samples failed to meet analysis criteria for interpretation Of the 79 samples that gave interpretable results 38 were normal 5 were heterozygous for F508del 3 were heterozygous for G551D 3 were heterozygous for W1282X 2 were heter
19. from an individual homozygous for the 2347delG mutation med 2347 deiG WT pa J il nt 3905insT Two peaks 1bp apart at position 26 S1251N WT will be observed in the B mix results from an individual heterozygous for the 3905insT mutation Peak 24 will drop out and peak 26 will be the expected height but shifted 1bp larger than the expected size in the results from an individual homozygous for the 3905insT mutation 200s T WT peat inl tty nN aT He Da HSS DSRNA PRM wR SMM S CF2EUBYEN 002 Sep 2014 Page 20 of 24 Elucigene CF EU2v1 Instructions for Use R1158X Reduced peak height at position 49 R1162X WT will be observed in the B mix results from an individual heterozygous for the R1158X mutation Peak 49 will drop out in results from an individual homozygous for the R1158X mutation Riset WT nee p AIN ATT Vy i f A MULT EUSE ON Tae amp ae amp I ie ee ee ee ee O new Ta 6 g yy SOUS amp aA SOs Se Oe TT a ROAD amp cas S i Wie i Polymorphism rs4148721 delAT in Intron 22 Two peaks 2bp apart at positions 45 3659delC WT 49 R1162X WT amp 50 R1158X WT will be observed in the B mix results from an individual heterozygous for the polymorphism rs4148721 deletion of AT in intron 22 Peaks 45 49 amp 50 will be the expected height but shifted 2bp smaller than expected size in the results from an individual homozygous for the polymorphism rs4148721 Othe
20. he CFTR gene in 1989 5 more than 1700 mutations and variants in the gene have been described 6 Many of these mutations are private having been described only in one patient and or family Routine testing for all possible mutations is neither feasible nor cost effective and is therefore confined to testing for the most common mutations CF EU2v1 is a cystic fibrosis testing kit designed specifically to address the most common mutations found in populations CF2EUBYEN 002 Sep 2014 Page 3 of 24 Elucigene CF EU2v1 Instructions for Use of European origin The assay identifies 50 mutations in total and also analyses the intron 9 polyT tract with accurate measurement of the adjacent TG repeat The polymorphic thymidine tract at the junction of intron 9 and exon 10 influences transcription The number of thymidine residues 5T 7T or 9T affects the splicing efficiency of exon 10 if the 5T allele is present a proportion of exon 10 transcripts will be absent resulting in non functional protein and variable CF symptoms It is reported that the number of TG repeats 5 to the polythymidine tract can also influence splicing of exon 10 7 If present on the same allele as the 5T variant the longer the number of TG repeats the higher the proportion of CFTR transcripts will lack exon 10 The number of TG repeats can be determined using CF EU2v1 by sizing the 5T amplicon peaks Principles of the Procedure The method employed by the Elucigene CF E
21. id Res 17 2503 2516 1989 9 Satsangi J et al Effect of heparin on polymerase chain reaction Lancet 343 1509 1510 1994 10 PCR Primer A Laboratory Manual 2 edition ColdSpring Harbour Laboratory Press Section 1 ELUCIGENE is a trademark of Delta Diagnostics UK Ltd ARMS is a trademark of AstraZeneca UK QIAAMP is a trademark of Qiagen Gmbh PICOGREEN is a trademark of Molecular Probes Inc GENEMARKER is a trademark of Softgenetics Corporation GENEMAPPER NED VIC PET POP 7 LIZ and HI DI are trademarks of Life Technologies Corporation NOTICE TO PURCHASER LIMITED LICENCE Polynucleotides labeled with VIC NED and PET dyes and or their use may be covered by one or more patents owned by Life Technologies Corp The purchase price of this product includes limited nontransferable rights under certain claims of certain patents owned by Life Technologies Corp to use only this amount of the product solely for activities of the purchaser in detection of Target s within the field of human diagnostics No other rights are conveyed Further information on purchasing licenses relating to the dyes mentioned above may be obtained by contacting the Director of Licensing outlicensing lifetech com Copyright 2014 Delta Diagnostics UK Ltd CF2EUBYEN 002 Sep 2014 Page 24 of 24
