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Kit Manual - Alere Technologies GmbH

1. 30 DNA QUALITY e 31 PHYSICAL DAMAGE TO THE ARRAY sisse eese eese sese esse sese ese esse e essi esse esa ese serere 31 AMBIGUOUS RES WETS 31 ADDITIONAL INFORMATION 4 esee eene ehe nnne ee 32 WARRANTY 32 DISCLAIMER mmm 32 i 32 LIST OF COMPONENTS FOR SEPARATE ORDER sese ess esses senes ese seen 33 CONTACT asc cosa Ven eun dabas E dude ded dedu DURER 33 Budd ull M 34 UPDATES amp SOFTWARE ees ni ate v o o en Sn nay EN p Eau ev vean SEN EY KY R a NER KY E Yan a Ro peo P Ya Ran aeu 35 APPENDIX 1 FLOW CHART eret etie Pk av In EE TERES RES MR 36 APPENDIX 2 IMAGES FOR TROUBLESHOOTING eee ee eee ee nenne nennen serus sues suene 37 APPENDIX GENE LIST ccsecssssccscnscenescesceessnsssessaesseessnessesseessessaesseessnessessaesseasenessusenessenss 39 www alere technologies com BACKGROUND The ALERE E c
2. ion ion ion ein LEE www alere technologies com UN Rccosiated with Genes Probes SPATE serin protease autotransporters eatA hp eatA 611 EatA serine protease autotransporter of EEZI Enterobacteriaceae SPATE AY163491 2 i Enterobacteriaceae SPATE AY258503 2 Enterobacteriaceae SPATE AF074613 1 Enterobacteriaceae SPATE U35656 1 Enterobacteriaceae SPATE AY552473 1 Enterobacteriaceae SPATE AY604009 1 sigA hp_sigA_611 SigA serine protease autotransporter of Enterobacteriaceae SPATE AF200692 2 tsh hp_tsh_611 Tsh hemoglobin binding protease SPATE AJ223631 1 vat hp_vat_611 Vat haemoglobin protease SPATE AF242872 1 ireA ireA_20 siderophore receptor iron regulated outer membrane protein AF320691 1 AF449498 2 Miscellaneous tir hp tir 4051 6 611 hp tir MPEC 611 translocated intimin receptor consensus hp tir O103H2 611 hp tir O111 611 hp tir O157H45 611 hp tir O157H7 611 hp tir NTH19 611 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 41
3. Always label your ArrayTubes with a laboratory marker at the recommended position Never label them on the bottom or across the data matrix barcode This may cause an error N label HERE Barcode keep it clean Avoid contact of data matrix barcode with organic solvents The ArrayMate needs the information encoded in the data matrix to perform the assay and the analysis afterwards Avoid touching the bottom of the microarray and keep it clean General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent reservoirs Please keep in mind the limited surplus of C1 100 96 We strongly recommend that the liquid is removed by pipetting Fine tipped soft disposable Pasteur pipettes are suited best such as VWR Cat 612 2856 Pasteur pipette plastic with a flexible tip Ce flexible tip Always place the pipette tip at the cavity between the array and the wall of the reagent well If you touch the array surface probes may be scratched off and this may cause an error E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 15 www alere technologies com pipette tip Use the cavity between array and the wall of the tube Do never touch the array array General Remarks the Substrate Precipitating Dye D1 An appropriate amount of substrate precipitating dye should be filled into an Eppendorf tube and taken out of the refrigerator when
4. Linear Amplification and Internal Biotin Labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 labelling reagent is 45 96 e Prepare a Master Mix by combining 4 9 ul of B1 labelling reagent and 0 1 ul of B2 DNA polymerase per sample Add 5 ul of E coli DNA cpna 0 1 0 4 ug ul prepared as described above to 5 ul of the Master Mix 1 2 Do not forget to label the vial e Perform amplification in a pre programmed thermocycler e g Eppendorf Mastercycler gradient with heated lid according to following protocol Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 50 C 50 cycles with 40 sec at 72 C 60 sec at 96 C Cool down to 4 C hold e The amplification products can be stored frozen until usage Please note When using another device some adaptations might be necessary Before starting routine use please test the protocol with a few known reference strains and the control DNA CM supplied with the kit Hybridisation General Remarks Handling of Arrays e Never touch the array surface e Avoid complete drying of the array surface during processing Do not allow it to stay without liquid for more than two minutes E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 14 www alere technologies com TM e Never rinse the wells with distilled water after the hybridisation step only use C2 Washing Buffer
5. e C3 100x HRP Conjugate Store at 2 C to 8 C Surplus 300 96 e C4 Conjugate Buffer Store at 18 C to 28 C Surplus 500 e C5 Washing Buffer 2 Store at 18 C to 28 C Surplus 140 e D1 Horseradish Peroxidase Substrate Store at 2 C to 8 C Protect against direct sunlight Surplus 200 e Optional cM Reference DNA from coli EDL933 GenBank accession number NC_002655 2 0 1 0 4 ug ul Store at 2 C to 8 C Sufficient for 5 6 tests Components required but not provided e Growth mediafor the cultivation of E coli The test should be performed with colonies harvested from 2xTY or Columbia blood agar Liquid media should not been used because contaminations or mixed cultures cannot be ruled out easily e Equipment and consumables needed for the cultivation of E coli incubator inoculation loops Petri dishes e DNA preparation kits The assay has been tested with the DNeasy Blood amp Tissue Kit from Qiagen 69504 and High Pure DNA Isolations Kit from Roche Cat 11796828001 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 8 www alere technologies com Please note The DNA specimen needs to be free of RNA Recommendation a pre treatment using the cell lysis components A1 A2 see below or a standard RNase A treatment during DNA preparation e 1 5 e RNAse A we recommend Qiagen s RNase A solution 100 mg ml Qiagen Cat 19101 e Equipment n
6. lt 37 C The assay components with limited stability are D1 and C3 The other kit components have proven to be stable six months post expiry Cell Lysis optional e 1 Lysis Buffer Cat 245101000 Store at 18 C to 28 C ambient temperature Surplus 200 e 2 Lysis Enhancer lyophilised Cat 245102000 Store at 18 C to 28 C ambient temperature Centrifuge A2 tubes shortly prior to opening Add 200 ul BufferA1 to Lysis Enhancer before use Mix well and store forlessthan 1 week at 2 C to 8 C Sufficientfor96 isolations DNA Labelling and Amplification e B1 Labelling Buffer Store at 2 C to 8 C Surplus 45 96 e B2 Labelling Enzyme Store at 2 C to 8 C Surplus 300 96 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 7 www alere technologies com Hybridisation and Detection e ArrayTubes 10 x 5 samples protected against light and sealed under inert gas Store at 18 to 28 After opening tubes are to be used within two weeks Close unused ArrayTubes protect them against humidity and dust and store in the dark Avoid ANY touching or scratching the microarray on the bottom of the vial Please note Do not store or handle unused wells above 60 relative humidity since this may irreversibly corrode the spots e C1 Hybridisation Buffer Store at 18 C to 28 C Protect against sunlight Surplus 100 96 e C2 Washing Buffer 1 Store at 18 C to 28 C Surplus 140 96
