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1. Pseudomonas aeruginosa Genotyping Kit 2 Array Hybridisation Kit for epidemiological typing of Pseudomonas aeruginosa isolates Kit order number 245600096 96 reactions ArrayStrip format For Research Use Only Not for Use in Diagnostic Procedures www clondiag com www alere com Content BACKOROU ND E ena ie oi e 1 GENERAL INSTRUCTIONS FOR USE zana aaa p Neubau a Mv E Fed O eva o uan EIN 2 NMG USE T 2 Sp cificatiO cR RENE Er 2 Technical SOUS OIE NR C rob AKEE Zone dejali ee ie elje 2 Safety PRE CaN ONA PTT PEE 3 Material Safety Data Sheets MSDS nana 3 Shipping PRECAUTIONS geste noj Lu aaa oa etu nad bn acus sun n bM Cun bU Nd on E dui itus 3 RERGENTS AND DEVICES us eR DRE ea ala Med pate oa Na vej ONE MOM a CU MALAE 4 Assay Components Storage and Stability ccecssssececsessececseseeeeceesenaeeeessaeeeceeseeeecesesaaeeeees 4 DNA Labelling and Amplification sessssssseeeeeeeee eene ennemis 4 Hybridisation and Detebtib i eise ab mra deduagesdeceiausdaddageddacdsaiaandoagendadedeundvadasedddediadededaageiies 4 Instrumentation amp SOIBWAFEB 4i de tret p nara Fa Dn Y ea ana Ua EE Oa a ocn a fue E EE 5 Components required but not provided 5 SOFTWARE INSTALLATION sd ueste Sensi analne kajo ROUES exui ae tutum 7 Assay Plugin and SDK for the ArrayMale ce eripe exor adeo nn Dua da nua cq uy ua
2. To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted P aeruginosa Genotyping Kit 2 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com Table 1 Example worklist Position samplelD assaylD comment 1 2013 12345 10418 2 2013 12346 10418 3 2013 12347 10418 4 2013 12348 10418 5 2013 12349 10418 6 2013 12350 10418 7 987654 10418 Isolate referred from Dr J Doe 8 P aeruginosa PAO1 10418 Control strain Table 2 Positions in the 96 well format 3 4 5 6 7 8 9 10 11 12 9 17 25 33 41 49 57 65 73 81 89 10 18 26 34 42 50 58 66 74 82 90 11 19 27 35 43 51 59 67 75 83 91 12 20 28 36 44 52 60 68 76 84 92 13 21 29 37 45 53 61 69 77 85 93 14 22 30 38 46 54 62 70 78 86 94 15 23 31 39 47 55 63 71 79 87 95 Z OJ I MmJOOonD gt o js lm ju R Ww N H HB 16 24 32 40 48 56 64 72 80 88 96 Data Acguisition in the ArrayMate Reader e Insert your memory stick containing the worklist into any of the USB ports down to the right side of the ArrayMate e Press the button a folder selection dialog will open e Select your worklist path My Computer Removable Disk e Open your selected worklist with Enter or the button Open e Press the button your imported worklist opens in a separate window Proofre
3. ampC 1 PAO PAO1 Sequence Stover et al updated 2006 ACGGCCGCCGGGTGACGCC a eee TCGTCGAGGCGCATCT K De Champs et al 2002 Kiewitz and Tummler J ACGGCCGCCAGGTGACGCC ampC 1 non PAO Bacteriol 2000 182 3125 35 G ampC 3 PAO PAO1 Sequence Stover et al updated 2006 CGACCTACGCGCCGGGCAG GGOGAGATAGCCGAA GACTTGCTGCTCCATG De Champs et al 2002 Kiewitz and Tummler J CGACCTATGCGCCGGGCAG ampC 3 non PAO Bacteriol 2000 182 3125 35 C ampC 4 PAO PIKO Sequence Stover et al updated 2006 ei cine heer a a a ampC 4 non PAO De Champs et al 2002 Kiewitz and Tummler J CGTTCGAACGACTCATGGAG p Bacteriol 2000 182 3125 35 CAGC ampC 5 PAO PAO1 Sequence Stover et al updated 2006 c CHAIR Hal Db e wilhipamers of De Champs et al 2002 Kiewitz and Tummler J TGGAGCAGCAACTGTTCCCG ampC 5 non PAO Bacteriol 2000 182 3125 35 ampC 6 PAO PAO1 Sequence Stover et al updated 2006 pos idi a Pacco Meet ampC 6 non PAO UCBPP PA14 complete genome Lee et al 2006 NE ampC 7 PAO PACH Sequanea Stover etal updated 2008 CGACCTGGGOCTGGTGATCO UE TE AE K De Champs et al 2002 Kiewitz and Tummler J GCGACCTGGGACTGGTGATC ampC 7non PAO Bacteriol 2000 182 3125 35 CTGG ica ATCC15691 Spangenberg et al 1998 GTCGCTGAACGGOACOTACT CGATCGCGATGTCGAC TGCCGATCGCGATGTC PAGI Sequence Stovet etal Updaied 2006 oo mi z mlaj a ani ss PAO t Sequence Stover et al updated 2006 GAGCOGAGTGAGGACGOGO CAGGGTCGOCAGCTO AGGGTOG
4. 100 96 Vortex the sample and centrifuge shortly Transfer the complete content of the tube including any precipitate into a spin column that is placed in a 2 ml collection tube 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 All liquid should be collected in the collection tube afterwards Discard collection tube with liquids Place the spin column in a new 2 ml collection tube provided with the Qiagen kit Add 500 ul Buffer AW1 P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com e Centrifuge at room temperature e g 1 min at 8000 rpm e Discard collection tube with liquids e Place the spin column in a new 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 min 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 mem
5. Detection e ArrayStrips P aeruginosa 4 12 x 8 samples protected against light and sealed under inert gas Store at 15 C to 28 C After opening to be used within two weeks Close the unused wells with caps protect them against humidity and dust and store them at a dark place Please note Avoid any touching or scratching of the surface of the microarray at the bottom of the well Do not store or handle unused wells at an air humidity of more than 60 since this may irreversibly corrode the spots e ArrayStrip Cover 24 strips e C1 Hybridisation Buffer P aeruginosa Genotyping Kit 2 4 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com Store at 18 28 C protect against sunlight Surplus 100 e C2 Washing Buffer 1 Store at 18 28 C protect against direct sunlight Surplus 100 e C3 HRP Conjugate 100x Store at 2 8 C protect against direct sunlight Surplus 100 e C4 Conjugate Buffer Store at 18 28 C protect against direct sunlight Surplus 200 e C5 Washing Buffer 2 Store at 18 28 C protect against direct sunlight Surplus 200 e D1 Horseradish Peroxidase Substrate Store at 2 8 C protect against direct sunlight Surplus 50 Instrumentation amp Software e ArrayMate Reader to be ordered separately for details see below The P aeruginosa Genotyping assay may be used on the ArrayMate reader only The alternative devices ATRO1 03 are not suitable for reading Array
6. Edit View Favorites Tools Help Qe v d Pp Search Folders EJ Address E Name Size Type BB arrayMate_SDKs_ALL _2012 12 10 exe 463KB Application 15 setup ArrayMateAssay R D 10418 P aeroginosa 2 AS 2013 03 18 exe 323KB Application File and Folder Tasks A O Make a new folder 6 The welcome screen of the setup appears ie Setup ArrayMateAssay_R_D 10418 P aeroginosa 2 AS Welcome to the ArrayMateAssay R D 10418 P aeroginosa 2 AS Setup Wizard This will install ArrayMateAssay_R_D 10418 P aeroginosa 2 AS 2013 03 18 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 Repeatthis process for the SDK Setup 9 Log off and log in again as User R amp D password abcde P aeruginosa Genotyping Kit 2 8 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com Experiment String Collector The Experiment String Collector is a tool to collect the main experiment information from a set of experiments from the Array Mate The use of this tool is described below 1 Download the Experiment String Collector from www alere technologies com to your computer 2 Start the Setup executable a welcome screen will appear Setup Alere Experiment String Collector Welcome to the Alere Experim
