E. coli Pathotyping Kit - Alere Technologies GmbH
Contents
1. Folder My Computer Ca re e Click on My Computer then on Removable Disk and choose an appropriate storage place 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 name Click on the Ok button data are exported now into the new folder on your memory stick Do NOT remove the memory stick as long as the hourglass symbol is visible Switch off the device Click on the Power button left down on the screen o Switch off the Screen There is no need to physically switch off the ArrayMate In case of any hang up restart the computer by pressing the on off button bottom left on the ArrayMate reader face FORMAT OF THE RESULTS ArrayMate The ArrayMate creates a test report that lists all markers that have been analysed along with an individual test result for each marker www identibac com 05_16_04 0006_V05 22 IDENT BAC FORMAT OF THE RESULTS ATRO3 From October 2011 after software update also ATRO3 will create a user friendly test report Up to then the result file that is exported from the ATRO3 may be transformed into an Excel compatible format like this Spot ID Substance Confidence Signal Valid Background Mean 0 866366 o 0 849185 __ 0 06769 2 Jasta consens_10 0 546296 _ 0 051432 o 0 85848 0 811578 3 bfpa_10 noo0000 _ 0 883014 o 0 862151 _ 0 053476 A cba 10 0 924457 _2. 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 Control Spots In case that the signals of the negative control spots are above 0 1 AND the spot is set valid column valid 0 please contact our customer service In case that the signals of the positive control spots are below 0 5 proceed as follows www identibac com 05 16 04 0006 V05 24 IDENT BAC e Horseradish Peroxidase Conjugate may have degraded during storage Add 1uL buffer C3 4 to 9 uL D1 substrate If the solution turns green within 3 5 seconds the Horseradish Peroxidase still has sufficient enzymatic activity 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 Physical damage of 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 spot is set invalid 1 and the other spot is positive and valid 0 We recommend to review section 4 for general precautions in array handling LITERATURE Pathotyping Escherichia coli using miniaturised DNA microarrays Anjum MF Mafura M Slickers P Ballmer K Kuhnert P Wood
3. Enter or the button Open e Press the button your imported worklist opens in a separate window Check for correctness If the new window is empty or the worklist is not the desired one repeat the import e Press the button ok worklist window is closing e Leave the memory stick attached to the ArrayMate if you intend to export test data afterwards www identibac com 05 16 04 0006 V05 16 IDENT BAC 7 Washing after Hybridisation Washing after hybridisation 3 washing steps Please keep in mind the limited surplus of C2 60 Remove the ArrayTubes from the thermoshaker Set the thermoshaker to 30 C for the following steps cooling will take some minutes Carefully open the tubes and remove the hybridisation mixture as completely as possible without touching the array surface 1 Washing step after hybridisation add 500 uL of buffer C2 remove and discard the washing solution WITHOUT TOUCHING THE ARRAY SURFACE on Washing step after hybridisation add 500 uL of buffer C2 incubate in the thermoshaker for 5 min at 30 C 550 rpm remove and discard the washing solution 3 Washing step after hybridisation repeat on Washing step NOTE a carryover of more than 1 of buffer C1 to the next step will denature the HRP 8 Addition of HRP conjugate and staining Addition of HRP conjugate NOTE the used wells do not need to be capped any more Reagent C3 contains Streptavidin Horseradish Peroxidase H
4. Purification of Total DNA from Animal Tissues next step add 200 uL of Ethanol IMPORTANT BEFORE CONTINUING Check for DNA integrity and absence of RNA agarose gel In case remove RNA using RNAse Measure DNA concentration A260 method 2 Worklist For the regular USER mode of the ArrayMate it is obligatory to up load a worklist onto the Reader before an analysis can be performed For future data mining we recommend the use of a worklist also for the R amp D mode The worklist may be exported from a LIMS or designed in EXCEL or any other appropriate software The final format must be a wordpad txt format that can be imported into the test specific ArrayMate software see below For setting