22. mix and the sample amplified with the wild type mix to reduce the potential for sample mix up a different STR profile in each of the two mixes indicates a sample mix up Absence of these STR markers indicates a failed sample Presence of the STR markers at very low rfus indicates a weak sample which should be analysed with caution Note See Troubleshooting Guide for more information CF2EUBYEN 002 Sep 2014 Page 15 of 24 Elucigene CF EU2v1 Instructions for Use Markers Detected The table below summarises the markers detected by CF EU2v1 mix Markers are listed according to the size range of PCR product observed Markers Detected Marker Marker Product bp Size Range Peak No 3130 POP7 data 01 R347H 110 5 116 5 02 R347P 117 123 03 2789 5G gt A 124 130 04 3120 1G gt A 132 5 138 5 05 7114 1G gt T 141 5 147 5 06 R334W 147 5 154 07 I507del 156 162 5 08 F508del 163 169 09 3849 10KbC gt T 172 178 10 1677delTA 180 188 5 11 1078delT 193 199 12 V520F 206 211 5 13 L206W 215 220 14 W1282X 224 5 230 5 15 R560T 234 5 240 5 16 2347delG 242 246 17 Q890X 250 252 18 R553X 255 5 261 5 19 G551D 265 267 20 S549R T gt G 267 5 269 5 21 S549N 275 280 22 M1101K 282 288 23 G542x 289 295 5 24 3905insT 297 303 5 25 Y1092X C gt A 308 314 26 1251N 315 321 27 444delA 323 326 28 18114 1 6kbA gt G 332 338 29 1717 1G gt A 341 5 347 5 30 R117H 349 355 31 R117C 357 360 32 N1303K 360 366 5 33 Y122X 367 373 34 394delTT
23. n B Mix Profile To obtain a copy of the Elucigene CF EU2v1 Troubleshooting Guide please contact our Technical Support Group T 44 0 161 669 8122 F 44 0 161 669 8129 E enquiries elucigene com E techsupport elucigene com Limitations of the Procedure 1 The results obtained from this or any other diagnostic test should be used and interpreted only in the context of the overall clinical picture Elucigene Diagnostics is not responsible for any clinical decisions that are taken 2 The absence of the mutations detected by this kit is no guarantee that other mutations in the CFTR gene are not present Other mutations are possible and are not detected by this kit 3 Mutations vary in frequency between different populations Population mutation frequency data is available from The Cystic Fibrosis Genetic Analysis Consortium 6 The user of this kit should emphasise these points when reporting results to the diagnosing Clinician Genetic Counsellor Disclaimer Results from this and other diagnostic assays should be interpreted in conjunction with other laboratory and clinical data available to the Clinician These Elucigene reagents are supplied for In Vitro diagnostic testing Further details for data interpretation are available in the Elucigene CF EU2v1 Guide to Analysis Software procedures www elucigene com products CF2EUBYEN 002 Sep 2014 Page 23 of 24 Elucigene CF EU2v1 Instructions for Use References 1
24. nsure optimal results Techniques such as PicoGreen fluorescence or UV absorbance are acceptable Due to variation in DNA quantification techniques the user should consider the following guidance Very high input DNA amounts will increase the likelihood that background peaks are labelled by the analysis software The following steps can be taken to reduce the likelihood that background peaks are labelled e Dilute DNA sample and re amplify e Reduce injection time see Capillary Electrophoresis section e Increase minimum peak amplitude threshold see Elucigene CF EU2v1 Guide to Analysis Software Low input DNA amounts will increase the likelihood that diagnostic peaks are weak and not labelled by the analysis software The following steps can be taken to increase signal e Increase injection time to 36 seconds strongly recommended for bloodspot extractions see Capillary Electrophoresis section e Re extract bloodspot sample and elute in reduced volume 50n of water e Increase number of bloodspots for extraction to 4 x 3mm discs CF2EUBYEN 002 Sep 2014 Page 8 of 24 Elucigene CF EU2v1 Instructions for Use Important Considerations DNA Quality CF EU2v1 is a highly multiplexed assay and requires efficient PCR amplification to function optimally Quality of DNA can determine the efficiency of the PCR process Inhibitors co extracted with the DNA sample may result in a sub optimal amplification leading to weak and poorly balanced