7. Reader section of this manual e Open the string collector by double clicking its icon on your desktop the main window appears Experiment String Collector e mJ path of the exported experiments e g USB Memory e path where to save the experiments csv CE gt quit open File generate e Select the path of the source folder the folder of the ArrayMate Data export by clicking P aeruginosa Genotyping Kit 2 28 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com into area 1 indicated by the first red circle on the image above e Select the path of the target folder the folder to save the csv file containing the collected data strings by clicking into area 2 indicated by the second red circle on the image above e Single click the generate button all csv files in the source folder will be scanned and the content will be collected within one csv file named Experiments YYYY MM DD HH MM SS csv e Click on the button open File the Experiments csv file created opens in the notepad text editor e The content of the new csv file may now be transferred into an Excel based Data Mining Tool by copy paste Excel Based Data Mining Tool An Excel Based Data Mining tool was developed together with the group of Lutz Wiehlmann The template for a user specific data sheet can be downloaded from our website www alere technologies com The Excel shee
8. X XX P aeruginosa Genotyping Kit 2 10 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com PROTOCOL Please note All protocol steps described here in detail are also available as a short flow chart protocol see Appendix 1 Culturing and Harvesting Bacterial Cells P aeruginosa 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 P aeruginosa on Columbia blood agar overnight at 37 C or 48 hrs at room temperature Make sure that you have a pure monoclonal culture of P aeruginosa Contamination with other bacteria especially with other non 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 P aeruginosa genotyping assay is 0 5 2 ug Cona 0 1 0 4 ug ul of intact genomic DNA from 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 P aeruginosa cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the P aeruginosa genotyping assay are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend following the protocols outlin
9. easier to comprehend However if the test result of some digit is unclear it is rated 2 in the 16 digit string If the test result of a marker is invalid it is rated X in the 16 digit string and then the four digit KinCode remains incomplete In these cases the respective alphanumeric digit of the KinCode is replaced by X For each complete KinCode background information regarding the frequency of the respective code is given based on a data base established by L Wiehlmann unpublished SNP pattern and KinCode were described by Wiehlmann et al 2007 see literature Marker Controls These controls are important for the rating of data quality Furthermore in case of handling mistakes they provide important information regarding the experimental step that may have failed P aeruginosa Genotyping Kit 2 26 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com e If the Staining Control failed and along with it all other controls this indicates failure of the Horseradish Peroxidase mediated conversion of D1 that means a failure of one of the steps after hybridisation e If all controls including the Hybridisation Control failed but not the Staining Control this indicates a failure of the Hybridisation step e If the Labelling Genus Species and DNA Stringency Control failed but not the Hybridisation Control and the Staining Control this indicates a failure of PCR e If only the Genus Species an
10. free of RNA as free RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation e DNA must be free of any traces of ethanol as ethanol strongly influences the amplification This is an important issue as e g in the top of QIAGEN tubes a drop of ethanol containing washing buffer might get trapped The tubes really need to be centrifuged on high speed Alternatively it is possible to heat the sample prior to adding it to the labelling mix some 5 minutes at 70 C Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples e All reagents for DNA extraction need to be within the recommended shelf life and stored in the appropriate way We have good experience with the manual QIAGEN DNeasy Kit and the automated device EZ1 Contrarily alkaline lysis mechanical bead beater extraction or ultrasonication in our hands resulted in poor DNA recovery poor DNA quality or both An alternative method that is fast and simple but that did work in the hands of several customers was described by Wiehlmann et al 2007 see literature PCR failed Labelling and DNA Extraction Controls failed but not the Hybridisation Control and the Staining Control In this case please carefully review the PCR process for correctness In some cases inhibitors of PCR are isolated along with DNA In these cases a rerun of DNA purificat
11. remove the memory stick as long as the hourglass symbol is visible e Switch off the device by clicking on the Power button left down on the screen D e Switch off the Screen There is no need to physically switch off the ArrayMate P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 25 www clondiag com www alere com Contents of the Test Report The ArrayMate creates a test report that lists all markers analysed along with an individual test result for each marker In addition the test report provides a rating of the data guality along with identification of a possibly erroneous step of the experimental process The html file html may be viewed with any browser software e g Internet Explorer Firefox etc and may be printed or converted into a pdf file for details please refer to the ArrayMate s user manual The contents of the test report are described in more detail in the following section Kinship Analysis Markers of the Core Genome KinCode SNP Pattern Sixteen different markers of the core genome were selected for genetic identification of P aeruginosa isolates In the test report these markers are named SNP Pattern and are formatted as a string of 16 digits Each digit may adopt the value 0 1 2 X respectively Strings containing 0 and 1 digits are used for deriving the four digit alphanumeric KinCode which contains all information of the 16 digit code but is