up a worklist e Create a list with at least three columns e The columns must have headers written into the first line e Each header MUST be one word different words may be linked by _ e Don t use special characters like etc e The following headers are obligatory in this order position samplelD assaylD where position position 1 to 6 of the metal frame to be used for reading in the ArrayMate List position numbers of wells that are to be analysed in a continuous fashion DO NOT leave empty lines in the worklist If you use EXCEL You need to type the position numbers into column A Note the raw numbers of the EXCEL spread template sheet CAN NOT be used to indicate well position samplelD
5. label HERE Barcode keep it clean e Avoid contact of data matrix barcode with organic solvents Avoid touching the bottom of the ArrayTube and keep it clean e Never rinse the tubes with water after hybridisation e Always cap the tubes before incubation in the thermoshaker Preparation of the hybridisation mixture e Pre heat the 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 www identibac com 05 16 04 0006 V05 14 e Incubate for one hour at 55 C and 550 rpm on a thermoshaker 5 Prepare reagents for detection and staining Dilute Streptavidin Horseradish Peroxidase C3 C4 IDENT BAC e Combine Reagent C3 Streptavidin Horseradish Peroxidase Buffer C4 1 100 gt C3 C4 The mixture is stable
6. pathogenicity markers of Escherichia coli THE RESULTING typing data provide information regarding genetic predisposition for pathogenicity of the analysed Escherichia coli bacterial isolates respectively THE ASSAY IS DESIGNED to be performed by personnel trained in microbiological and molecular biological laboratory methods www identibac com 05 16 04 0006 V05 3 IDENT BAC GENERAL INSTRUCTIONS FOR USE Intended Use For Investigational Use Only Not Intended for Use in Clinical Diagnostics The kit is manufactured for research use only The kit is intended for use by personnel that are well trained in molecular biology Preparation of DNA from E coli colonies clones requires expertise in microbiology and the local regulations for handling of potentially infectious organisms are to be obeyed 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 unless otherwise specified Technical Support If you require any further information on this product please contact Alere GmbH Am Wassermann 28 D 50829 K ln Fon 49 221 271 43 392 391 Fax 49 221 271 43 491 e mail info identibac com or visit our website www identibac com Safety Precautions DNA preparation must be performed under the biosafety conditions as defined by your local authorities for wor
7. your sample ID as exported from your LIMS or assigned in any different way assaylD 50202 ID number of the E coli Genotyping assay DO NOT use a different assay ID since this could lead to loss of data www identibac com 05 16 04 0006 V05 11 IDENT BAC You may add further columns and headers at your convenience Example position samplelD assayID comment 1 12345 50202 Dr Jones 2 12346 50202 Dr Miller 3 12347 50202 Dr Yale 4 12348 50202 Dr Palmer 5 12349 50202 Dr Jones 6 12350 50202 Dr Jones We recommend using a printout of the worklist as guidance for pipetting Always convert your file into a wordpad txt format tabstop delimited version Safe the wordpad txt file of the worklist onto the memory stick provided along with the ArrayMate To avoid confusion make sure that earlier versions of the worklist have been deleted from the memory stick 3 Linear amplification and biotin labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 is 40 Suspend 0 5 1 5 microgram of clonal and intact E coli DNA in 5 uL of ultrapure water ina reagent tube suitable for PCR concentrate DNA if necessary by evaporation in a vacuum centrifuge or by precipitation Do not forget to label the vial Prepare a Master Mix by combining 4 9 uL of B1 2x Labelling Buffer and 0 1 uL of B2 DNA Polymerase per specimen Add