7. 14 09 46 2015 01 26 14 18 28 2015 01 30 13 03 40 2015 01 30 13 24 09 EJ ED EH gt Archive 5 8 8 If you click on the plus symbol left on the the folder opens and you will see a list of the individual arrays ordered by Sample ID Browse i Search experiment sample ID position assay image raw data results AT 06 ad 2015 03 23 15 06 34 E coli Bio E coli Bio D 1 050202 x x x 9 2015 03 23 15 09 07 P E coli Bio E colBio D 2 050202 x x x New Run 2015 03 23 17 06 30 E coli Bio E coli Bio D 3 050202 x x x 89 2015 03 23 17 59 20 E coli Bio E coli Bio D 4 050202 X x x 2015 03 27 09 37 26 E coli Bio E coli Bio D 5 050202 x x x 10 00 41 E coli Bio E coli Bio D 6 050202 x x x Archive AT 06 E coli Bio DNA virgen 018 UI 007807 E coli Bio DNA virgen 038 UI 007961 2 E coli Bio DMA virgen 084 BIK 014064 21 E coli Bio DNA virgen 73 260315 4 E coli Bio DNA virgen EDL933 ATCC7009 E coli Bio DN virgen E2348 69 261 E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 24 www alere technologies com Activate the tab results top left and click onto the position of an individual experiment the report of this particular array will appear on the right side of the window T New Run Archive Please Note Browse A Search 2015 03 23 15 06 34 2015 03 23 15 0
8. hp iha 611 nfaE 10 hp saa 611 bfpA 10 cfa c 10 cofA 10 f17 A 40 f17 A 50 f17 A 60 f17 G 20 fanA 10 K88ab 10 ngA 20 hp IpfA 611 perA 10 perA 20 prfB 30 5 gt Co I to I i sfaS 10 hp cif 611 p espA 2 611 hp espA O hp espA O127H7 611 espA 157 11 611 hp espA O49H12 611 hp espA O55H7 611 hp espA O8 611 hp espA Crod 611 7 hp_espC_611 hp_espF_611 hp_espF_612 hp_espF_Crod_611 hp espF O103H2 611 hp espF O103H2 612 hp espl 611 hp 5 611 hp esp 612 hp etpD 611 hp n hp n hp n eA 611 612 hp nleA 613 eA 614 eB 611 nleB O157H7 611 nleB Styp 611 o _ _611 _ _611 _612 hp eaaA 611 05 16 04 0006 VO6 Manual E coli Genotyping Kit 40 an outer membrane protein important for the attachment to host cells pathogenesis factor M58154 1 lymphocyte inhibitory factor A adherence factor AF159462 2 EspB protein type lll secretion system 0157 H7 BA000007 2 EspB protein type Ill secretion system 26 and O15 H AJ287768 1 chaperone protein required for the expression of aggregative adherence fimbria S61968 1 STEC autoagglutinating adhesin AF399919 3 BfpA protein essential for apoptosis signalling 24946 1 outer membrane usher protein 5566 1 1 major pilin subunit CFA III pilin D37957 1 major fimbrial subunit protein L770
9. D1 substrate If the solution turns green within 3 5 seconds the horseradish peroxidase still has sufficient enzymatic activity Enzymatic reaction is inhibited by carryover of buffer C1 Ensure proper washing with buffer C2 of the wells to remove all of buffer C1 prior to adding horseradish peroxidase conjugate If the staining control has Passed referto the following hints Image Quality In case of poor image quality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel In order to determine whether any problems originated from the DNA preparation you might perform an experiment with the Control Material This is genomic DNA from E coli EDL933 GenBank accession number NC 002655 2 It is be provided free of charge upon request If the control experiment yields a valid result and a correct identification of probes gapA ihfA and gad there was probably an issue with DNA preparation If the control experiment also fails an error affecting later steps a degradation of reagents from later steps is likely See also Appendix 2 Images for troubleshooting p 35 and 36 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 30 www alere technologies com DNA Quality The amount of DNA is crucial because of the linear kinetics of amplification see Introduction DNA should be free of RNA as free RNA reduces the efficiency of amplification and
10. The Software installation can be tested with the unprocessed Array by the following steps 1 Log on to the ArrayMate in User R amp D Mode default password abcde and start a New Run 2 Choose automatic detection in Experiment Infos and press Next Place the ArrayTube rack with an unprocessed ArrayTube into the ArrayMate than press Next After the Experiment Run the ArrayMate automatically enters the Archive mode and displays the results of the last experiment 3 Each cell of the columns image raw data and results must contain an X Otherwise please retry the installation process of the AssayPlugin and the installation software SDK E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 6 www alere technologies com expe sample ID position assay image raw data results 01 A 1 050202 x x x 01 2 050202 x x x 01 3 050202 x x x 01 0 4 050202 x x x D1 E 5 050202 x x x D1 F 6 050202 X x x Kit Components Storage and Stability All reagents are provided in surplus see below If necessary all components may be ordered separately please refer to the catalogue reference numbers Cat at the end of this manual For pricing please contact your local representative or our customer service respectively The expiry date can be found on each bottle and on the outer packaging All components have been stability tested for short term shipment lt 1 week at ambient temperature
11. User for full access to test specific software default password abcde If you log in as User you will obtain only raw values but neither positives negatives interpretation nor strain assignment The Administrator log in default password 12345 will allow the installation of a new assay specific plug in which can be downloaded at http alere technologies com e The user interface will be loaded ArrayMate performs internal testing This will require slightly less than a minute e Click on the icon New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience Worklist A Worklist file allows an identifier such as laboratory or sample number to be linked to the respective array position on the ArrayStrip For privacy reasons arrays should not be identified by patient names Worklists can be generated using spreadsheet software such as EXCEL see below but must be saved in the txt file format that can be imported into the test specific ArrayMate software Do not use special characters such as V etc e Create a list with at least three columns that have headers written in the first line The following headers are obligatory in this order position samplelD assayID Table 1 e Positions are consecutively numbered from 1to a maximum of 6 Do not leave empty
12. coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 37 www alere technologies com Signal intensity is too low This could be due to low DNA concentration fragmented DNA ethanol trace contaminations in DNA sample or expired reagents The experiment should be repeated with a new DNA preparation If this also fails try an experiment with EDL933 control DNA CM available on request Chip was not in focus during Repeatimage acquisition after fitting the image acquisition E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 38 ArrayTube in the frame www alere technologies com APPENDIX 3 GENE LIST Accosiated with Description Family Genus and Species Genes gad 10 glutamate decarboxylase AE014075 1 Family Genus and Species Genes gapA prob gapA 611 glyceraldehyde 3 phosphate dehydrogenase A i cil jM crates Family Genus and Species Genes ihfA prob_ihfA_611 integration host factor subunit alpha i Family Genus and Species Genes ipaH9 8 ipaH9 8 20 invasion plasmid antigen AF047365 1 pue so 7979 astA astA_consens_10 heat stable enterotoxin consensus sequence Toxin cdtB cdtB_40 cdtB_50 cdtB_60 cytolethal distending toxin subunit B AJ508930 1 celB celb_10 colicin lysis protein X03632 1 Toxin cnf1 20 cytotoxic necrotizing factor type 1 CP000243 1 dd ItcA ItcA 20 heat labile enterotoxin subunit A AB011677 1 Toxin mchF