12. the correct buffers Did you incubate at the correct temperature Is your thermoshaker working correctly i e at the temperature it is supposed to work Alternatively these options may be checked e The C3 C4 may not have been washed away correctly In this case the D1 turns dark green whilst incubating and this may result in nonspecific background e The array may have dried out during hybridisation if the cap had not been closed properly Plausibility controls failed This may either indicate a failure of the test protocol in this case the additional failure of other controls see above is expected In any case we recommend to review the conditions of hybridisation especially the real hybridisation temperature and the washing steps after hybridisation Alternatively failure of the plausibility controls may indicate the mixture of different P aeruginosa clones in this case we recommend subcloning and repeat of the experiment P aeruginosa Genotyping Kit 2 32 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com Physical damage to the array Scratching of the array surface with a pipette tip can lead to damage of an array spot that prohibits the acguisition of a valid signal In this case the respective spot is set invalid 1 the corresponding spot may be positive and valid lt 0 We recommend reviewing section 4 for general precautions in array handling LITERATURE e Wiehlmann et al 2004 JO
13. to your computer by using the Experiment String Collector and the Excel Based Data Mining Tool see page 29 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 www alere technologies com 2 Copy downloaded files Plugin and SDK setup to an USB Memory Stick and connect it to the ArrayMate 3 Log on as user admin to the ArrayMate default password 12345 4 Open the Windows Explorer and navigate to the downloaded setup files My Computer File Edit View Favorites Tools Help c 27 Pp Search Folders Hi Address 9 My Computer Name Type System Tasks Files Stored on This Computer View system E Shared Documents File Folder information k B WE Admin s Documents File Folder programs user s Documents File Folder g Change a setting O User R amp D s Docu File Folder Q Eject this disk Z Hard Disk Drives Se System C Local Disk lt Data D Local Disk Other Places amp My Network Places E My Documents Shared Documents j STICK E Removable Disk E Control Panel Devices with Removable Storage P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 7 www clondiag com www alere com 5 Start installation double click on setup file of the AssayPlugin USB STICK E File
14. 1 0 fax 49 0 36 41 3111 120 info identibac com cct home clondiag com P aeruginosa Genotyping Kit 2 2 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com Safety Precautions e The assay is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from pure P aeruginosa colonies clones reguires expertise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed e Isolated cell free P aeruginosa DNA may be processed without further biosafety precautions although contamination with P aeruginosa or other bacteria needs to be ruled out e Always wear protective clothes as required for laboratory work by your local regulations Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest amendments to the European Union Directives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet MSDS They do not contain more than 196 of a component classified as hazardous and do not contain more than 0 1 of a component classified as carcinogenic MSDS therefore are not provided 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 pr
15. A based assignment of unknown P aeruginosa isolates to phylogenetic lineages RNA free unfragmented genomic DNA from pure and monoclonal P aeruginosa 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 biotin labelled 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 99 probes for different genetic markers plus controls All of them are printed in five spots each The array contains three different kinds of markers and each marker is represented by one or more probes 16 markers for kinship analysis represented in total by 36 different probes 39 markers of the so called accessory genome represented in total by 39 different probes which may be used for the analysis of microevolutionar
16. Genotyping Report for this array is shown in the window on the right Browse A Search results results b raw data segmentation image image ARCHIVE 2013 03 18 14 04 32 P aeruginosa 2 Genotyping Report 1 4 01 C oLD AssayiD 10418 01 E BET For Research Use Only Not For Use In ore Diagnostic Procedures 01 H Strip ID Date of Result Mon Mar 18 14 05 13 2013 Experiment ID D1 A Well Position 01 01 A Operator Software Version 2013 03 18 Device 01a0001 KinCode Export of Test Reports Two result files in html format will be generated The shorter report gives a summary on genotyping information A longer html result sheet result B res htmi provides information on all probes Possible error messages in these reports are explained below see Troubleshooting Other files that are generated and can be exported include e A csv file containing the experimental result as a string which can be imported into an Excel based list for data mining see chapter 3 below e A txt file with the raw measurements e Animage file bmp showing 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 and e A xml file providing the same information as the html result sheet for future export into databases etc e A log file containing specific data of the evaluation of spots and substances e An out file conta
17. In case it is intended to enter the result of more than one array into the database we recommend the use of the String Collector see below TROUBLESHOOTING For training and gualification purposes we recommend our Control L Kit Alere order number 205500010 In case of doubt this kit is the perfect tool to make sure that your laboratory processes are working correctly It may also be used for the functional testing of individual kit components Poor DNA Quality Only the DNA Extraction Control failed In these cases we recommend to re check DNA quantity and quality by loading leftover DNA on an agarose gel e The amount of DNA is crucial A260 readings will cover RNA and other contaminants as well Therefore pure DNA preparation without bulk amounts of RNA is prerequisite for proper DNA concentration measurement RNAse treatment prior to A260 reading therefore is necessary component A2 contains RNAse P aeruginosa Genotyping Kit 2 30 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com e Some P aeruginosa colonies protect themselves with thick layers of slime It may help to wash the bacterial pellet 3x in 1 ml of 5 mM EDTA pH 7 0 re suspend and centrifuge at 3 000 x g for 6 min at room temperature discard the supernatant e DNA must not be fragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites e DNA should be