8. Thermocyler Thermoshaker we strongly recommend the BioShake iQ from Quantifoil Instruments http www ginstruments com equipped with a specialised platform for the warming of Alere s ArrayTubes Alternatively you may use Eppendorf s Thermomixer Comfort equipped with a platform for the warming of reaction tubes Pipettes suitable for 1uL 5uL volumes 90uL 100uL 200uL 1000uL For removal of liquid from the ArrayTubes we recommend the use of plastic Pasteur pipettes with flexible tips Pasteur pipette plastic with a flexible tip Ce n flexible tip Reagent tubes suitable for PCR Ultrapure water www identibac com 05 16 04 0006 V05 7 IDENT BAC SOFTWARE AND READER Please keep in mind that you need to install a kit specific software on your reader Image analysis for any IDENTIBAC test kit and on any IDENTIBAC reader is performed by the iconoclust software that has been delivered along with the reader In addition some test kit specific information tied to some reader specific information needs to be installed separately This information like spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is provided by the specific iconoclust package Whenever you want to work with a kit OR a kit version you have never worked before please install the appropriate iconoclust package first The appropriate package you may download from our website www iden
9. due to insufficient amounts of DNA We therefore strongly recommend to follow the protocol outlined below From several DNA isolation methods tested so far only the DNA extraction Kits from Qiagen yielded sufficient DNA CAUTION E coli strains bearing genetic information of special virulence factors are potentially pathogenic All procedures prior to complete death of the bacteria need to be performed by properly trained staff in a biosafety Level 2 facility Harvest of bacteria From a bacterial clone grow enough cells to fill an inoculating loop diameter 1 mm as pictured below Add an inoculating loop of bacteria to 0 18 mL to reagent ATL Qiagen kit loop empty loop full It is important to harvest enough bacteria this is a prerequisite for extraction of sufficient DNA Take an inoculating loop of 1mm diameter filled with bacteria as shown in the left picture Extraction of DNA Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit Add 20 uL proteinase K Qiagen Kit Add 200 uL buffer AL Qiagen Kit Vortex shortly or shake vigorously www identibac com 05_16_04_0006_V05 10 IDENT BAC Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker important add 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing From the Qiagen kit manual follow Qiagen protocol
10. for 1 day at room temperature C3 is delivered with a surplus of 300 C4 is delivered with a surplus of 500 e 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 5 uL 3 5 uL 7 uL 11 uL 16 uL 21 uL 32 uL 42 uL C4 150ul 350uL 7OOuL 1100 uL 1600 uL 2100 uL 3200 uL 4200 uL e put aside at room temperature until use Pre warm the staining reagent D1 e Transfer enough reagent D1 into a separate vessel e g a clean and sterile centrifuge tube 100 uL for each well and a surplus of not more than 20 e put aside at 20 to 25 C until use Please note cold D1 may yield in weak signals Centrifuge D1 immediately prior to staining see below otherwise clots within D1 may contaminate your array 6 Setup of the ArrayMate Reader We recommend to set up the ArrayMate Reader after having started the hybridisation this allows you a restart without time pressure in case of any software problems 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 www identibac com 05 16 04 0006 V05 15 IDENT BAC e Login as User R amp D password to be defined by the instrumen
11. suspended DNA to a 5 uL aliquot of the MasterMix Perform the following thermocycler protocol required time approximately 2 hours Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 50 C 45 cycles with 30 sec at 72 C 20 sec at 96 C Cool down to 4 C hold www identibac com 05 16 04 0006 V05 12 IDENT BAC 4 Hybridisation General Precautions e For removal of liquid from the ArrayTubes we recommend the use of plastic Pasteur pipettes with flexible tips Pasteur pipette plastic with a flexible tip a H t flexible tip e Never touch the Array surface Remove liquid with a pipette always place the pipette tip at the cavity between the array and the wall of the tube If you touch the array surface probes may be scratched off and this may cause an error use the cavity between array andthe wall of the tube do never touch the array Array e Avoid any complete drying of the array surface during processing i e do not allow it to stay without liquid for more than two minutes e We strongly recommend that the liquid is removed by pipettes instead of inverting the tubes and flicking them out e Please keep in mind the limited surplus of C1 100 www identibac com 05 16 04 0006 V05 13 IDENT BAC e 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 5