25. ozygous for D1152H and 2 were heterozygous for G542X Four compound heterozygotes 3120 1G gt A F508del R117H F508del R1162X F508del and R553X F508del were also determined Additionally the following heterozygous samples were each observed on a single occasion E60X G85E 394delTT Y122X 621 1G gt T 1078delT R334W R347P A455E 1507del 1717 1G gt A R553X 1811 1 6kbA gt G 1898 1G gt A 2184delA W846X 3272 26A gt G Y1092X 3659delC 3849 10kbC gt T S1251N and N1303K Forty six DNA samples representing all 50 mutations detected by the CF EU2v1 kit were amplified on five separate occasions All samples gave the expected results with no false negative or false positive results demonstrating 100 clinical specificity and sensitivity CF2EUBYEN 002 Sep 2014 Page 22 of 24 Elucigene CF EU2v1 Instructions for Use Troubleshooting These Instructions for Use are supplied to ensure optimal performance of the assay Users must not deviate from the procedures provided Any deviation may result in sub optimal performance yielding poor quality data A Troubleshooting Guide is available on request from Elucigene Diagnostics and provides examples and solutions to some of the most common observations with Elucigene CF EU2v1 these include No Diagnostic or STR Peaks Weak Diagnostic or STR Peaks Excessive Breakthrough Background Peaks Unlabelled Peaks Weak FAM Mutant Peaks Unbalanced A Mix Profile Unbalanced B Mix Profile Split Peaks i
26. peaks Elucigene Diagnostics has validated the use of QlAamp 96 DNA Blood Kit and QlAamp DNA Mini Kit QIAGEN GmbH with the CF EU2v1 kit Other DNA extraction kits or methods should be validated and optimised for use with CF EU2v1 Note See Elucigene CF EU2v1 Troubleshooting Guide for more information CF2EUBYEN 002 Sep 2014 Page 9 of 24 Elucigene CF EU2v1 Instructions for Use Test Protocol Control of PCR contamination The PCR process generates a large number of amplified products amplicons and carries a high risk of contamination which may lead to false results Contamination can arise from two sources Cross contamination contamination of the sample with non amplified material from the environment or other samples which contain the target sequence End product contamination contamination of the sample with amplicons from previous PCR s leading to amplification of both target and contaminant amplicons Laboratories performing PCR should be aware of these sources of contamination and have procedures in place that significantly reduce the risk of contamination Methods to control contamination are well documented 10 and include the physical design of laboratories workflow sample handling procedures and chemical and enzymatic approaches Well defined and good laboratory procedures are essential to control PCR contamination and should be implemented prior to testing clinical samples Amplification Procedure Note To
27. r Observations V520F Polymorhism 1584G gt A in exon 12 The 1584G gt A polymorphism has been identified to interfere with amplification of the V520F mutant and wild type sequence Reduced peak height at position 12 V520F will be observed in the B mix in an individual heterozygous for the 1584G gt A polymorphism Peak 12 will drop out of the B mix in an individual homozygous for the1584G gt A polymorphism An individual who is heterozygous for the F508del mutation and also heterozygous for the 1584G gt A mutation will exhibit only one peak 12 in the B mix rather than the expected two peaks at position 12 normally seen in a F508del heterozygote see example below Peak 12 will drop out of the A mix in an individual heterozygous for the V520F mutation and the 1584G gt A polymorphism on the same allele such a result has not been observed to date and is likely to be a rare combination CF2EUBYEN 002 Sep 2014 Page 21 of 24 Elucigene CF EU2v1 Instructions for Use Cross Reactivity Every effort has been taken during test development to avoid interference of test function by the presence of other reported polymorphisms and mutations in the CFTR gene The rare mutation R1283M has been evaluated for cross reactivity and was not detected by the Elucigene CF EU2v1 kit In addition the following polymorphisms were not detected by the test 1655T G F508C 1651A G Evaluation of known mutations and polymorphisms in the CFTR gene has highlighted the