18. OCAGOTOG exoU UCBPP PA14 complete genome Lee et al 2006 E Ma rr ue e ev PAGI Sequanacdisverabal updated 2006 COTQAATCCGAGCATTCGCG CATICAGGTCGTAGAG oza TCGGACTGTACTCCTACGAA TGCCGAAGGTGAATGG CCTGATGGTCCGATCC pvA type Ila de Chial et al 2003 GCAGC CTTGCC CAGC P aeruginosa Genotyping Kit 2 05 16 04 004 VO3 Manual P aeruginosa pdf 37 www clondiag com www alere com Antisense Antisense Probes Reference Probes primer 1 primer 2 CCAATCCCTATCGCTGGAAC GCCGAGGGTCAAGAA TCTTGGCCCAGTCATA fpvA type IIb Spencer et al 2003 CGTACC CCACTGG GCGGC fpvA type Ill de Chial et al 2003 Seer quida ai oe eae fpvB PAO1 Sequence Stover et al updated 2006 pipe pete UTD ee eee LES LES400 personal communication C Winstanley P re ere Naa bed v cdd PA0636 PAO1 Sequence Stover et al updated 2006 en ee ic a PAO722 PAO1 Sequence Stover et al updated 2006 COTO TCAGQAACTOGCATOG TO TOOCGGAATGAGGT a RV PA0728 PAO1 Sequence Stover et al updated 2006 een ni oe o ener PA2185 PAO1 Sequence Stover et al updated 2006 NI RRNA are SECAT ee PA2221 PAO1 Sequence Stover et al updated 2006 Ga ae D OR acre Hte PA3835 PAO1 Sequence Stover et al updated 2006 ee oi ERU Je a ee fla island Arora et al 2001 ae a E Fere DUE orfA Arora et al 2001 ar ee ee JE EL am CCTGGACCTCTCCAAGGT
19. PAO UCBPP PA14 complete genome Lee et al 2006 Go po Reed oprL 2 PAO PAO1 Sequence Stover et al updated 2006 eres mane oprL 2 non PAO UCBPP PA14 complete genome Lee et al 2006 GCTGCAGGGCGTTTCGCCG fiCa 1 PAK PAK Totten and Lory 1990 flagellin type a2 Giske et al CAAGATCGCCGCAGCGGTCA AGCTGATGGTATCGCC CTAGTGATCGCACCGG 2006 A GTCGC AGCC i ATCC15691 Spangenberg et al 1998 flagellin type a1 CAAGATCGCCGCTGCGGTCA fica 1 non PAK Giske et al 2006 AC fliCa 2 PAK PAK Totten and Lory 1990 flagellin type a2 Giske et al CAAGATCGCCGCAGCGGTCA 2006 ACGAG i s ATCC15691 Spangenberg et al 1998 flagellin type a1 CAAGATCGCCGCTGCGGTCA fliCa 2 non PAK Giske et al 2006 ACGAC alkB2 PAO PAO1 Sequence Stover et al updated 2006 oe ey alkB2 non PAO ATCC 15691 Morales et al 2004 o A TCGAGCAACTGGCAGAGAAA GCAGGTAGCAGGTTTC AACTGTTCCTTCTGCG citS 1 PAO PAO1 Sequence Stover et al updated 2006 TCCG CAGG CGGCG cit 1 non PAO UCBPP PA14 complete genome Lee et al 2006 o am S SAO PAGT Sequence Stoveret al updated 2006 GCOGAAAACTTCCTGCACAT EM SEIS a citS 2 non PAO Kiewitz and Tummler J Bacteriol 2000 182 3125 35 APOO MAC ns opri 1 PAO PAGi Sequence Stover tal updated 2008 San o mo URI oprl 1 non PAO UCBPP PA14 complete genome Lee et al 2006 a ee oprl 2 PAO PAO1 Sequence Stover et al updated 2006 oo ee oprl 2 non PAO UCBPP PA14 complete genome Lee et al 2006 oma a ANE
20. Strip based assays In case of any questions please contact us e conoclust software provided with the reader e Test specific software plug in can be downloaded from Alere CLONDIAG s website check periodically for updates for details see below Information such as spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is delivered with the reader or can be downloaded from our website These test specific plug ins will occasionally be updated Please check the NEWS section of our website www alere technologies com Support is available under cct home clondiag com Components required but not provided e Growth media for the cultivation of P aeruginosa The test should be performed with colonies harvested from Columbia blood agar Other nutrient rich media e g 2xTY or LB may also suffice but have not systematically been tested Liquid media should not been used because contaminations or mixed cultures cannot easily be ruled out P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 5 www clondiag com www alere com e Eguipment and consumables needed for the cultivation of P aeruginosa incubator inoculation loops Petri dishes e DNA preparation kits The assay has been tested with the DNeasy Blood amp Tissue Kit from Qiagen cat 69504 and High Pure DNA Isolations Kit from Roche Cat No 11796828001 Please note The DNA sp
21. TC AAACTGCCCCGCCCCC GGAAAAACTGCCCCGC orfl Arora et al 2001 GCCT CATCC CCCCC ortJ Arora et al 2001 GOCATTOCGAGGAGCAAAGA ree rM dd Eon eve PAO980 PAO1 Sequence Stover et al updated 2006 Ce ee nep Ee are INIMA XF1753 UCBPP PA14 complete genome Lee et al 2006 a ona MR ADU DAS Ero ML acetyltransferase UCBPP PA14 complete genome Lee et al 2006 o o a HANS NIE oe pKL 1 Klockgether et al 2004 ao a eae RAM EI pKL 3 Klockgether et al 2004 TOOM a tk a ee iioc nici TB C47 1 P aeruginosa TB pKLC102 related gene island integrated GCAGGCGTCCAAGTTGGAGC GCCTGTTGGACCCCTT TACTCCTGCCTGTTGG in tRNA Lys PA4541 1 TCTCC TGACC ACCCC TB C47 2 P aeruginosa TB pKLC102 related gene island integrated TCCAACAGGCAGGAGTACAG TCTGTCAATCCCCTIT AGCCCCTTTCTGTCAA in tRNA Lys PA4541 1 GGTG GGGG TCCCC PAPI 1 pili chaperone UCBPP PA14 complete genome Lee et al 2006 Cac HORNO oe nes o o Ue Ua PAPI 1 luminal CCAGTTGGCACCACCATGCT CGGTAGAGAGCTGGG AACCTGGAGCTAGGGC binding protein UCBPP PA14 complete genome Lee et al 2006 TGC TTGGC AGAGC pKLC conserved GCCTGCCTACTTGTTCCCAA CTACCCAGCTTGGGCG AAGCGATAGCCGTGCT hypothetical mlbergether eral 2004 CGC TAGC CCTGC pKLC adhesin Klockgether et al 2004 c UE e E AL UHR pKLC fatty acid CGACAGACAGAAAGGGTTCT GGTGGCGTCGGGTTTT AGGTCGTAGCGGAAG synthase Klockgether et al 2004 TGCGC TCTGC GTGGTGG PAGI 2 3 4 Larbig et al 2002 o mene Z NE RKG PAGI 2 3 5 Larbig et al 2002 aa ace ee AD can
22. URNAL OF BACTERIOLOGY Vol 186 No 13 p 4228 4237 e Wiehlmann et al 2007 PNAS vol 104 no 19 p 8101 8106 P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 33 www clondiag com www alere com ADDITIONAL INFORMATION Warranty Alere guarantees the performance as described in this manual Usage of the Kit was successfully tested at ambient temperatures up to 37 C a guarantee is limited to ambient temperature in the laboratory between 18 C and 28 C Kit components comprise the ArrayStrips 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 reason than misuse contact Alere for replacement or refund Terms and conditions apply If you have any problem or question please contact our technical service Quality Control Each batch is stringently tested with the use of standard DNA preparations for good performance and correctness of results List of Components for Separate Order If required these reagents for the P aeruginosa Genotyping Kit 2 may be ordered separately component name category amount order no storage B1 Labelling buffer Buffered reagents 700 ul 245103002 2 8 C B2 Labelling Enzyme Buffered enzyme 20 ul 245104000 2 8 C C1 Hybridisation buffer Buffered reagents 30 ml 245105000 18 28 C C2 Was