13. freeware programs are e Alere Result Collector for the conversion of multiple result xml files from the ArrayMate into spreadsheet tables This should make it easier to compare isolates or to determine relative abundances of genes or strains etc Alere Worklist Generator is a tool which helps you to create a well formatted worklist for the Arraymate e Alere Report Generator is a software tool to create reports using the assay software normally used and installed on the ArrayMate It uses an image taken by the ArrayMate or a txt file raw signal data file and generates a report from the raw signal data E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 35 www alere technologies com APPENDIX 1 FLOW CHART prepare ArrayTubes prepare DNA pro cessing time over Grow CLONAL bacteria isolate GENI night not part of the kit m isolate genomic DNA not part of the kit label RNA free DNA in thermocycler rinse ArrayTubes 5 ul DNA 0 1 0 4 ug ul 500 ul water 55 550 rpm 2 min plus MM 4 9 ul B1 0 1 ul B2 i i discard water preparing labeled DNA 500 ul Buffer C1 55 550 rpm 4 min to 10 ul of labeled DNA add 90 ul of discard C1 process promptly Buffer C1 transfer 100 pl labeled DNA to ArrayTube Barcode keep it clean hybridise 55 C 550 rpm 60 min i discard labeled DNA incubate twice in 500 ul Buffer C2 30 C 550 rpm
14. lines in the worklist If you use EXCEL position numbers should be entered into column A e Sample IDs are strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patients names should not be used as sample IDs e Assay ID allows the system to identify the current test and to correctly use information on layout spot number and identity etc The E coli Genotyping Kit has the Assay ID 050202 Please note When entering assay IDs manually make sure to enter the correct number as entering wrong numbers could lead to errors or loss of data E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 21 www alere technologies com e Werecommend using a printout of the worklist as template for pipetting e Save the worklist as tab separated txt file on the memory stick provided together with the ArrayMate e To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted e You may use the software tool Worklist Generator to create a worklist easily http alere technologies com en products lab solutions software tools worklist generator html Table 1 Example worklist Please note Table header must be written exactly as shown EDL933 ATCC700927 260315 050202 CFTO73 260315 050202 084 BIK 014064 260315 050202 038 UI 007961 260315 050202 6 EPEC E2348 69 260315 050202 E 0140
15. lose its activity at 55 C Do NEVER incubate above 30 C Make sure that the thermoshaker has cooled down before mounting the ArrayTubes Please keep in mind the limited surplus of 300 96 e Add 100 ul of prepared C3 C4 mixture to each tube e Incubate in the thermoshaker at 30 550 rpm for 10 minutes e Remove and discard C3 C4 mixture completely Washing step after binding of conjugate addition of HRP conjugate e Please keep in mind the limited surplus of C5 140 96 e 1 Washing step after conjugation add 500 ul of buffer C5 incubate in the thermoshaker at 30 550 rpm for 1 minute remove and discard the washing solution e 24 Washing step after conjugation Repeat 1 Washing step Please note A carryover of more than 0 5 of C3 C4 into the following staining reagent will create black particles which in the worst case may mimic signals hybridised spots On the same time real signals may appear pale due to competition of soluble Horseradish Peroxidase with the DNA bound enzyme for substrate molecules E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 19 www alere technologies com Staining of bound HRP conjugate Please note Do not move ArrayTubes during staining The reagent D1 contains a substrate for Horseradish Peroxidase Please keep in mind the limited surplus of D1 200 96 e Setthe thermoshaker to 30 C for the following steps e Add 100ul pre warmed reagent D1 to each
16. mchF 10 putative microcin L transport protein AJ515252 1 Toxin stb stb_10 heat stable enterotoxin Stb enterotoxinB M35729 1 hp subA 611 subtilase cytotoxin subunit A AF399919 3 hp toxB 611 hp toxB 612 hp toxB 613 cytotoxin B 11549 2 Toxin virF virF_20 transcriptional regulator required for transcription of virB and icsA AF348706 1 Shiga Toxin stx1A_10 shiga toxin 1 stx2A_10 or shiga toxin 2 variant d X61283 1 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 39 o ajs 8 www alere technologies com Accosiated with adhesins eae consensus adhesins saa dac cofA HT A imbrae imbrae imbrae espB_0157 espB_026 fasA fedA fedF fim41a ih fanA K88ab ngA pfA perA imbrae imbrae imbrae imbrae secretion systems cif secretion systems m X sfaS secretion systems secretion systems espA C rodentium espC espF 5 pF C rodentium espF O103H2 secretion systems secretion systems secretion systems secretion systems secretion systems etpD 2 R R o c n nleB 0157 H7 secretion systems eB Salmonella secretion systems secretion systems SPATE serin protease autotransporters n tccP eaaA oO E coli Genotyping Kit eae_consensus_10 eae consensus 20 eae consensus 30 eae consensus 40 hp 1 611 espB 157 20 espB O26 40 fasA 10 fedA 10 fedF 10 fim41a 10
17. results b is not active with this assay Activate the tab raw data top left and the raw signal results of this particular array will appear on the right side of the window Browse Q Search New Run Archive E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 25 results results b raw data segmentation image image Spot ID Substance Background Confidence Mean Signal Valid 2015 03 23 15 06 34 1 K88ab 10 0 867286 0 613636 0 871648 0 004735 0 2015 03 23 15 09 07 10 cfa c 10 0 871896 0 821385 0 871714 0 000196 0 2015 03 23 17 06 30 100 hp nle 612 0 861177 1 000000 0 344314 0 565015 0 2015 03 23 17 59 20 101 hp nleA 613 0 853595 1 000000 0 081250 0 851798 0 2015 03 27 09 37 26 102 hp nleA 614 0 868093 1 000000 0 660599 0 225017 0 i e 10 00 41 103 hp nleB 611 0 851541 1 000000 0 072371 0 861398 0 E col io DNA virgen 018 I 007807 M 104 hp nleB O 0 854489 1 000000 0 110279 0 819910 0 E col Bie DNA virgen 038 UI 007961 2 105 hp nleB St 0 868421 0 821385 0 872383 0 004295 0 E coli Bio DNA virgen 084 014064 2 106 hp 611 0 839869 1 000000 0 076425 0 855742 0 E coli Bio DNA_virgen_CFT073 260315 107 hppic 611 0 870417 0 710477 0 869528 0 000961 0 E coli Bio DNA virgen EDL933 9 108 hp rpeA 611 0 874510 0 892006 0 874778 0 000288 0 E coli Bio DNA viraen EPEC E2348 69 261 109 hp saa 611 0 872097 0 910006 0 8749
18. than version 4 4 Software Installation For analysis of the final image of the DNA microarray on the ArrayMate specific software plug in is required This software plugin can be downloaded from our website www alere technologies com under Downloads plug ins Please install it on your reader according to the following instructions E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 4 www alere technologies com Assay Plugin and SDK for the ArrayMate The following instruction describes the installation of the AssayPlugin and ArrayMate installation software SDK 1 Download the AssayPlugin and the ArrayMate SDK from http alere technologies com en products lab solutions e coli e coli genotyping kit html 2 Copy downloaded files Plugin and SDK setup to an USB Memory Stick and connect it to the ArrayMate 3 Log as user admin to the ArrayMate default password 12345 4 Open the Windows Explorer and navigate to the downloaded setup files T My Computer File Edit View Favorites Tools Help Q amp 2 Search Folders Address 3 My Computer Al Name System Tasks Files Stored on This Computer Mesi nd Shared Documents File Folder B Caadmin s Documents File Folder programs user s Documents File Folder g Change a setting user R amp D s Docu File Folder 559 Eject this disk Hard Disk Drives Se System C Local Disk Data D Local Disk