12. 0 000794 o 0 87347 __ 0 874207 5 Jed_10 Cf 0 900066 _ 0 003228 fo 0 878431 0 875419 6 cdte ao 0 95421 0 857459 o 0 876036 __ 0 078118 z cdtBiso SS o 95421 0 572145 o _ o ss86699 _ 0 347803 8 cdte 6o 0 891279 0 000566 0 0 891359 0 891895 9 celb_10 0 731481 _ 0 001272 o 0 897874 0 896661 10 cacao a ooo000 _ 0 001629 o 0 895425 _ 0 893875 11 ema20__ f 000000__ 0 872491 o _ 0 890287_ 0 065173 12 enft_20_ f 1 000000 _ 0 000422 0 0 904902 _ 0 905307 13 cofa_10 0 856323 0 004607 Jo 0 909804 _ 0 905352 141 0 1M NaPP Standard pH9 0 941429 _ 0 00168 o 0 897958 0 89956 142 __ Biotin Marke_2 54M___ 1 000000_ 0 835682 o 0 887522 _ 0 099673 145 K88ab_10 1 000000 0 846823 o 0 900338 _ 0 090458 0 146 _ astA_consens 10 ___ 0 546296 _ 0 089045 o __ 0 903499__ 0 818039 o fo 072136 The meaning of the columns of the raw data list has been outlined above Please note that each substance is represented by two spots e g substance K88ab_10 is represented by the spots 1 and 145 respectively Spots 141 and 283 respectively contain substance 0 1M NaPP Standard pH 9 which is a negative control i e Signal of these spots should be far below 0 1 Spots 142 and 284 respectively contain substance Biotin Marke_2 5uM which is a positive control i e Signal of these spots should be above 0 5 For the meaning of the other substances spots please refer t
13. 34 2009 06 23 15 06 20 fr 2009 06 23 15 12 58 2009 06 23 15 28 39 2009 06 23 16 23 31 S 2009 06 24 13 31 15 2009 06 24 13 47 25 2009 06 24 13 55 22 2009 06 24 14 01 07 lt e Click onto the plus symbol of one defined reading the folder opens and you will see a list of the individual ArrayTube s sorted by position within the adapter E ArrayMate Browse Search results results b raw data segmentation image image Spot ID Substance Confidence Signal Valid Background Mean 25678_20_Var2_lightint_66_ueber A amp 25678_20_Mar3_lightint_41_zu dur E qer94433 01 4 01 6 01 C 01 D 01 E 01 F 01 G 01 H e Activate the Flag raw data top left and click onto the position of an individual tube e g 01 A the results of this particular array will appear on the right side of the window E ArrayMate Browse Search results results b raw a segmentation image image SpotID Substance Confidence Signal Background Mean 25678_20_Var2_lightint_66_ueber 1 s_aur_rrn_ 1 000000 0 525380 0 0 767417 0 339137 S 25678_20_Var3 _lightint_41_zu dur 10 agrC I_12 1 000000 0 531283 0 765924 0 333674 qcr94433 100 setl var2_11 1 000000 0 390218 0 767611 0 449432 0A 101 setl var4_11 1 000000 0 412722 0 766248 0 430317 102 set2 varl_11 1 000000 0 480295 0 767491 0 375926 103 set2 var2_11 1 000000 0 444688 0 766040 0 404189 104 set2 var
14. 3_11 1 000000 0 542246 0 768450 0 325826 nie 105 set2 var4_11 1 000000 0 484461 0 767739 0 372650 oit 106 set3 varl_11 1 000000 0 423567 0 766600 0 421684 01 H 107 set3 var2_11 1 000000 0 463712 0 766925 0 389158 108 set4 varl_11 1 000000 0 435425 0 764688 0 411000 inn fob nawa 44 4 annn naaca n azennon n anccoe 01 B 01 C 01 D 01 E Note the flags results and results b are not active with this assay www identibac com 05_16_04_0006_V05 20 e Interpretation of the raw data list results results b it wd ta segmentation image image Spot ID gt Substance Confidence Signal Valid Background Mean a 1 s_aur_rrn_ 1 000000 0 525380 0 0 767417 0 339137 10 agrC I_12 1 000000 0 531283 0 0 765924 0 333674 100 seti var2_11 1 000000 0 390218 0 0 767611 0 449432 101 seti var _11 1 000000 0 412722 0 0 766248 0 430317 102 set2 vari_11 1 000000 0 480295 0 0 767491 0 375926 103 set2 var2_11 1 000000 0 444688 0 0 766040 0 404189 104 set2 var3_11 1 000000 0 542246 0 0 768450 0 325826 105 set2 var4_11 1 000000 0 484461 0 0 767739 0 372650 106 set3 varl_11 1 000000 0 423567 0 0 766600 0 421684 107 set3 var2_11 1 000000 0 463712 0 0 766925 0 389158 108 set4 vari_11 1 000000 0 435425 0 0 764688 0 411000 anaba sard 1 1__1_ NADA Q AALS 27 aa azenon ANGE DE Spot ID numerical identifier of the spot on the array Substance name of the DNA probe Confidence an intrinsic
15. Fon 49 221 271 43 392 391 Fax 49 221 271 43 491 e mail info identibac com or visit our website www identibac com www identibac com 05 16 04 0006 V05 26