23. ad If the new window is empty or if it was the wrong worklist repeat the import e Press the button OK the worklist window will close P aeruginosa Genotyping Kit 2 05 16 04 004 VO3 Manual P aeruginosa pdf 21 www clondiag com www alere com e Leave the memory stick attached to the ArrayMate if you intend to export Test Reports afterwards e Press the button Next bottom right on the screen reader opens e Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply any strong force Assure proper fit otherwise the images may be out of focus e Carefully insert the white frame with the array strips into the metallic adapter Assure the correct orientation Position A1 in the frame next to the data matrix barcode on the adapter and proper fit otherwise the images may be out of focus ArrayStrip frame with Strips inserted in accordance with the worklist Please note ArrayStrips must be clean They should not contain any liquids during analysis Barcodes must be clean There must be no ArrayStrip Covers on the wells to be analysed however unused wells should remain covered e Press the button Next bottom right on the screen reader closes analysis program starts reading takes 2 10 min dependent on the number of strips reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analys
24. al PAGI 2 3 6 Larbig et al 2002 a eco ee pen 1 AGGAGTTTI Al T TCATCCAGCAA AT TGGAGT TTT C 45 Larbig et al 2002 De G CGGACCCGC Dana GCAAGCC Ed CGCTTTCCGC C 46 Larbig et al 2002 a cae ee np Mena dest C 47 Larbig et al 2002 SOLO AG TANT SA CORPORUM ao eee PAGI 2 Larbig et al 2002 PoE ee eee a PAGI 2 3 1 Larbig et al 2002 a a eal ps TRAD UDINE or d PAGI 3 1 Larbig et al 2002 oo eee POS oe NAME EEG TO RESTE PAGI 3 8 Larbig et al 2002 mou UI m oa tRNA Pro island 1 P aeruginosa TB gene island integrated into tRNA Pro GTGTCACGGCCCATGTCTAG TCCACGCCGAGGGAC GCTCCACGCCGAGGG PA2736 1 CAGC GTGCC ACGTGCC tRNA Pro island 2 P aeruginosa TB gene island integrated into tRNA Pro AGGCCATGGGCTAGCCGGAT AGGAGGCCGATGACA TGCCGATTCCATGCTC PA2736 1 GC ACACCC ACGCC PAGI 1 Liang et al 2001 o ao a NCGRIAG nee P aeruginosa Genotyping Kit 2 38 05 16 04 004 V03 Manual P aeruginosa pdf
25. brane 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 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 A gt 60 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 vac centrifuge P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 13 www clondiag com www alere com Linear Amplification and Internal Biotin Labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of Bi labelling reagent is 25 e Prepare a Master Mix by combining 4 9 uL of B1 labelling reagent and 0 1 uL of B2 DNA polymerase per sample e Add 5 ul of P aeruginosa DNA Cpwa 0 1 0 4 ug ul prepared as described above to 5 ul of the Master Mix B1 B2 Do not forget to label the vial e Perform amplification in a p
26. d DNA Stringency Control failed this indicates poor quality of DNA Failure of Negative Controls indicates a high nonspecific signal background Please refer to the troubleshooting section below for further information Plausibility Controls Some combinations of markers in nature are impossible to occur for biological reasons The software checks for those combinations and returns a warning if they are diagnosed In these cases the test result is implausible Please refer to the troubleshooting section below for further information Accessory Genome and Microevolution Features of the Isolate In addition to the markers that characterise a clone additional markers of the accessory genome were added to the array Originally Wiehlmann et al 2007 41 markers of the accessory genome had been selected but meanwhile two of them TB C47 1 and TB C47 2 have been removed because of faulty results The remaining 39 markers are listed in the test report as Features of the Isolate and they may be exported along with the KinCode as a 39 digit code in the 0 1 2 or X format as described above This information may be used to characterise microevolutionary events within a clone that is characterised by a uniform core genome pattern The markers of the accessory genome have been described in detail by Wiehlmann et al 2007 see literature P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 27 www cl
27. dentified 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 Va etc 20 Create a list with at least three columns with obligatory headers in the order position sample ID assay ID Table 1 Positions are continuously numbered from 1 to a maximum of 96 Position 1 would correspond to A1 8 to H1 9 to A2 and 96 to H12 Table 2 Do not leave empty lines in the worklist If you use EXCEL position numbers should be typed into column A Sample ID is strain sample laboratory number such as exported from your LIMS or assigned in any different way Patient name should not be used as Sample ID The Assay ID enables the system to identify the current test and to correctly use information on layout spot number and identity etc P aeruginosa Genotyping 2 Assay has the Assay ID 10418 Assay ID numbers must not be confused as this could lead to errors or loss of data You may add further columns and headers with notes and comments at your convenience Information from these columns will not appear on the result screen or in the Test Report We recommend using a printout of the worklist as template for pipetting Safe the worklist as tab separated txt file on the memory stick provided together with the ArrayMate
28. dud ten adus ki ru du Read 7 Experiment String Collector RERUM 9 H EDIECCOAJIl Me p M 10 l9 pss MTM A 11 Culturing and Harvesting Bacterial Cells eese eene ener 11 Extraction of DNA qe C S 11 Via Spin Columns e g Qiagen DNeasy Blood amp Tissue eeeeeeeeennenns 12 Linear Amplification and Internal Biotin Labelling eeseeeeeeeeeeennneennn 14 HybridisatiOn ont pe EEE e E TEE T 14 General Remarks Handling of Arrays toe trs ep rt Ripa Vie ov aaa FU MU cu AE 14 General Remarks Handling of Liquids ccsccccssssececesssceceeseaececseseeecesseeeecesseueeeeseseeees 15 General Remarks the Substrate Precipitating Dye D1 ssseeeeeeeeeennnen 16 General Remarks Thermoshakers eseesse esses eee enne 16 Protocol for Quantifoil s BioShake iQ and Eppendorfs Thermomixer Comfort with Microtitre Plate Adapter aaa 17 ee ps a ME Po a AA DNE MN MA RAME RK MARI aa ar 18 Starting the ArrayMate REAAEV sn sa naivno je eelo eaa udis ua do cut akne aUe 18 Worklist ep O OE O SREDNO 20 Data Acquisition in the ArrayMate Reader sess 21 LCD ne c 23 Export of Test RepOP tes cu edd irse eo nee ele en ae naknadna na qiu
29. e Ya Ais 24 Contents of the Test Report iens aoro qai mir ease Pet Ret mx bear SUN A Ra edi M ae Ur AN ME lenih 26 Kinship Analysis Markers of the Core Genome KinCode SNP Pattern 26 Marker Controls creerii PP 26 Plausibility Gontrols 22 1 aet dra ee ab a E 27 Accessory Genome and Microevolution Features of the Isolate 27 Recommendation for a Project Specific Database ssesssssseseseseeeeeene nennen 28 String CONG CON QUON RE c 28 P aeruginosa Genotyping Kit 2 2 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com Excel Based Data Mining Tool t 29 TROUBLESHOOTING si ia io o HQ 30 LITERATURE c 33 ADDITIONAL INFORMATION iuiiiee span retrace o ke endet tetti e cenae educ Lupa anl 34 Waranty kivi ine eat ae ane aaa V reak od kaja da baba ve excedacs cvedng od bana zobna va aaa zajla ed evanje da dela a aaa 34 ei ipse aaa ee ole ea kko E a k ea bah 34 List of Components for Separate Order nana 34 CONTACT 35 APPENDIX Flow CIENT m 36 APPENDIX 2 Probes and Primers for KinCode Genotyping and the Accessory Genome 37 BACKGROUND The ALERE P aeruginosa Genotyping Kit 2 is a guick and simple method for the DN