19. the individual markers on the E coli Genotyping Test Report the result can also be ambiguous In cases affecting virulence factors no definitive answer with regard to this specific marker can be given This can be caused by poor sample quality poor signal quality and especially in some resistance associated genes by the presence of plasmids in low copy numbers E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 31 www alere technologies com TM Allelic variants of some markers differ only in single or few nucleotides This can cause the effect that the actual allele yields a positive signal while other mismatching probes give ambiguous rather than negative results ADDITIONAL INFORMATION Warranty Alere guarantees the performance as described in this manual Usage of the Assay was successfully tested at ambient temperatures up to 37 A guarantee is limited to ambient temperatures in the laboratory between 18 and 28 Kit components comprise the arrays the Lysis Enhancer the reagents for DNA labelling and for detection of labelled DNA products on the array the ArrayMate reader and its software In case one of these components fails within the expiry date due to other reasons other than misuse contact Alere for replacement or refund Terms and conditions apply If you have any problem or question please contact the technical service Disclaimer This system is for research use only
20. well supernatant of centrifuged D1 without precipitate e Incubate at 25 C WITHOUT agitation for 10 min e Read out WITHOUT removing D1 removal of D1 would leave air bubbles within the tubes Please note The ArrayTubes as used in this kit do have a different geometry than the 8 well ArrayStrips that are used in other kits Therefore unlike the directive for ArrayStrips D1 is NOT to be removed from the ArrayTubes before reading Check immediately all images for cleanliness i e absence of dust particles residual liquids and for good focus Dust particles and residual fluids inside the vial can be removed by cautiously washing twice with 200 ul PCR grade distilled water If necessary scan and process again Data Analysis Starting the ArrayMate Reader e We recommend starting the ArrayMate Reader after starting the hybridisation this allows the convenience of starting the device and importing the worklist file see below e Please note that this is a short instruction only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate main switch on the rear below the electric cable plug operating switch on the bottom left corner of the front side e Switch on the screen switch right hand below the screen E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 20 www alere technologies com TM e Log in as R amp D User Research and Development
21. 0 n Folder My Computer E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 28 www alere technologies com TM e Click My Computer then Removable Disk and choose the folder where to save or click Make New Folder on the bottom a new folder icon appears e Rename the new folder e g with the experiment name or date e Click Ok data are exported into the new folder on your memory stick e Do NOTremove the memory stick as long as the hourglass symbol is visible e Switch off the device by clicking Power at the bottom left on the usen e Switch off the screen There is no need to physically switch off the ArrayMate Reader E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 29 www alere technologies com TROUBLESHOOTING In case of trouble always make sure that reagents are within the recommended shelf life and stored under appropriate conditions Should you encounter a problem we will always be happy to support you Please e mail to cct home Qclondiag com and include a description of the problem as well as the array images bmp files in question Staining Control A staining control is included to check whether possible problems originate from the hybridisation or the staining procedure If the staining control has Failed proceed as follows Horseradish peroxidase conjugate may have degraded during storage Add 1 ul mixture C3 C4 to 9 ul
22. 103 105 07749 Jena Germany Contact If you require any further information on this product please contact cct home clondiag com E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 33 www alere technologies com LITERATURE 1 Development of a miniaturised microarray based assay for the rapid identification of antimicrobial resistance genes in Gram negative bacteria Batchelor M Hopkins KL Liebana E Slickers P Ehricht R Mafura M Aarestrup F Mevius D Clifton Hadley FA Woodward MJ Davies RH Threlfall EJ Anjum MF Int J Antimicrob Agents 2008 Feb 1 2 Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain Muna F Anjum Suman Choudhary Victoria Morrison Lucy C Snow Muriel Mafura Peter Slickers Ralf Ehricht and Martin J Woodward J Antimicrob Chemother 2011 For further literature please referto http alere technologies com en science technologies publications E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 34 www alere technologies com UPDATES amp SOFTWARE Notifications on database software updates and freeware tools can be found at http alere technologies com en products lab solutions e coli e coli genotyping kit html http alere technologies com en products lab solutions software tools html and or http alere technologies com en news html Currently available
23. 37 0 003066 0 www alere technologies com Interpretation of the raw data list e SpotlID numerical identifier of the spot on the array e Substance name of the DNA probe e Confidence an intrinsic estimate of spot confidence based on size and shape of that particular spot where 1 high confidence and 0 no confidence e Signal spot signal intensity grey scale value where 1 black and 0 white e Valid valid 1 invalid confidence below 0 75 e Background luminous intensity of the background where 1 maximum brightness and 0 maximum darkness e Mean luminous intensity of the signal spot where 1 maximum brightness and 0 maximum darkness Please note The correlation between mean background signal is roughly 1 mean background however there are some correction factors that depend on the statistics of pixel distribution E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 26 www alere technologies com TM Activate the tab segmentation image and the analysed picture of this particular array will appear on the right side of the window p Browse Q Search results results b raw data segmentation image image 2015 03 23 15 06 34 2015 03 23 15 09 07 NewRn 2015 03 23 17 06 30 A 2015 03 23 17 59 20 2015 03 27 09 37 26 Archive 2015 03 27 10 00 41 AT 06 E col
