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Kit Manual - Alere Technologies GmbH

1. Activate the tab results top left and click onto the position of an individual experiment the report of this particular array will appear on the right side of the window 9 Browse Q Search results results b raw data segmentation image image 2015 03 23 17 59 20 8 print 2015 03 27 09 37 26 For Research Use Only Not For Use In Diagnostic Procedures New Run 5 2015 03 27 10 00 41 Operator ines E 55 2015 03 27 10 04 06 5 2015 03 27 11 16 35 Sample ID C freu Bio DNA AMR CH Nord49 436 NDM 1 L5O 010415 2015 03 27 12 07 29 Experiment ID C freu Bio DNA AMR CH Nord49 R435 NDM 1 L50 010415 Archive 2015 03 30 15 56 45 239695A47 6C48 4E3B A2DA C439100E263C 2015 03 30 16 51 19 oa 2015 03 31 16 01 50 Date of Result Thu Apr 02 11 23 19 2015 2015 03 31 17 06 11 Assay Name AMR ve Genotyping AT 1 2015 04 02 11 07 29 Assay ID 050103 5 2015 04 02 11 19 28 C freu Bio DNA CH Nord49 R436 I Well Position D5 01 E c CH No j Software Version 2014 04 08 E coli Bio DNA Device 0430023 E coli Bio DNA 102 vLW 81 50 01 E coli Bio DNA DD EPEC 348 68 E coli Bio DNA DD EPEC E2348 69 Controls 55 2015 04 02 11 24 15 2015 04 08 17 20 48 veut explanation 2015 04 10 15 32 02 control result explanation 2015 04 10 16 37 25 Failed indicates possible pro
2. Barcode keep it clean hybridise 55 C 550 rpm 60 min discard labeled DNA incubate twice in 500 ul Buffer C2 30 C 550 rpm 5 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrat D1 25 C 60 min Omin 15 min 10 min discard Buffer C2 10min 2min incubate in 100 pl C3 C4 conjugate 30 C 550 rpm 10 min discard C3 C4 conjugate Simin Siti incubate twice in 500 ul Buffer C5 30 C 550 rpm 1 min discard Buffer C5 incubate with 100 ul Substrate D1 25 C 10 min Quantifoil BioShake iQ or Eppendorf Thermomixer 10 min 2min take image WITHOUT removing 01 analyse 5 min 2min MM MasterMix PM lt Beiner Mik total time requirement over night app 120 min 7 8h a with heating block for 1 5 ml Eppendorf tubes AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 35 www alere technologies com APPENDIX 2 IMAGES FOR TROUBLESHOOTING Comment Handling Valid experiment Valid results no error messages The bottom of the AT is contaminated with dust particles Please clean the bottom of the well scan and process again The microarray surface is contaminated with dust particles If the microarray surface is contaminated with particles wash the microarray with double distilled water pipetting water carefully up and down remove scan and process again The bottom of the AT is contaminated with a liquid
3. Intended Use For Research Use Only Not for Use in Diagnostic Procedures This kit allows the detection of resistance associated genes in isolates of Gram negative bacteria Enterobacteriaceae for research and epidemiological applications It must not be used as a substitute for phenotypic susceptibility tests and for the guidance of antibiotic therapy Please note It cannot be used for Gram positives 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 Email cct home clondiag com Phone 49 0 36 41 3111 155 Fax 49 0 36 41 3111 120 For up to date information regarding the kit please visit our website at http alere technologies com en products lab solutions amr ve genotyping html Safety Precautions e The kit is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from bacterial colonies clones requires expertise in microbiology and the local regulations for handling of pathogenic microorganisms usually biosafety level 2 are to be obeyed e Isolated cell free DNA may be processed without further biosafety precautions although bacterial contaminations need to be ruled out e Always wear protective clothes as required for laboratory work according to your local regulations AMR ve