30. ecimen needs to be free of RNA Recommendation a pre treatment with RNase A while DNA preparation e 1x PBS e RNAse A we recommend Qiagen s RNase A solution 100 mg ml Qiagen order 19101 e Equipment needed 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 We strongly recommend the BioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips Alternatively you may use Eppendorf s Thermomixer Comfort equipped a heating block for microtitre plates e Pipettes suitable for 1uL 5pL volumes 90uL 100uL 200uL 1000uL e Multichannel Pipettes for 100 200 uL e Reaction vials suitable for PCR e Ultrapure PCR grade water P aeruginosa Genotyping Kit 2 6 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com SOFTWARE INSTALLATION For analysis of the final image of the DNA microarray on the ArrayMate a specific software plug in is reguired 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 Further analysis can be made after transferring the data from the ArrayMate
31. ed 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 P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 11 www clondiag com www alere com Via Spin Columns e g Oiagen DNeasy Blood amp Tissue Add an inoculating loop full of monoclonal colony material of the P aeruginosa isolate to 0 2 ml 1xPBS and vortex thoroughly Loop empty Loop full It is important to harvest enough bacteria this is a prereguisite for extraction of a sufficient amount of DNA Take an inoculating loop of 1mm diameter filled with bacteria as shown in the left picture 12 Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit that is as follows Add 20 uL proteinase K Qiagen Kit or equivalent and add 200 uL buffer AL Qiagen Kit Vortex shortly or shake vigorously Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker Add 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing Add 200 ul ethanol 96
32. ent String Collector Setup Wizard This will install Alere Experiment String Collector 1 0 on your computer It is recommended that you dose all other applications before continuing Click Next to continue or Cancel to exit Setup 3 Follow the instructions and press Finish to complete The software will start automatically after the installation Please Note The Experiment String Collector is designed to work on Windows 7 P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 9 www clondiag com www alere com Test the AssayPlugin 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 strip rack with the unprocessed ArrayStrip into the ArrayMate than press Next 3 After the Experiment Run the ArrayMate automatically enters the Archive mode and displays the results of the last experiment 4 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 expe v sample ID position assay results 01 4 10418 01 6 10418 01 C 10418 01 D 10418 01 E 10418 01 F 10418 01 G 10418 01 H 10418 x xX X X X X XX x x x x x x x x O SOM A0N EH x xX X KX X
33. he bottom or across the data matrix barcode This may cause an error Barcode d label HERE keep it clean A 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 strip 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 rather than by inverting the strips and flicking the liquids out Fine tipped soft disposable Pasteur pipettes are suited best such as BRANDT Cat No 612 2856 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 Pasteur pipette plastic with a flexible tip Cee T flexible tip P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 15 www clondiag com www alere com ho Pipette tip usethe cavity between array andthe wall ofthe tube donevertouch 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 starting the procedure allowing it to pre
34. hing buffer 1 Buffer 120 ml 245106000 18 28 C C3 HRP Conjugate 100x Buffered enzyme 200 ul 245107000 2 8 C C4 Conjugate buffer Buffered reagents 30 ml 245108000 18 28 C C5 Washing buffer 2 Buffer 120 ml 245109000 18 28 C D1 HRP Substrate Buffered reagents 15 ml 245110000 2 8 C ArrayStrips AS P aeruginosa 4 microarrays 12 ST 240009724 15 28 C ArrayStrip Cover ArrayStrip Cover plasticware 24 ST 245112000 For pricing please contact your local representative or our customer service respectively 34 P aeruginosa Genotyping Kit 2 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com LEGAL MANUFACTURER Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena Germany CONTACT info identibac com cct home clondiag com P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 35 www clondiag com www alere com APPENDIX 1 Flow Chart Pro Hands Prepare ArraysStripes Prepare DNA cessing on time time ZN Grow pure and monoclonal over 5 min P aeruginosa isolate night Isolate genomic DNA 3 4h 40 min not part of the kit Label RNA free DNA in 2h 5 min thermocycler 5 ul DNA cpya 0 1 0 4 ug ul Rinse ArrayStripes P2 in 200 ul water pipette up and down 4x plus MM 4 9 3 a di Discard water Dilute labelled DNA 200 ul Buffer C1 60 C 550 rpm 2 min 10 uL of labeled DNA in 90 uL of 2 min 2 min discard C1 process promptly Buffer C1 T
35. ining output log data which help our service to trace image evaluation errors P aeruginosa Genotyping Kit 2 24 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com Please note Only complete runs can be exported The export of individual Test Reports is not possible e Right click on the selected run a menu appears with the option Export Run 2013 03 18 10 51 44 D H OLA Export Run Di p Export Run Reports Di C Edit Run 01 0 Delete Run 01 E 01 F 01 G 01 H e Right click on Export Run a file browser opens zm Browse Search ig s ARCHIVE s 2009 02 05 09 00 42 expe v sample ID order37x order37x23 order37x order37x78 order37x order37x82 ouderd2x order42x01 Browse For Folder mA s order42x02 s order42x03 vee order42x06 order42x07 Choose a directory order78x01 Desktop A vee Order78x01 t7 B My Documents order78x08 Power Y My Computer HB Se Local Disk C 4 Se Local Disk D E s Removable Disk E 0 2 w K Folder My Computer e Click on My Computer subseguently on Removable Disk and choose the folder to save or click on the button Make New Folder on the bottom a new folder icon appears e Rename the new folder e g with the experiment run name or date e Click on the OK button data are exported into the new folder on your memory stick e Do NOT