28. sualised as red amplicon peaks CF2EUBYEN 002 Sep 2014 Page 4 of 24 Elucigene CF EU2v1 Instructions for Use Warnings and Precautions 1 The DNA Control supplied with this kit is of human origin and has been independently tested using a PCR based assay and found to be negative for Hepatitis B Virus HBV Hepatitis C Virus HCV and Human Immunodeficiency Virus 1 HIV1 Care should be taken when handling material of human origin All samples should be considered potentially infectious No test method can offer complete assurance that HBV HCV HIV 1 or other infectious agents are absent Handling of samples and test components their use storage and disposal should be in accordance with the procedures defined by the appropriate national biohazard safety guideline or regulation In line with current good laboratory practice laboratories should process their own internal QC samples of known genotype in each assay so that the validity of the procedure can be assessed If kit box is damaged there maybe be damage to the contents do not use the kit contact Customer Service CF2EUBYEN 002 Sep 2014 Page 5 of 24 Elucigene CF EU2v1 Instructions for Use Symbols Used on Labels The symbols used on all labels and packaging conform to the harmonised standard ISO 15223 Manufacturer Number of tests E lt E See Instructions for Use Store below temperature shown Use before date shown Catalogue code a K LOT
29. the mutants amplified by the A primer mix with the exception of F508del the normal allele which is amplified by primers included in the A primer mix This mix also contains primers for identification of 2 hypervariable STR markers 404474 450044 2 x 400ul 75ul vials of PCR Master Mix containing HotStart Taq DNA Polymerase and deoxynucleotide triphosphates in buffer 404480 450045 1 x 50ul vial DNA Control at 6ng ul normal for the mutations detected by Elucigene CF EU2v1 404489 Materials Required but not Provided Laboratory consumables gloves screw capped microfuge tubes 0 2m PCR vials or microtitre plates recommended by the manufacturer of the thermal cycler used pipette tips DNA Preparation QlAamp DNA Mini Kit Qiagen GmbH Cat No 51304 51306 or equivalent Capillary Electrophoresis GeneScan 600 LIZ size standard ABI Cat No 4866589 GeneScan 600v2 LIZ size standard ABI Cat No 4408399 Multi capillary DS 33 dye set G5 matrix standard ABI Cat No 4345833 POP 7 Polymer ABI Cat No 4352759 10x Genetic Analyzer Buffer ABI Cat No 402824 and Hi Di Formamide ABI Cat No 4311320 Note Ensure that all materials used are within the manufacturer s stability dating Equipment Required Laboratory equipment precision pipettes 2 sets 1 for pre amplification and 1 for post amplification handling protective clothing vortex mixer microfuge 96 well microtitre plate centrifuge PCR Amplification
30. time e g CFEU2 DDMMYYYY On the 3130 the Results Destination parameters should be set so that the sample name is included in the fsa file name e g CFEU2 DDMMYYYY_1234 5 A_A01 CF2EUBYEN 002 Sep 2014 Page 13 of 24 Elucigene CF EU2v1 Instructions for Use Meere ronpi r Sample File Name Format Example 123456789_Sample3_A12 lt ext gt Filename is greater than 13 characters Prefix Name Delimiter _ Format z ID El sample Name E well Position E lt none gt Suffix File Extension lt None Run Folder Name Format Example PADiagnostics DevelopmentiDiag Dev 3100 Projects CF HNCF UK3130 runsisinstr Prefix Name Delimiter _ v Format snone gt On the 3500 the file name convention should be set so that the sample name is included in the fsa file name e g CFEU2 DDMMYYYY_1234 5_ A_A01 Fine Crosa thoa Nis Pane Coamererary Ga Seay a Fie Name Comontios Ss hiom n a roparmd taht Prosts s urepe vera e o 4 At a Linton vate ta avadabte strba toprak Ekme Interpretation of Results During data collection PCR fragments will be observed as either Blue mutant or Green wild type peaks on the Raw Data electropherogram An individual has two copies of the CFTR gene Where these copies have the same sequence for any given site an individual is described as being homozygous for this site Where the copies differ in sequence at a giv