4. Washing step after hybridisation repeat 13 Washing step Please note Acarryover of more than 1 of buffer C1 to the next step will denature the HRP Addition of HRP conjugate Please note Reagent C3 contains Streptavidin Horseradish Peroxidase HRP 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 Add 100 uL of prepared C3 CA mixture to each tube e Incubate in the thermoshaker at 30 C 550 rpm for 10 minutes e Remove and discard C3 C4 mixture completely Washing step after binding of conjugate Addition of HRP conjugate e Please keep in mind the limited surplus of C5 140 96 e 13 Washing step after conjugation add 500 uL of buffer C5 incubate in the thermoshaker at 30 C 550 rpm for 1 minute remove and discard the washing solution e 2 Washing step after conjugation Repeat 1 Washing step Please note A carryover of more than 0 5 of C3 C4 into the following staining reagent will create black particles which in the worst case may mimic signals hybridised spots On the same time real signals may appear pale due to competition of soluble Horseradish Peroxidase with the DNA bound enzyme for substrate molecules AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 18 www alere technologies com Staining of bound HRP conjuga
5. with the actual photo of the array e A second image file png in which the coordinate grid is superimposed and the recognised spots are circled segmentation image and e A XML file xml that contains the same information like the html result sheets for future export into databases and for using the Result Collector tool Please note Only complete runs can be exported The export of individual E coli Genotyping Test Reports is not possible e Right click on the selected run a menu appears with the option Export Run Reports e Right click on Export Run Reports a file browser opens E ArrayMate Browse 4 Search expe v order37x Ea ARCHIVE order37x 5 2009 02 05 09 00 42 order37x NewRun we Order42x01 Browse For Folder vee Order42x02 4 Order42x03 Choose a directory ve order42x06 order42x07 we Order78x01 order78x01 t B My Documents we Order78x08 My Computer H Se Local Disk C S Local Disk D H s Removable Disk E 0 Folder My Computer AMR ve Genotyping Kit 05 16 04 0005 07 Manual AMR ve Genotyping Kit 27 www alere technologies com TM Click My Computer then Removable Disk and choose the folder where to save or click Make New Folder on the bottom a new folder icon appears e Rename the new folder e g with the experiment name or date e Click Ok data are exported into the new folder o
6. 16 04 0005 VO7 Manual AMR ve Genotyping Kit 13 www alere technologies com 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 only use C2 Washing Buffer 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 label HERE Barcode keep it clean Avoid contact of data matrix barcode with organic solvents The ArrayMate needs the information encoded in the data matrix to perform the assay Avoid touching the bottom of the microarray and keep it clean General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent reservoirs Please keep in mind the limited surplus of C1 100 We strongly recommend that the liquid is removed by pipetting Fine tipped soft disposable Pasteur pipettes are suited best such as VWR Cat 612 2856 Pasteur pipette plastic with a flexible tip Ce T flexible tip AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 14 www alere technologies com 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 use the cavit
7. and speed need to be determined depending on viscosity of the sample and type of centrifuge used All liquid should be collected in the collection tube afterwards e Discard collection tube with liquids AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 10 www alere technologies com e Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 uL Buffer AW1 e Centrifuge at room temperature e Discard collection tube with liquids e Place the spin column in a new 2 ml collection tube provided with the 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 Discard collection tube with liquids e Place the spin column in a clean 1 5 ml tube provided with the 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 1 min to elute DNA e Centrifuge e Optional add another 50 uL Buffer AE or PCR grade distilled water directly onto the membrane incubate at room temperature for 1 min and centrifuge again e Discard the spin column Please note Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay A contamination with washing Buffer might occur during elution of prepared DNA by drops adhering to the funnel of the spin columns Thus these funnels should be gently