36. ion may help Hybridisation failed All controls including the Hybridisation Control failed but not the Staining Control In this case please carefully review the hybridisation step Was the buffer C1 used correct Was the hybridisation temperature used correctly P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 31 www clondiag com www alere com Staining failed All controls failed This indicates a failure of one of the steps after hybridisation The most likely reasons are e The component D1 was too cold when administered Please carefully follow the steps that are outlined in section 8 e Horseradish Peroxidase Conjugate C3 or its substrate D1 may have degraded during storage If this may be an option we recommend testing C3 and D1 with our Control L kit e Enzymatic reaction is inhibited by carryover of buffer C1 Ensure proper washing of the wells to remove all of Buffer C1 prior to adding Horseradish Peroxidase Conjugate C3 C4 e Horseradish Peroxidase may have degraded because of an incubation temperature gt gt 30 C Therefore please review the incubation step with C3 C4 for correctness Make sure that the shaker indeed has cooled down to 30 C before incubation Negative control failed In this case a high nonspecific background may result in false positive markers In this case please carefully check the hybridisation step and the subsequent washing steps for correctness Did you use
37. is ready e The reader indicates the end of the entire process with an acoustic signal beep e Press the button Next bottom right on the screen reader opens e Remove the white frame with the ArrayStrip s e Press the button Next bottom right on the screen reader closes P aeruginosa Genotyping Kit 2 22 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com Results On the left hand side of the screen you will see a list showing all runs stored on the Array Mate s hard disk A run contains the results from all arrays analysed together within one frame If this list is not displayed e Press the button Archive left hand and activate the flag Browse top left The runs are organised like folders in Windows Explorer and named by default according to the date of acquisition Example There is one experiment run in this archive Browse Q Search gd z ARCHIVE 28 2013 03 18 14 04 32 Archive If you click on the plus symbol left on the run name the folder opens and you will see a list of the individual arrays ordered by Sample ID Browse Search ig S ARCHIVE fe 2013 03 18 14 04 32 cond UES 01 A 01 B pj 01 C 01 D Archive D1 E 01 F 01 G 01 H P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 23 www clondiag com www alere com Click on a Sample ID and the P aeruginosa 2
38. ondiag com www alere com Recommendation for a Project Specific Database The CSV file csv which contains the experimental result as a string For comparison of the results and for data mining each of these strings may be imported into an Excel Based Data Mining Tool see 3 1 as suggested by Wiehlmann et al 2007 Alere Technologies provides a drawing of this Excel sheet that has been developed from the sheet suggested by Wiehlmann et al Further Alere Technologies provides a tool the Experiment String Collector see 3 2 that enables the user to collect the strings from all csv files within one folder in one single step Both the Excel datasheet and the string collector are described in the following section String Collector The tool called Experiment String Collector enables the user to collect all data strings from csv files found within one folder into one output csv file CommaSeparatedValue files being generic files for export into spreadsheet programs As outlined above and in the ArrayMate manual the results of each experimental run can be exported from the ArrayMate to an USB stick into a folder In order to collect all data strings found within this folder into a single csv file proceed as follows p e Download the string collector from our website www alere technologies com and install it on your office computer according to the instructions you will find in the Software and
39. otective goggles gloves and lab coat and avoid contact with the reagents In case any liquids are spilled clean with 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 P aeruginosa Genotyping Kit 2 05 16 04 004 VO3 Manual P aeruginosa pdf 3 www clondiag com www alere com REAGENTS AND DEVICES Assay Components Storage and Stability All reagents are provided in a certain surplus amount see below In case of need all components may also be ordered separately please refer to the order numbers at the end of this user guide 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 package All components have been tested for stability for short term shipment lt 1 week at ambient temperature lt 37 C The assay components with a rather limited stability are D1 and C3 The other components have proven to be stable for up to six months after the assay expiry date has passed DNA Labelling and Amplification e B1 2x Labelling Buffer Store at 2 8 C Surplus 25 e B2 Labelling Enzyme Store at 2 8 C Surplus 50 Hybridisation and
40. ransfer 100 ul labeled DNA to ArrayStripes 2min 2 min Barcode psl here Hybridise 60 C 550 rpm 60 min 60min Omin l Discard labeled DNA l Add 200 ul Buffer C2 discard C2 repeat this step twice prepare C3 C4 conjugate C3 C4 1 100 preheat Substrate D1 25 C 5 min 5 min i Incubate in 100 ul C3 C4 conjugate 30 C 550 rpm 10 min 10min 2min Discard C3 C4 conjugate add 200 ul Buffer C5 pipette up and down 4x repeatthis step once 2min 2min Quantifoil BioShake iQ or Eppendorf Thermomixer Discard Buffer C5 6 min 6 min incubate with 100 ul Substrate D1 30 C 5 min Discard Substrate D1 analyse ArrayMate 10 min 10min Total time requirement overnight ca 120 min MM MasterMix 7 8h a with heating block for microtitre plates P aeruginosa Genotyping Kit 2 36 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com APPENDIX 2 Probes and Primers for KinCode Genot ing and the Accessor Genome Antisense Antisense Probes Reference Probes s primer 1 primer 2 oriC PAO PAO1 Sequence Stover et al updated 2006 pM M Es IN eee oriC non PAO UCBPP PA14 complete genome Lee et al 2006 EIS aka oprL 1 PAO PAO1 Sequence Stover et al updated 2006 dug S A alain Ero roe oprL 1 non
41. re programmed thermocycler e g Eppendorf Mastercycler gradient with heated lid VWR cat No 460 0108 according to following protocol Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 62 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 Hybridisation General Remarks Handling of Arrays e Never touch the array surface e Avoid complete drying of the array surface during processing e Do not allow it to stay without liquid for more than two minutes e Never rinse the wells with distilled water after the hybridisation step use only C2 Washing Buffer P aeruginosa Genotyping Kit 2 14 05 16 04 004 VO3 Manual P aeruginosa pdf www clondiag com www alere com Unused wells should be capped during the whole procedure The strips may be processed up to three times without a loss of guality of properly capped unused arrays Close all wells that will not be used with a cap und leave it there until you use these wells for storage conditions after use see section Assay components storage and stability Hybridisation and Detection Always label your array strips with a laboratory marker at the recommended position Never label them on t