31. tions and sample collection devices are compatible with this test Blood samples should be stored at 20 C prior to preparation of DNA Avoid repeated freezing and thawing Preparation of DNA from Whole Blood EDTA Samples Results are consistently obtained with DNA extracted using the QlAamp 96 DNA Blood Kit or the QlAamp DNA Mini Kit using Proteinase K following the protocol as described in the QlAamp Handbook starting with 200ul liquid whole blood and eluting in 200ul of molecular biology grade water Preparation of DNA from Dried Bloodspots Results are consistently obtained with DNA extracted using the QlAamp DNA Mini Kit following the protocol as described in the QlAamp Handbook but starting with 2 x 3mm discs from a dried blood spot and eluting in 100ul of molecular biology grade water It is recommended that alternative DNA extraction methods and sample types are thoroughly evaluated with the Elucigene CF EU2Vv1 test prior to the results being used for diagnostic purposes Important Considerations DNA Amount Under optimal PCR conditions and using the recommended sample injection settings see note in Capillary Electrophoresis section given in the capillary column run modules acceptable results have been routinely obtained from DNA extracted using the above methods at concentrations between 1 5ng ul and 25ng ul The quantification of DNA is very important the concentration of every DNA sample to be tested should be measured to e
32. wild type genotype this is seen as a green peak at about 167bp There will also be an additional green peak observed at about 164bp 1507del heterozygote sample An 1507del F508del sample will yield a shifted green F508del WT peak at approximately 164bp 3bp less than normal a blue F508del M peak at 166bp and a blue 1507del M peak at about 159bp An 1507del I507del sample will yield a blue peak at approximately 159bp and a single green peak at approximately 164bp 3bp smaller than the normal F508 peak observed when 1507del is not present CF2EUBYEN 002 Sep 2014 Page 17 of 24 Elucigene CF EU2v1 Instructions for Use F508del The presence of an F508del mutation prevents the I507del WT primer working Therefore an individual homozygous for F508del will have no I507del WT peak in the WT mix see below E iji iiil iiif A reduced height 1507del WT peak and 2 peaks 3bp apart at positions 10 and 12 in the WT mix see below will be observed in the B mix results from an individual heterozygous for F508del m 7 Insertions and Deletions Due to the nature of the design of the CF EU2v1 kit the presence of insertions or deletions between two opposing primers will result in size changes to all the amplicon produced between these two primers Therefore in addition to the 50 mutations detected by the CF EU2v1 kit any insertions and deletions within the amplified target sequences can be detected by the change in expected amplicon
33. y progressive obstructive lung disease during adolescence and increasing frequency of hospitalisation for pulmonary disease in early adulthood to recurrent sinusitis and bronchitis or male infertility in young adulthood Most commonly the diagnosis of cystic fibrosis is established in individuals with one or more characteristic phenotypic features of CF plus evidence of an abnormality in CFTR function based on one of the following presence of two disease causing mutations in the CFTR gene or two abnormal quantitative pilocarpine iontophoresis sweat chloride values gt 60 mEq L or transepithelial nasal potential difference NPD measurements characteristic of CF The CFTR mutation detection rate varies by test method and ethnic background In some symptomatic individuals only one or neither disease causing mutation is detectable in some carriers the disease causing mutation is not detectable CFTR related disorders are inherited in an autosomal recessive manner Siblings of a proband with cystic fibrosis have a 25 chance of being affected a 50 chance of being asymptomatic carriers and a 25 chance of being unaffected and not carriers Molecular genetic testing for disease causing mutation s in the CFTR gene is used for carrier detection in population screening programs Prenatal testing is available for pregnancies at increased risk for CFTR related disorders if the disease causing mutations in the family are known Since the discovery of t
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