8. be prepared by disrupting bacterial cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols We made good experiences with the manual QIAGEN DNeasy kit and the automated device EZ1 DNA must be free of any traces of ethanol as ethanol strongly influences the amplification It is possible to heat the sample prior to adding it to the labelling mix 5 10 minutes at 70 C Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples see above AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 29 www alere technologies com Physical Damage to the Array Scratching of the array surface with a pipette tip can lead to the damage of array spots that prohibits the acquisition of a valid signal In this case the respective marker is not assigned as negative but instead the message none appears next to the marker name Ambiguous Results Apart from a positive or negative result for the individual markers on the AMR ve Genotyping Test Report the result can also be ambiguous In cases affecting markers for genes associated with resistance no definitive answer with regard to this specific marker can be given This can be caused by poor sample quality poor signal quality and especially in some resistance associated genes by the presence of plasmids in low copy numbers Allelic variants of some markers differ only in
9. blaCMY 9 MOX 4 lactamase precursor associated with resistance to cephalosporins AF381617 1 prob 1 21 blaOXA 1 blaOXA beta lactamclass D beta lactamase OXA 1 30 blaOXA 31 AY458016 1 blaOXA 33 prob_oxa2_11 blaOXA 2 blaOXA 3 beta lactamase OXA 2 oxacillinase U63835 1 blaOXA 15 blaOXA 20 blaOXA 21 blaOXA 22 blaOXA 32 blaOXA 34 blaOXA 36 X X X prob oxa7 11 blaOXA 35 class D beta lactamase OXA 7 AY866525 1 Xx prob oxa9 11 blaOXA9 beta lactamase OXA 9 M55547 1 e a a a a beta lactam resistance beta lactam resistance beta lactam resistance m m beta lactam resistance i beta lactam resistance per prob_per2_1 class A beta lactamase PER 2 extended spectrum beta lactamase X93314 1 prob_psel_1pm_ blaCARB 2 PSE 1 carbenicillinase 218955 1 prob_pse1_1mm blaCARB 2 PSE 1 carbenicillinase 218955 1 x x a a a a beta lactam resistance shv prob shv1 11 blaSHV PIT 2 class A beta lactamase consensus sequence for blaSHV genes including extended spectrum beta lactamases EF035566 1 m t t t t beta lactam resistance t prob tem1 1 blaTEM class A beta lactamase consensus sequence for blaTEM genes including extended spectrum beta lactamases Y12694 1 chloramphenicol prob catA1 11 chloramphenicol acetyltransferase group A resistance V00622 1 chloramphenicol acetyltransferase type AJ249249 1 chloramphe
10. e g buffer Please clean the bottom surface with a cleanroom wipe scan and process again AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit 36 www alere technologies com Signal intensity is too low This could be due to low DNA concentration fragmented DNA ethanol trace contaminations in DNA sample or expired reagents The experiment should be repeated with a new DNA preparation If this also fails try an experiment with EDL933 control DNA CM available on request Chip was not in focus during image acquisition Repeat image acquisition after fitting the ArrayTube in the frame AMR ve Genotyping Kit 05 16 04 0005 07 Manual AMR ve Genotyping Kit 37 www alere technologies com APPENDIX 3 GENE LIST acetyltransferase vatE hp vatE 611 vatE 3 vatE acetyltransferase streptogramin A 4 vatE 5 vatE acetyltransferase associated with resistance to 6 vatE 7 vatE 8 streptogramin A AF242872 1 satG acetyltransferase vatE hp vatE 612 vatE 3 vatE acetyltransferase streptogramin A 4 vatE 5 vatE acetyltransferase associated with resistance to 6 vatE 7 vatE 8 streptogramin A AF242872 1 satG resistance aminoaminoglycoside aac prob_aac3la_1 aacC1 3 N aminoglycoside acetyltransferase associated with resistance to astromicin gentamicin sisomicin U90945 1 aminoaminoglycoside aac resistance prob aac3lVa 1 aacC4 3 N ami