42. ruginosa Genotyping Kit 2 16 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com Protocol for Quantifoil s Bioshake iQ and Eppendorf s Thermomixer Comfort with Microtitre Plate Adapter Switch on the thermoshaker and let it pre heat to 60 C Remove the ArrayStrip s from the pouch Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode close to row A and proper fit Pre wash the array in two steps e First PCR grade distilled water 200 ul per well just pipette 4x up and down remove and discard e Second C1 Hybridisation Buffer 200 ul per well at 60 C 2 min and 550 rpm in the thermoshaker Add 90 uL of buffer C1 to each tube with 10 uL labelled amplification product mix gently Remove the buffer from the well with the array and add the mixture of C1 and labelled amplification product Incubate at 60 C 60 min and 550 rpm in the thermoshaker Meanwhile prepare conjugate for each experiment add 1 ul conjugate 100x HRP to 100 ul C4 Conjugation Buffer This mixture is stable for around one working day at room temperature C3 is delivered with a surplus of 100 C4 is delivered with a surplus of 200 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells C3 1 5ul 3 5 ul 7 ul 11 ul 16 ul 21 ul 32 ul 42 ul C4 150 ul 350 ul 700 ul 1100 jul 1600
43. t draft is separated into two areas e The first area columns A to M is meant for entering data like source of the specimen manually e The second area columns N to BS is meant for pasting experiment results provided by the ArrayMate in the csv files in a copy paste manner These data consist of the Identifier of the result the date of test the KinCode and the strings for SNP pattern and Features of the Isolate as a 16 t 39 digit string that may be used for Kinship Analysis In order to copy the data from an individual csv file into the data mining list proceed as follows P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 29 www clondiag com www alere com e Open the csv file of the experiment with any editor program Editor Notepad etc select and copy al Bo 1 A 10418 1 result csv Editor e paste the string into the data mining list in the Pseudomonas test results table column N Identifier of result 5 P GIRISITIUIVIWIXIY Z ANAB ACAD AE AF AG AH Al AJ AK ALJAM ANAO AP AG AR AS ATTAUTAVIAW AX AYI AZ BA BE BC BD BE BF BG BH BI BJ BK BLJEM BN BO BP EG BR BS Area for pasting test results non PAO non PAO non PAO Identifier of result Date of test prl PAO non PAO mpC 1 PAD 01 4 26925091041 20TL 12 7 15 52 40100 01 G 2692509104 2011 12 7 16 3 5 4300 e Enter the appropriate additional data into columns A to M manually
44. 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 P aeruginosa Genotyping Kit 2 18 05 16 04 004 V03 Manual P aeruginosa pdf www clondiag com www alere com e Log on as R amp D User Research and Development User for full access to test specific software a default password will be provided together with the ArrayMate device If you log on as User you will obtain only raw values but no interpretation as positives negatives and no strain assignment Administrator log in will allow manipulation of file folders and software and this should be done only upon direct advice of Alere s IT team e The user interface will be loaded ArrayMate performs internal testing This will require 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 e Type in your operator ID optional e You may enter a comment into the memo field optional P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 19 www clondiag com www alere com Worklist A Worklist file allows linking an identifier such as a laboratory sample number to a position of an array within the ArrayStrip For privacy reasons arrays should not be i
45. ul 2100 ul 3200 ul 4200 ul e Remove liquid from the well and add 200 ul C2 Washing Buffer discard C2 e Repeat this step twice P aeruginosa Genotyping Kit 2 05 16 04 004 V03 Manual P aeruginosa pdf 17 www clondiag com www alere com e The thermoshaker needs some minutes to cool down from 60 C to 30 C To speed up use the cooled cover for Quantifoil s BioShake iQ can be ordered from Quantifoil or an ice pack Eppendorf s Thermomixer Comfort e After removal of C2 Washing Buffer add 100 ul diluted conjugate to each well incubate at 30 C 10 min and 550 rpm in the thermoshaker e Remove liquid and wash with 200 ul C5 Washing Buffer pipette 4x up and down remove and discard e Repeat this step e Add 100 ul of D1 substrate precipitating dye at 25 C see above per well e Incubate at 30 C 5 min in the thermoshaker but do not shake e Remove liquid completely e The bottom of the ArrayStrips may be cautiously be cleaned with wipes bubbles may be removed by removing and adding D1 e Scan and process see below Data Analysis Starting the ArrayMate Reader We recommend starting the ArrayMate Reader after having started the hybridisation this allows you to conveniently start the device and to import the worklist file see below 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
46. warm to room temperature 25 C Cold D1 may yield weak signals D1 should shortly be centrifuged 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 remove and discard reagent D1 as completely as possible 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 ArrayStrips and Eppendorf s Thermomixer Comfort equipped with a heating block for microtitre plates When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference strains or with the control DNA P aeruginosa PAO1 P ae
47. y events 24 probes for internal control of the analytic process These probes test for proper DNA preparation using conserved genes of P aeruginosa PCR using a synthetic template hybridisation using control spots with biotinylated antisense partners and staining reaction using biotinylated control spots respectively Oligonucleotide probes are directly spotted onto the array primers and control oligonucleotides are present in the MasterMix B1 The resulting typing data provide information for kinship analysis Strain assignment using this information was described in detail by Wiehlmann et al 2007 PNAS vol 104 no 19 p 8101 8106 P aeruginosa Genotyping Kit 05 16 04 004 VO3 Manual P aeruginosa pdf 1 www clondiag com www alere com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not for Use in Diagnostic Procedures This assay allows genotypic characterisation of P aeruginosa isolates for research and epidemiological applications It cannot be used for other bacteria than P aeruginosa 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 Alere Technologies GmbH L bstedter Stra e 103 105 D 07749 Jena Germany phone 449 0 36 41 311
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