16. IDENT BAC E coli Pathotyping Kit Array Hybridisation Kit for DNA based detection of genetic pathogenicity markers of Escherichia coli For Investigational Use Only Not Intended for Use in Clinical Diagnostics www identibac com 05 16 04 0006 V0O5 IDENT BAC Content BACKGROUND ietecsere ceccencedecacdedeececacesevscqanceteeasedecscgedgeceeuecsecseuansecee ndedeceeds REEE SE E EES deceacveuenecesacedeceeueceecees 3 GENERAL INSTRUCTIONS FOR USE ccccceeesseesseesesesesesesenesesesseseseeeseseessesesesesesesesesesesessseseseseeeeeseseseseeeseseseeeseeenenens 4 KIT COMPONENTS STORAGE AND STABILITY cc ccsesssescssecececececececececececeeaeaeaeaeaeaeseaeaeaeaeaeaeaeaeaeececeeeeeeeeeeeeeeesaeaeaeaes 6 SOFTWARE AND READER cccecececececececececececeeeceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeesesesESEEEEESESESESESEEEEEEEEEEESEEEEEEEEEEEEE EE EE ES 8 PROTOCOL roe se r eE EEE ERE E EE E EEA 9 MSIE COU DNA ereraa E E Ee TENE EE E eE E EA 10 2 Yo 4 T eee E E E T rere errr re 11 3 Linear amplification and biotin labelling 0 cccccccccccccccessssscecececeesenseeececececseseaeceeceesesesecececsesensaaeeeeeceeens 12 A Hybridisation cccccsccccessseceesssceceeseeecssssaececseccecsessesecsesaeeeceessecsesueecsesaeeeccseaaecessseeecsesaesecseaaesessuseeessnreeennas 13 5 Prepare reagents for detection and Staining ccccccccesscecesssececeesececseccecessaeeeceesaece
17. RP that would denature and 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 C3 300 Combine Reagent C3 HRP Buffer C4 1 100 gt C3 C4 see section 5 the mixture is stable for 1 day at room temperature both reagents are delivered with a surplus of more than 100 each Add 100uL of C3 C4 to each tube www identibac com 05 16 04 0006 V05 17 IDENT BAC e Incubate for 10 minutes at 30 C and 550 rpm on a thermoshaker e Remove and discard C3 C4 completely Washing step after binding of conjugate Addition of HRP conjugate 2x e Please keep in mind the limited surplus of C5 140 e add 500 ul of buffer C5 Shake for 1 minute at 30 C and 550 rpm on a thermoshaker remove and discard the washing solution e Repeat this step once 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 Staining of bound HRP conjugate NOTE a carryover of more than 0 5 of C3 C4 will cause unwanted effects see above donot move ArrayTubes during staining Reagent D1 contains a substrate for Horseradish Peroxidase Please keep in mind the lim
18. cseeeeeeesneeeeseaeeeeseaaees 15 6 Setup of the ArrayMate Reader 0 0 c ccccscececciscoscccsscovesssstocessconcvessnontcesssctevsctscussentseesvuctecessestecbenoedsevsnects 15 7 Washing after Hybridisation 0 0 0 0 ccc cccsccecssceceessececeesseececseneecessaeeecsesaeeecsesaeceseeeecsesaeeecsecaeceseneeeceesueeeeneaaes 17 8 Addition of HRP conjugate and staining ccccccccsscccccecsessssesecececeessaaeceeeecesensaaesesececeeseasseeeeeesensssaeseeees 17 9 Data Acquisition in the ArrayMate Reade ccccccccssscecsseeceeseececeesaeeecssaeeceseneeeesssaeeecsesaeeesseeeeeeesaeeeesaas 19 FORMAT OF THE RESULTS ArrayMate ccccceccccssccsscssccsececscsvsvsesesecscacscecevevevcucecessvavarescesessvavavavsesesasacacesevavavsaeseenens 22 FORMAT OF THE RESULTS ATROS f0 5 c t3ecccsec sie kel tance E E cana set E A E E 23 TROUBLESHOOTING oi csecccestece cs cas ire ee Er veaees netedeadectckeidetecccav EE EEEE EE eE EAEN 24 ITERATURE mae E E EE EE E E E O EE E caret venyonens ees 25 ADDITIONAL INFORMATION cccccecececececececececececececeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeees 25 Warranty eee E A E E tes cast ceaucele ceteets 25 Quality Control ees so ise co5 os ss ek secs coe ses nea ee a See aaa sk her ee cai aaa eggs E AE A sn aca laden ban Tab ok EEEa 25 List of components for Separate order ce ccecccccesssccecsesececseseeceenececsesa
19. ececseaeecessaeeecsesaeescseseeeesseeeeseaaeeesseeaeees 26 LEGAL MANUFACTURER sivcccc cnceccecd eweccecstccacecetonccd conecacdeaeaescsece bee cdeeneescedcesce cones nee cebee bee cdneneeee been aces cned suiec buch Sueacuentenaueeus 26 CONTAC eee ss otc ec cee tice fern chn sete cs Seon neu cone tea ae So ee see etna eee sion ete seen catalase oe E E E 26 www identibac com 05 16 04 0006 V05 2 IDENT BAC BACKGROUND The IDENTIBAC E coli Pathotyping Kit is a quick and simple method for pathotyping Escherichia coli Virulence genes present in E coli clinical isolates of both human and animal origin can be determined using the ArrayTube system For Investigational Use Only Not Intended for Use in Clinical Diagnostics TEST PRINCIPLE DNA isolated from