11. html Table 1 Example worklist Please note Table header must be written exactly as shown position samplelD assaylD 1 DD 018 UI 007807 MK L50 010415 050103 2 DD 038 UI 007961 L50 010415 050103 3 VLW B2162 00 419 L50 010415 050103 4 102 VLW 81 L50 010415 050103 5 DD EPEC E2348 69 L50 010415 050103 6 14050313 501 L50 010415 050103 Data Acquisition in the ArrayMate Reader e Insert your memory stick containing the worklist into any of the USB ports down to the right hand side of the ArrayMate e Press a folder selection dialogue will open e Select your worklist path My Computer Removable Disk e Open your selected worklist by pressing Enter or Open e Press your imported worklist opens in a separate window Proofread If the new window is empty or if it was the wrong worklist repeat the import e Press OK the worklist window will close AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 21 www alere technologies com photographed Leave the memory stick in the ArrayMate if you intend to export AMR ve Genotyping Test Reports afterwards check the memory stick for computer viruses and malware using an appropriate program on a regular basis Press Next at the bottom right on the screen reader is opening Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply strong force Assure proper fit otherwise the i
12. into spreadsheet tables This should make it easier to compare isolates or to determine relative abundances of genes or strains etc e Alere Worklist Generator is a tool which helps you to create a well formatted worklist for the Arraymate e Alere Report Generator is a software tool to create reports using the assay software normally used and installed on the ArrayMate It uses an image taken by the ArrayMate or a txt file raw signal data file and generates a report from the raw signal data AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 34 www alere technologies com APPENDIX 1 FLOW CHART The figure on this page summarises the test procedure However please refer to the text section of this user guide at any step of the test protocol for further important details prepare ArrayTubes prepare DNA pro hands cessing on time time over Grow CLONAL bacteria isolate night 10 min not part of the kit v d i isolate genomic DNA not part of the kit 3 4h 40 min label RNA free DNA in thermocycler rinse ArrayTubes 5 ul DNA Conga 0 1 0 4 ug ul 2 3h 10 min 500 ul water 55 C 550 rpm 2 min plus MM 4 9 uL B1 Mf 0 1 uL B2 discard water preparing labeled DNA o 10min 10min 500 ul Buffer C1 55 C 550 rpm 4 min to 10 uL of labeled DNA add 90 discard C1 process promptly of Buffer C1 transfer 100 pl labeled DNA to ArrayTube 2 min 2 min label HERE
13. 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 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 AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 11 www alere technologies com concentration A so 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 Extraction of DNA by Automated Device The assay has been tested with Qiagen s EZ1 Other systems also can be used However performance should be checked with some known reference strains prior to routine use e Add 10 uL proteinase K and add 100 uL buffer AL to harvested bacteria e Vortex shortly or shake vigorously e Incubate sample 45 60 min at 56 C and 550 rpm in the thermomixer e Add 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing e When the cells are lysed proceed by performing the tissue lysis protocol Bacteria card for Qiagen s EZ1 e For Qiagen s EZ1 Front row empty elution tubes 1 5 ml Second row tip holder with tips Third row empty Back row sample tube with conical tip 2 ml with the 200 uL sample volume Set tissue lysis p
14. 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 89 probes for different genetic markers plus controls All of them are spotted in duplicates The target set consists of a variety of antibiotic resistance genes of different Gram negative bacteria family specific markers for Enterobacteriaceae and different genus markers for Salmonella Escherichia and Shigella The array contains markers for genes associated with e family identification streptogramin A B resistance e aminoaminoglycoside resistance e beta lactam resistance e chloramphenicol resistance e macrolide resistance e quinolone resistance e sulphonamide resistance e tetracycline resistance e trimethoprim resistance e integrases AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 1 www alere technologies com GENERAL INSTRUCTIONS FOR USE