24. 5 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrat D1 25 i discard Buffer C2 incubate in 100 ul C3 C4 conjugate 30 C 550 rpm 10 min i discard C3 C4 conjugate incubate twice in 500 ul Buffer C5 30 C 550 rpm 1 min discard Buffer C5 incubate with 100 ul Substrate D1 25 C 10 min Quantifoil BioShake iQ or Eppendorf Thermomixer take image WITHOUT removing D1 analyse 5 min 2 min MM MasterMix total time requirement over night app 120 min 3 with heating block for 1 5 ml Eppendorf tubes 7 8h The figure on this page summarises the test procedure However please refer to the text section of this manual at any step of the test protocol for further important details E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 36 www alere technologies com APPENDIX 2 IMAGES FOR TROUBLESHOOTING Valid experiment Valid results no error messages The bottom of the AT is contaminated with dust Please clean the bottom of the well scan particles and process again Ifthe microarray surface is contaminated with particles wash the microarray with double distilled water pipetting water carefully up and down remove scan and process again The microarray surface is contaminated with dust particles The bottom of the AT is contaminated with a liquid Please clean the bottom surface with a e g buffer cleanroom wipe scan and process again E
25. 64 B Data Acquisition in the ArrayMate Reader e Insert your memory stick containing the worklist into any of the USB ports down to the right hand side of the ArrayMate Press folder selection dialogue will open e Select your worklist path My Computer Removable Disk e Open your selected worklist by pressing Enter or Open e Press your imported worklist opens in a separate window Proofread If the new window is empty or if it was the wrong worklist repeat the import e Press OK the worklist window will close E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 22 www alere technologies com TM e Leave the memory stick in the ArrayMate if you intend to export E coli Genotyping Test Reports afterwards check the memory stick for computer viruses and malware using an appropriate program on a regular basis e Press Next at the bottom right on the screen reader is opening e Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply strong force Assure proper fit otherwise the images may be out of focus e After having inserted the adapter carefully insert the Array Tubes into the adapter e ArrayTubes need to be open with tube lid connections placed into appropriate notches e Assure proper fit otherwise the images may be out of focus e Barcodes ArrayTubes and holder must be clean e Press Next at the bottom right on the sc
26. 9 07 2015 03 23 17 06 30 2015 03 23 17 59 20 2015 03 27 09 37 26 2015 03 27 10 00 41 AT 06 E coli Bio DNA virgen 018 UI 007807 E coli Bio DNA virgen 038 UI 007961 2 E coli Bio DNA virgen 084 014064 21 E coli Bio DNA virgen CFT073 260315 EI 9 E c io DNA virgen E2348 69 261 2015 03 27 10 04 06 AT 02 E coli Bio DNA virgen EDL933 ATCC7009 E coli Bio DNA virgen LW 260315 565 2015 03 27 11 16 35 01 A 01 01 01 0 01 01 01 6 O1 H 02 4 results results b raw data segmentation image image print For Research Use Only Not For Use In Diagnostic Procedures Operator ines Sample ID E coli Bio DNA virgen EDL933 ATCC7UD927 260315 Experiment ID E coli Bio DNA virgen EDLS33 ATCC7UD927 260315 CFO1C784 8117 46A9 A14A 47 C239 C85AB2 Date of Result Fri Mar 27 10 01 59 2015 Assay Name Ecoli Genotyping AT 3 Assay ID 050202 Well Position 01 01 4 Software Version 2014 04 08 Device 0440023 Controls ee Ss Sa ary control result explanation Failed indicates possible problems originating staining control passed from the hybridization or the staining procedure see troubleshooting section in the manual Failed indicates possible problems originating negative control passed from the staining procedure see troubleshooting section in the manual the flag
27. 91 1 major fimbrial subunit protein pilin G L43372 1 L4 ma or pilu subunit operon regulatory protein adhesin minor Shigella fimbriae subunit cell cycle inhibiting factor type IIl secretion EspA protein type Ill secretion system AF054421 1 EspA protein type Ill secretion system associated with Citrobacter rodentium AF311901 1 EspC extracellular serine protease type Ill secretion system AF297061 1 EspF effector protein type Ill secre system AE005174 2 EspF effector protein type Ill secre system AF311901 1 EspF effector protein type Ill secre system AJ277443 1 Espl non LEE encoded effector prot EPEC Locus of Enterocyte Effacemen AJ278144 1 EspJ non LEE encoded effector protein LEE EPEC Locus of Enterocyte Effacement AE005174 2 EtpD type Il secretion pathway related NleA non LEE encoded effector protein A type Ill secretion system AM42 1997 1 NleB non LEE encoded effector protein B NleB non LEE encoded effector protein B type Ill secretion system assodated with serotype O157 H7 BA000007 2 NleB non LEE encoded effector protein B type Ill secretion system assodated with Salmonella enterica AE008894 1 NleC non LEE encoded effector protein C type Ill secretion system AY485823 1 Tir cytoskeleton coupling protein type Ill secretion system AB275113 1 EaaA serine protease autotransporter of Enterobacteriaceae SPATE AF151674 1
28. Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 3 www alere technologies com DEVICES SOFTWARE AND REAGENTS e ArrayMate Reader to be ordered separately for details see below e Alternatively Reader ATRO3 to be ordered separately for details see below e conoclust software provided with the reader e Report Generator optional Whilst the E coli Genotyping assay runs both on the ArrayMate Reader and the ATRO3 reader respectively this manual describes the reading of processed AT on the ArrayMate reader only If you want to use ATRO3 please refer to the latest version of the ATRO3 manual or contact us Assay specific software plug in is delivered with the reader or can be downloaded from our website where it will occasionally be updated The ArrayMate Reader by default has all software on board However the E coli Genotyping assay specific package might be missing e g if you obtained the device for the use with another assay Then you may needto install it separately It will be provided upon kit order and can also be downloaded from our website as discussed above No issues regarding compatibility of software have been observed with the ArrayMate device The ATRO3 reader requires several pieces of software to be installed on an external PC please refer to the latest version of the ATRO3 manual for details The E coli Genotyping assay specific software is not compatible with iconoclust versions older
29. Other Places My Network Places E My Documents Shared Documents Removable Disk E Control Panel Devices with Removable Storage 5 Start installation double click on setup file of the AssayPlugin fi Genotyping File Edit View Favorites Tools Help c a yo Search Folders EJ Address e Conanitemp_public InesE Genotyping x Name Size Type Date Modified A lArrayMate SDKs exe 468KB Application 4 1 2015 1 03 PM 8 My Documents SSetup ArrayMateAssay D 050202 Ecoli Genotyping AT 3 2014 04 08 exe 322 KB Application 4 1 2015 12 45 PM a 3 My Computer i w System C E C3 Documents and Settings C3 Admin idlerc Application Data NE s E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 5 www alere technologies com 6 The welcome screen of the setup appears 18 Setup ArrayMateAssay R D 050202 virgen3 ecoli m Welcome to the ArrayMateAssay D 050202 virgen3_ecoli Setup Wizard This will install ArrayMateAssay_R_D 050202 virgen3_ecoli 2014 04 08 on your computer It is recommended that you close all other applications before continuing Click Next to continue or Cancel to exit Setup 7 Follow the instructions and press Finish to complete the installation 8 Repeat this process for the SDK Setup 9 Log off and log in again as User R amp D default password abcde Test the AssayPlugin