bacterial clones is amplified approximately 40 fold and labelled with biotin dUTP adopting a so called linear PCR protocol In linear PCR only one primer is used instead of a primer pair producing single stranded reaction products only therefore limiting the degree of amplification limited risk of cross contamination Labelled single stranded DNA is transferred and hybridised to microarrays with 124 probes representing different genetic markers plus a staining control plus a negative control single redundant each Spot recognition as well as geometric layout is performed automatically based on scans of the arrays and advanced image processing The target set consists of a variety of genetic
20. efahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods www identibac com 05 16 04 0006 V05 5 IDENT BAC KIT COMPONENTS STORAGE AND STABILITY Please keep in mind the limited surplus of each reagent when pipetting Arrays and B1 respectively are by far the most expensive and hence the limiting components of the kit All components may be ordered separately please refer to the order numbers at the end of this manual For pricing please contact your local representative or our customer service respectively DNA labelling and amplification e Bik Labelling Buffer Store at 2 8 C Surplus 40 e B2 Labelling Enzyme Store at 2 8 C Surplus 200 Hybridisation and Detection e ArrayTubes 10 x 5 samples protected against light and sealed under inert gas Store at 18 C to 28 C After opening to be used within two weeks Close the unused tubes protect them against humidity and dust and store them at a dark place Avoid ANY touching or scratching the microarray on the bottom of the well CAUTION 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 28 C protect against sunlight Surplus 100 e C2 Washing Buffer 1 Store at 18 C 28 C protect against direct sunlight Surplus 60 e C3 HRP Conjugate 100x Store at 2 8 C protect against direct sunlight Surplus 300 e C4 Conjugate Buff
21. er Store at 18 C to 28 C protect against direct sunlight Surplus 500 e C5 Washing Buffer 2 Store at 18 C to 28 C protect against direct sunlight Surplus 140 e D1 Horseradish Peroxidase Substrate Store at 2 8 C protect against direct sunlight Surplus 200 Expiry date is to 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 kit components with limiting stability are D1 and C3 respectively www identibac com 05 16 04 0006 V05 6 IDENT BAC The other components have proven to be stable even six months after the kit expiry date has passed Components required but not provided DNA preparation kit The assay has been tested with the DNeasy Blood amp Tissue Kit from Qiagen cat 69504 according to Qiagen s instructions for DNA preparation of gram negative bacteria RNAse we recommend Qiagen s RNase A solution 100 mg mL Qiagen order number 19101 Instrumentation provided by Alere to be ordered separately ArrayMate Reader or ATRO3 reader respectively Materials and instruments required but not provided by Alere Equipment growth media and consumables needed for the cultivation of E coli Equipment needed for DNA isolation e g pipettes centrifuge thermoshaker or robot Photometer for quality control of DNA Equipment for DNA gel electrophoresis for quality control of DNA
22. estimate of spot confidence based on size and shape of that particular spot where 1 high confidence and 0 no confidence Signal spot signal intensity grey scale value where 1 black and 0 white valid O valid 1 invalid confidence below 0 75 Background luminous intensity of the background where 1 maximum brightness and 0 maximum darkness Mean luminous intensity of the signal spot where 1 maximum brightness and 0 maximum darkness The correlation between mean background and signal is roughly 1 mean background however there are some correction factors that depend on the statistics of pixel distribution Export of E coli Genotyping Test Results e Note only complete readings can be exported The export of Test results for individual ArrayTubes of a reading is not possible e Right Click on the desired reading a menu appears with the option Export Run www identibac com 05 16 04 0006 V05 21 e Right Click on Export Run a file browser opens E ArrayMate Browse Search expe sample ID order37x E ARCHIVE order37x 2009 02 05 09 00 42 order37x New Run gov order42x01 Browse For Folder we Order42x02 dEl order42x03 Choose a directory a order42x06 order42x07 vee order78x01 Desktop ve order78x01 i B My Documents ve order78x08 My Computer H Se Local Disk C H Se Local Disk D E Se Removable Disk E i
23. ips MUST be clean and free from air bubbles and liquid Barcodes on ArrayTubes and holder must be clean Press the button Next bottom right on the screen reader is closing analysis program starts it takes ca 2 5 min dependent on the number of ArrayTubes reader takes images AND automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analysis ready The reader indicates the end of the entire process with an acoustic signal beep Press the button Next bottom right on the screen reader is opening Remove the ArrayTube s Press the button Next bottom right on the screen reader is closing www identibac com 05_16_04 0006_V05 19 DENT BAC Results e On the left hand of the screen you will see a list showing all readings stored on the ArrayMate A reading contains the results from all arrays analysed together in one frame If this list is not visible press the button Archive left hand activate the Flag Browse top left e The readings are organised like folders in Windows 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 E ArrayMate BEE Browse a Search iL F expe sample ID position raw data results is E ARCHIVE 2009 06 05 10 28 06 _NewRun 2009 06 23 14 57
24. ited surplus of D1 200 e D1needs to be pre warmed and centrifuged see section 5 e Add 100uL of reagent D1 to each well supernatant of centrifuged D1 without precipitate e Incubate at room temperature WITHOUT agitation for 10 min e Read out without removing D1 removal of D1 would leave air bubbles within the tubes CAUTION the strips MUST be clean underneath the arrays and there MUST NOT be air bubbles or remains of liquid in the wells Strips may be cleaned with lint free wipes bubbles may be removed by adding and removing D1 again 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 www identibac com 05 16 04 0006 V05 18 IDENT BAC 9 Data Acquisition in the ArrayMate Reader The reading process The setup of the reader is briefly described in the section 6 Setup of the ArrayMate Reader Press the button next bottom right on the screen reader is opening Carefully insert the appropriate metallic adapter for ArrayTubes into the ArrayMate Do not apply any strong force After having inserted the adapter carefully insert the Array Tubes into the adapter ArrayTubes need to be open with tube lid connections placed into appropriate notches Assure proper fit otherwise the images may be out of focus CAUTION the str
25. k with E coli strains that are suspected to be pathogenic In most countries Biosafety Level 2 is obligatory for this kind of work E coli DNA may be processed in laboratories below this biosafety level if potential contamination by living E coli bacteria can be definitively eliminated Always wear protective clothes as regulated for laboratory work by your local authorities www identibac com 05 16 04 0006 V05 4 IDENT BAC Material Safety Data Sheets MSDS Per 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 1 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 protective goggles gloves and lab coat and avoid contact with the reagents In case buffers or solutions are spilled clean with 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 G
26. o hands cessing on prepare Arrays prepare DNA time time grow bacteria over not part of the kit night 5 min Ve isolate DNA not part of the kit 3 4h 10 40 min Ti v label DNA in Thermocycler e rinse arrays 5 pl panera 2h 5 min 0 5 ml water 55 C 550 rpm 2min 43 HLBI 01 uL B2 e incubate in buffer C1 0 5 ml 55 C 550 rpm 4 min discard C1 process promptly to 10 uL of labelling product 10min 10min add 90 uL of Buffer C1 transfer DNA to ArrayTube 2 min 2 min hybridise 55 C 550 rpm 60 min 60min Omin discard DNA add and remove 0 5 ml Buffer C2 15 min 15 min incubate twice in 0 5 ml Buffer C2 30 C 550 rpm 5 min vy incubate in 0 1mL C3 C4 1 100 30 C 550 rpm 10 min 10min 2min discard conjugate 5 min 5 min incubate twice in 0 5 ml Buffer C5 30 C 550 rpm 1 min v incubate with substrate 0 1 ml Buffer D1 RT 10 min 10min 10min take image WITHOUT removing D1 analyse 15min 15min total time requirement overnight 80 110 min 7 8h www identibac com 05 16 04 0006 V05 9 IDENT BAC 1 E coliDNA The required specimen for the E coli Genotyping test is 0 5 2 ug of intact DNA from a single clone of E coli The DNA specimen needs to be virtually free of RNA as determined by agarose gel electrophoresis Please note This assay requires much more DNA than common PCR applications Most performance problems with the E coli Genotyping Kit are