30. We do not accept any liability for damages caused by misuse Furthermore we do not accept any liability for damages caused by inappropriate use of the device as a personal computer for instance related to the use of additional software to network connections or to a breach of privacy related to the storage of confidential information such as names of patients on its hard disk and or to the use of external storage devices that might be contaminated with spyware Quality Control Each batch is stringently tested with the use of standard DNA preparations for good performance and correctness of results E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 32 www alere technologies com List of Components for Separate Order If required these reagents may be ordered separately amount component Lysis Buffer 245101000 18 28 Lysis Enhancer 245102000 18 28 Labelling Buffer 360ul 245403000 Labelling Enzyme 20 ul 245104000 Hybridisation Buffer 245105000 18 28 Washing Buffer 1 120ml 245106000 18 28 HRP Conjugate 100x 200ul 245107000 Conjugate Buffer 245108000 18 28 C Washing Buffer 2 120ml 245109000 18 28 Control Material 30 yl onrequest 2 8 C E coli EDL933 DNA ArrayTubes Identibac Ec 03 201009288 15 28 For pricing please contact your local representative or our customer service respectively Legal Manufacturer Alere Technologies GmbH Loebstedter Str
31. a single clone The DNA specimen needs to be free of RNA and it should not be fragmented This can be determined by agarose gel electrophoresis DNA should not be prepared by disrupting E coli cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the E coli Genotyping Kit are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend following the protocols outlined below The use of automated systems for DNA preparation EZ1 Qiacube Magnapure etc has not yet been systematically evaluated with this assay While there are positive experiences with some of our other assays we recommend testing some known strains for evaluation prior to routine use of these systems Lysis steps and addition of RNase should be performed as described below before loading the samples in an automated system for DNA preparation DNA Extraction via Spin Columns e g Qiagen DNeasy Blood amp Tissue e Add an inoculating loop full of monoclonal colony material of the E coli isolate to 0 2 ml 1xPBS and vortex thoroughly E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 10 www alere technologies com Loopempty Loop full It is important to harvest enough bacteria this is a prerequisite for extraction of a sufficient amount of DNA Take an inoculating loop of 1 mm diameter filled with bacteria as shown in the left pict
32. control spots E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 1 www alere technologies com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not for Use in Diagnostic Procedures This assay allows genotypic characterisation of important virulence genes of E coli It cannot be used for other bacteria than E coli Specifications Upon receipt the assay components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 C to 28 C Technical Support If you require any further information on this product please contact Email cct home clondiag com Phone 49 0 36 41 3111 155 Fax 49 0 36 41 3111 120 For up to date information regarding the kit please visit our website at http alere technologies com en products lab solutions e coli e coli genotyping kit html Safety Precautions e The assay is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from pure E coli colonies clones requires expertise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be adhered to isolated cell free E coli DNA may be processed without further biosafety precautions although contamination with E coli or other bacteria needs to be ruled out e Always wear protective clothing as require
33. d for laboratory work according to your local regulations E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 2 www alere technologies com Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest regulations EC 1272 2008 CLP and 1907 2006 REACH the enclosed reagents do not require a Material Safety Data Sheet MSDS except Hybridisation Buffer C1 The MSDS can be downloaded via our website from any lab solutions product page e g http alere technologies com en products lab solutions html All other reagents do not contain more than 1 9 of a component classified as hazardous and do not contain more than 0 1 of a component classified as carcinogenic Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents If liquid is spilled clean with a disinfectant and or laboratory detergent and water Alere assumes no liability for damage resulting from handling or contact with these products If you have any questions please contact our Technical Support see above Shipping Precautions RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods E coli Genotyping
34. delivered with a surplus of 500 96 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 17 www alere technologies com Pipetting scheme ArrayTubes AT 2 3 ATs 4 6 ATs 7 10 ATs 11 15 ATs 15ul 35 11 ul 16 yl 150 ul 350 700ul 1100 1600 pl e Putaside at room temperature until use Pre warm the staining reagent D1 e Transfer enough reagent D1 intoa separate vessel e g a clean and sterile centrifuge tube 100 ul for each well and a surplus of not more than 20 96 e Putaside at 20 C 25 C until use Washing after hybridisation e Please keep in mind the limited surplus of C2 140 96 e Remove the ArrayTubes from the thermoshaker e Set the thermoshaker to 30 C for the following steps e Carefully open the tubes and remove the hybridisation mixture as completely as possible without touching the array surface e 1 Washing step after hybridisation add 500 ul of buffer C2 and incubate in the thermoshaker at 30 C 550 rpm for 5 min remove and discard the washing solution 24 Washing step after hybridisation repeat 1 Washing step Please note Acarryover of more than 1 of buffer C1 to the next step will denature the HRP E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 18 www alere technologies com Addition of HRP conjugate Please note Reagent C3 contains Streptavidin Horseradish Peroxidase HRP that would denature and
35. eeded for DNA isolation e g pipettes centrifuge thermoshaker or automated device see above e Photometer OD 260 nm for measuring the concentration of DNA e Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA e Thermocycler for PCR e Thermoshaker Please note We recommend the Eppendorf s Thermomixer Comfort equipped with a heating block for 1 5ml tubes e Pipettes suitable for 1 5 ul volumes 90 ul 100 ul 200 ul 1000 ul e Multichannel Pipettes for 100 200 ul e Sterile reaction vials suitable for PCR VWR Cat 732 0098 e Ultrapure PCR grade water e Pasteur pipettes VWR Cat 612 2856 E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 9 www alere technologies com PROTOCOLS Culturing and Harvesting Bacterial Cells E coli strains are potential pathogens All procedures for cultivation of the bacterium and DNA preparation need to be performed by properly trained staff in a biosafety level 2 facility Grow E coli on 2xTY or Columbia blood agar overnight at 37 or 48 h at room temperature Make sure that you have a pure monoclonal culture of E coli Contamination with other bacteria especially with othernon fermenting Gram negative rods needs to be strictly avoided If necessary sub clone and incubate again Extraction of DNA The required sample type for the E coli Genotyping Kit is 0 5 2 ug 0 1 0 4 ug l of intact genomic DNA from
36. es com TM Alternatively prepared DNA can shortly be heated to evaporate ethanol e g 10 min at 70 C with open lid e Check for DNA integrity and absence of RNA e g agarose gel If necessary you might perform another digestion step with additional RNase A not provided Measure DNA concentration method it shouldn t be less than 0 1 ug ul The concentration might be increased by heating and evaporation of water or by using a speed vaccentrifuge DNA Extraction by Heat Lysis Please note Only a fresh overnight culture can be used After DNA extraction by heating the linear amplification must be done immediately Storage of extracted DNA is not recommended e Add 1 inoculating loop Please Note do not use too much culture material see Figure below of a monoclonal E coli isolate to 50 ul PCR grade distilled water and vortex thoroughly e Incubate at 99 C 15 min at 550 rpm in a thermoshaker e Centrifuge for 5 min at 13 600 rpm at room temperature e Carefully pipette 25 ul supernatant into a new 1 5 ml tube and discard the old tube with the pellet Add 0 25 ul RNase A not provided see above with a stock concentration of 1 mg ml e Incubate at 37 5 min at 550 rpm in thermoshaker e Use 5 ul of this DNA suspension for the linear amplification and internal Biotin labelling process E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 13 www alere technologies com
37. i Bio DNA virgen 018 UI 007807 E coli Bio DNA virgen 038 UI 007961 2 E coli Bio DN virgen 084 014064 21 E coli Bio DNA virgen 73 260315 E coli Bio DNA virgen EDL933 ATCC7009 E coli Bio DNA virgen 2348 69 26 8 2015 03 27 10 04 06 AT 02 E coli Bio DNA virgen EDL933 ATCC7009 E coli Bio DNA virgen LW 260315 565 5 2015 03 27 11 16 35 01 01 01 01 01 01 01 6 O1 H 02 A 02 Activate the tab image and the picture of this particular array will appear on the right side of the window Browse 8 Search results results b raw data segmentation image image aj 2015 03 23 15 06 34 8 2015 03 23 15 09 07 NewRun 2015 03 23 17 06 30 v 2015 03 23 17 59 20 25 2015 03 27 09 37 26 E3 2015 03 27 10 00 41 Archive AT 06 E coli Bio DNA virgen 018 UI 007807 E coli Bio DNA virgen 038 UI 007961 2 E coli Bio DNA virgen 084 014064 21 E coli Bio DNA virgen 73 260315 E coli Bio DNA virgen E2348 69 261 S 2015 03 27 10 04 06 AT 02 E coli Bio DNA virgen EDL933 ATCC7009 E coli Bio DNA virgen LW 260315 565 2015 03 27 11 16 35 01 A 01 B 01 C 01 D 01 01 01 6 02 02 8 02 C 02 D 02 E 02 02 6 02 H E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 27 www alere technologies com Export of Te
38. labelling by effectively removing primer from the reaction mix due to competitive hybridisation A260 readings will cover RNA and other contaminants as well Therefore pure DNA preparations without RNA contaminations are a prerequisite for proper DNA concentration measurement RNAse treatment prior to reading therefore is necessary component A2 contains RNAse DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites DNA should for this reason not be prepared by disrupting bacterial cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols We made good experiences with the manual QIAGEN DNeasy kit and the automated device EZ1 DNA must be free of any traces of ethanol as ethanol strongly influencesthe amplification It is possible to heat the sample prior to adding itto the labelling mix 5 10 minutes at 70 Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples see above Physical Damage to the Array Scratching of the array surface with a pipette tip can lead to the damage of array spots that prohibits the acquisition of a valid signal In this case the respective marker is not assigned as negative but instead the message none appears next to the marker name Ambiguous Results Apart from a positive or negative resultfor
39. nown reference strains or the control DNA supplied with the kit E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 16 www alere technologies com Preparation of the hybridisation mixture e Pre heatthe thermoshaker to 55 C e Add 90 ul of buffer C1 to each labelling product mix gently vigorous mixing results in foaming and put aside Pre washing of the arrays 2 washing steps e Remove the ArrayTube from the bag open the bag at its predetermined breaking point e Add 500 ul of ultrapure water to each tube e Incubate in the thermoshaker at 55 C 550 rpm for 2 minutes e Remove and discard the water WITHOUT TOUCHING THE ARRAY SURFACE e Add 500 ul buffer C1 to each tube e Incubate in the thermoshaker at 55 C 550 rpm for 4 minutes e Remove and discard buffer C1 e Proceed promptly hybridisation mixtures must be ready when buffer C1 is removed Hybridisation e Transfer each hybridisation mixture 100 ul to a prepared ArrayTube avoid extensive foaming e Incubate in the thermoshaker at 55 C 550 rpm for 60 minutes Note Meanwhile login to the ArrayMate device and prepare your worklist see section worklist S 21 Dilute Streptavidin Horseradish Peroxidase C3 C4 HRP conjugate e Combine reagent C3 Streptavidin Horseradish Peroxidase and Buffer C4 in a ratio of 1 100 the mixture is stable for 1 day at room temperature C3 is delivered with a surplus of 300 96 C4 is
40. oli Genotyping Kit allows a quick and simple method to detect important virulence genes of Escherichia coli E coli RNA free unfragmented genomic DNA of a pure and monoclonal E coli colony material is amplified approximately 50 fold and internally labelled with biotin 11 dUTP using a linear amplification protocol In contrast to standard PCR a multiplex primer extension reaction is performed with two nested primers per target in each cycle Two versus one primer for each target increase and synchronize the yield of biotinlabelled single stranded ss DNA product for all markers This allows a simultaneous sequence specific labelling and amplification of an essentially unlimited number of targets However sensitivity is lower than in a standard PCR whereas contamination with amplicons is nearly impossible and for that reason the method is restricted to clonal colony material and cannot be performed on samples such as swabs or other patient samples Resulting biotin labelled ssDNA is transferred and hybridised to DNA oligonucleotide microarrays with 124 probes for different genetic markers plus controls of them are spotted in two spots each The array contains markers for family genus and species identification toxins i e shiga toxin fimbrial associated proteins i e bacterial adhesins secretion systems i e type Ill secretion system Serine protease autotransporters SPATE staining controls using biotinylated
41. reen reader closes analysis program starts it takes about 2 10 min depending on the number of ArrayTubes the reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analysis ready e The reader indicates the end of the entire process with an acoustic signal beep e Press Next at the bottom right on the screen reader is opening e Remove the adapter with the ArrayTubes E coli Genotyping Kit 05 16 04 0006 06 Manual E coli Genotyping Kit 23 www alere technologies com e Press Next at the bottom right on the screen reader is closing Results On the left hand side of the screen you will see a list showing all runs stored on the ArrayMate s hard disk A run contains the results from all arrays analysed together within one frame If this list is not displayed e Press Archive left hand side and activate the flag Browse at the top left e runs are organised like folders in Windows Explorer and named by default according to the date of acquisition Example there are several readings in the archive by default they are named by date and time of day of creation which you may have changed see section setup of the ArrayMate reader Browse Q Search TotalSize of D 107 GB FreeSapce on D 98 GB g New Run 2013 12 06 13 25 51 2015 01 26 09 59 25 2015 01 26 13 47 41 2015 01 26