27. o our gene list www identibac com 05_16_04_0006_V05 23 IDENT BAC TROUBLESHOOTING Data Quality In case of poor data quality we recommend to re check DNA quantity and quality first 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 are a prerequisite for proper DNA concentration measurement RNAse treatment prior to A260 reading therefore is necessary component A2 contains RNAse e DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites e DNA should be 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 need to be within the recommended shelf life and stored in the appropriate
28. t s administrator on the customer s site and to be initially implemented by the supplier s technical service The user interface is loaded ArrayMate performs internal testing for lt 1 minute e Click on the icon New Run left upper edge of the screen a suggestion for a run name for the new run appears in the top line of the screen e You may now modify or change the experiment name at your convenience e Type in your operator ID e If desired you may enter a comment into the memo field e Insert your memory stick containing the worklist use any of the USB ports down to the right side of the ArrayMate e Press the button fa a folder selection dialog opens e Select your worklist path My Computer Removable Disk Choose a file Strips Eea Caution Make sure to chose the Look in worklist examples D Wy ReceniDecureni correct worklist nes B My Documents My Recent My Computer Documents lt Local Disk C amp Local Disk D s 4 Ej q eiaa Caution By default an example file Desktop gt i shed Documents My Documents 7 amp User s Documents for a worklist is selected This file is User R amp D s Documents My Documents My Network Places only for training purposes Do NOT Ss My Computer i e z use it for your experiments d My Network Files of type worklist txt e Open your selected worklist with
29. tibac com and install it on your reader according to the instructions you will find on this website Please note You should NOT use a package for ArrayMate that has been designed for ATRO3 and vice versa The ArrayMate Reader by default has all software on board except the E coli Pathotyping kit specific package 50202 icpck that may be missing ant that you may need to install as outlined above Unlike ATRO3 ArrayMate is not linked to problems with compatibility of software The ATRO3 reader is a research tool that is highly versatile but it 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 Pathotyping kit specific package 050202 icpck is not compatible with iconoclust versions older than version 3 1 Whilst the E coli Pathotyping test runs both on the ArrayMate Reader and on 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 your local distributor or Alere s technical service respectively www identibac com 05 16 04 0006 V05 8 IDENT BAC PROTOCOL The graphic on this page summarises the test procedure but due to limited space it is missing important details Therefore it is crucial to refer to the text section of this manual at any step of the test protocol pr
30. ward MJ Ehricht R Appl Environ Microbiol 2007 Jul 13 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 temperatures in the laboratory between 18 C and 28 C Kit components comprise the ArrayTubes 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 www identibac com If you have any problem or question please contact the technical service Quality Control Each batch is stringently tested with the use of standard DNA preparations for good performance and correctness of results www identibac com 05 16 04 0006 V05 25 IDENT BAC List of components for separate order If required these reagents for the E coli Genotyping Kit may be ordered separately component name category amount order no storage arayecoh o S CO c For pricing please contact your local representative or our customer service respectively LEGAL MANUFACTURER Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena Germany CONTACT If you require any further information on this product please contact Alere GmbH Am Wassermann 28 D 50829 K ln
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