42. so 552285 10 CULTURING AND HARVESTING BACTERIAL CELLS 10 EXTRACTION Wodph nem 10 DNA Extraction via Spin Columns e g Qiagen DNeasy Blood amp 10 DNA Extraction by Heat 13 LINEAR AMPLIFICATION AND INTERNAL BIOTIN eene remesas 14 wife enm 14 General Remarks Handling of ansa ann nnns 14 General Remarks Handling of 1 rne nnn rns 15 General Remarks the Substrate Precipitating Dye 01 16 General Remarks ans ans ann nnns 16 PPS rmt 20 Starting the ArrayMate 20 21 Data Acquisition in the ArrayMate nennen nares nnne 22 imc N A A E E E S E AAA A A A AEE 24 Export Of Test Res lts t b el entia Ooa doa dei iE ATARE 28 TROUBLESHOOTING 5 s eno o ena naue scene conse scssdescesceucavetassveccaesocsocnactocsessosaderersesdascosiss 30 STAINING CONTROL mr ELE 30 eere Aic
43. st Results The generated result files in an html format will show information of all target genes Possible invalid controls that might display inthis report will be explained below see Troubleshooting Other files that are generated and that can be exported include e Atextfile txt with the raw measurements raw data e Animage file bmp with the actual photo of the array e A second image file png in which the coordinate grid is superimposed and the recognised spots are circled segmentation image and e A XML file xml that contains the same information like the html result sheets for future export into databases and for using the Result Collector tool Please note Only complete runs can be exported The export of individual E coli Genotyping Test Reports is not possible e Right click on the selected run a menu appears with the option Export Run Reports e Right click on Export Run Reports a file browser opens ArrayMate Browse 5 expe sample ID order37x order37x23 8 ARCHIVE order37x order37x78 2009 02 05 09 00 42 order37x order37x82 New Run 2x order42x01 dig Browse For Folder ve Order42x02 order42x03 Choose a directory Order42x06 Order42x07 Order78x01 O Desktop Order78x01 j B My Documents Order78x08 3 My Computer Se Local Disk C Local Disk D H S Removable Disk E
44. starting the procedure allowing it to pre warm to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged 1 min 13 000 rpm prior to use to remove bubbles as well as possible precipitates Triggered by peroxidase in case of positive reactions the dye precipitates but it is not covalently bound The precipitate can be dissolved by vigorous shaking Thus the arrays must not be shaken dropped or moved abruptly during the staining procedure and afterwards After completion of staining do not remove reagent D1 and scan immediately The dye precipitate fades slowly in presence of liquids General Remarks Thermoshakers The correct temperature within the vessels is essential therefore always use the appropriate equipment for heating Because of a possibly inhomogeneous distribution of the temperature within the heating block and because of possible differences between displayed and actual temperatures the use of different brands of thermoshakers might affect test performance We tested the assay with BioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayTubes and Eppendorf s Thermomixer Comfort equipped with a heating block for 1 5 ml Eppendorf tubes Thus we recommend the use of either device When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few k
45. terwards e Discard collection tube with liquids e Place the spin column in anew 2 ml collection tube provided with the Qiagen kit e Add 500 ul Buffer AW1 e Centrifuge at room temperature e g 1 min at 8000 rpm e Discard collection tube with liquids e Place the spin column in anew 2 ml collection tube provided with the Qiagen kit e Add 500 ul Buffer AW2 e Centrifuge at room temperature the membrane of the spin column should be dry and all liquid should be in the collection tube e g 3 min at 14000 rpm e Discard collection tube with liquids e Place the spin column in a clean 1 5 ml tube not provided with the Qiagen kit e Add 50 ul Buffer AE or PCR grade distilled water directly onto the membrane of the spin column e Incubate at room temperature for 5 to elute DNA e Centrifuge e g 1 min at 8000 rpm e Optional add another 50 ul Buffer AE or PCR grade distilled water directly onto the membrane incubate at room temperature for 1 5 min and centrifuge again e Discard the spin column Please note Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay Such contamination might occur during elution of prepared DNA by drops adhering to the funnel of the spin columns Thus these funnels should be gently touched and tried with sterile filter paper or wipes prior to the elution step E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 12 www alere technologi
46. ure Optional cell lysis with A1 A2 reagent instead of 1xPBS e Centrifuge A2 tube shortly open it add 0 2 ml of Lysis Buffer A1 to Lysis Enhancer A2 and dissolve e Add an inoculating loop full of monoclonal colony material of the E coli isolate to this A1 A2 reagent and vortex thoroughly e Incubate the colony material of the E coli isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker e Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit that is as follows e Add 20 ul proteinase K Qiagen Kit or equivalent and add 200 ul buffer AL Qiagen Kit e Vortex briefly or shake vigorously e Incubate for 30 60 min at 56 and 550 rpm in the thermoshaker e important If A1 A2 reagent not used add now 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing e Add 200 ul ethanol 96 100 96 e Vortex the sample and centrifuge shortly e Transfer the complete content of the tube including any precipitate into a spin column that is placed in a 2 ml collection tube E coli Genotyping Kit 05 16 04 0006 VO6 Manual E coli Genotyping Kit 11 www alere technologies com TM e Centrifuge at room temperature time and speed need to be determined depending on viscosity of the sample and type of centrifuge used e g 1 min at 8000 rpm AII liquid should be collected in the collection tube af
47. www alere technologies com www alere com Manual E coli Genotyping Kit Array Hybridisation Kit to detect important virulence genes of Escherichia coli Kit order number 205400050 50 reactions ArrayTube format For Research Use Only Not for Use in Diagnostic Procedures www alere technologies com www alere com CONTENT BACKGROUND 1 GENERAL INSTRUCTIONS FOR sese russes on 2 disaster re 2 SPECIFICATIONS 2 MEGHNICALS UPPORT I 2 er idzdud ize noc sessed sewaed ses 2 MATERIAL SAFETY DATA SHEETS 505 3 SHIPPING PRECAUTIONS 4 542 i nda ge creo rab api e ceps eee T E a ES EEEE SEEE ERE 3 DEVICES SOFTWARE AND REAGENTS ccsscscasccccoscaccencodsencasccnscectaccvctescnsavetnceessndecncssccanceesvaseccvesoses 4 SOFTWARE INSTALLATION M L 4 Assay Plugin and SDK for the 5 Test the AssayPlUg N mE 6 KIT COMPONENTS STORAGE AND esses ene 7 COMPONENTS REQUIRED BUT NOTPROVIDED cssseseseee emen nemen ese ese sese ese esi sss sese senes 8 iore

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