15. 0 106 prob_qnr_11 0 862581 1 000000 0 852517 0 010984 0 2015 09 31 17 06 11 107 prob sul2 11 0 857627 1 000000 0 085454 0 847605 0 E 2015 04 02 11 07 29 108 prob sul 11 0 867512 0 750000 0 867671 0 000172 Q0 E 2015 04 02 11 19 28 109 prob tet 11 0 860784 1 000000 0 066443 0 868741 0 C freu Bio DNA AMR CH Nord49 R436 11 prob tet 11 0 872906 1 000000 0 858946 0 015055 C freu Bio DNA_AMR_CH_Nord49 R436 110 prob tetB 11 0 871501 0 750000 0 874785 0 003547 O E coli Bio DNA AMR 102 VLW 81 L45 01 111 prob_tetC_11 0 874377 0 750000 0 872961 0 001525 0 E coli Bio DNA AMR 102 VLW 81 L50 01 112 prob tetD 1 0 875713 1 000000 0 837477 0 041104 0 E coli Bio DNA AMR DD EPEC E2348 69 113 prob tetE 11 0 877526 1 000000 0 857274 0 021727 0 E coli Bio DNA DD EPEC E2348 69 114 prob tetG 11 0 876887 0 756639 0 874602 0 002452 0 15 2 11 24 10 aac eee anman A e Spot ID numerical identifier of the spot on the array e Substance name of the DNA probe e Confidence an intrinsic estimate of spot confidence based on size and shape of that particular spot where 1 high confidence and 0 no confidence e Signal spot signal intensity grey scale value where 1 black and 0 white e Valid 0 valid 1 invalid confidence below 0 75 e Background luminous intensity of the background where 1 maximum brightness and 0 maximum darkness e Mean luminous i
16. 54 1 quinolione resistance nr prob qnrS 11 quinolone or fluoroquinolone resistance protein AM234722 1 ul prob qnr 11 quinolione resistance prob qnr 12 qnrA1 quinolone or fluoroquinolone resistance protein AY931018 1 sulphonamide 5 dihydropteroate synthetase type 2 DQ464881 1 resistance sulphonamide prob sul3 11 dihydropteroate synthetase type 3 AJ459418 2 resistance sulphonamide prob sul1 11 prob sul2 11 in in e e q q q q q 5 dihydropteroate synthetase type 1 AJ698325 1 AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 41 www alere technologies com resistance tetracycline resistance tet prob tetA 11 tetA2 tetA3 tetracycline resistance protein A tetracycline efflux protein CP000971 1 tet prob tetB 11 EE tetracycline resistance protein A class B V00611 1 tetracycline resistance tet prob tetC 11 tetracycline resistance protein A class C EU751612 1 prob tetD 1 tetracycline resistance protein A class D X65876 1 prob tetE 11 tetracycline resistance protein A class E L06940 1 tetracycline resistance tet prob tetG 11 tetA G tetracycline resistance protein A class G AF261825 2 tetracycline resistance tet prob tetG 12 tetA G tetracycline resistance protein A class G AF261825 2 resistance trimethoprim dfr prob dfrA1 22 dhfrl dihydrofolate reductase type AJ884723 1 resistance trimethoprim dfr prob_dfrA7_11 dfrA9 dhfrlX dhfrVI
17. Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 2 www alere technologies com Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest regulations EC 1272 2008 CLP and 1907 2006 REACH the enclosed reagents do not require a Material Safety Data Sheet MSDS except Hybridisation Buffer C1 The MSDS can be downloaded via our website from any lab solutions product page e g http alere technologies com en products lab solutions html All other reagents do not contain more than 1 of a component classified as hazardous and do not contain more than 0 1 26 of a component classified as carcinogenic Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents If liquid is spilled clean with a disinfectant and or laboratory detergent and water Alere assumes no liability for damage resulting from handling or contact with these products If you have any questions please contact our Technical Support see above Shipping Precautions RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 3 www alere techn
18. I dihydrofolate reductase type 7 AB161450 1 resistance trimethoprim dfr prob_dfrA7_12 dfrA9 dhfrlX dhfrVII dihydrofolate reductase type 7 AM237806 1 resistance trimethoprim dfr prob_dfr12_11 dhfrxil dihydrofolate reductase type 12 AB154407 1 resistance trimethoprim dfr prob_dfr13_11 dfrA21 dihydrofolate reductase type A13 dihydrofolate resistance reductase type A21 250802 3 trimethoprim dfr prob dfrA14 21 dhfrib dihydrofolate reductase type 14 313522 1 resistance trimethoprim dfr prob dfrA15 1 dihydrofolate reductase type 15 283311 1 trimethoprim dfr prob dfrA17 11 dihydrofolate reductase type 17 AF169041 1 trimethoprim dfr prob dfrA19 1 dihydrofolate reductase type 19 310778 1 trimethoprim prob dfrV 21 dihydrofolate reductase type 5 AB188269 1 resistance 1 http ardb cbcb umd edu index html 2 http www uniprot org AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 42
19. N 30 DISCLAIMER 31 QUALITY CONTRO Saasboceagssnapsdansnisossobnassdbosnabasaednasss 31 LIST OF COMPONENTS FOR SEPARATE ORDER 55 2 PER y SPA RENE ERE REM ERR AERE ERE T SIRE a Reis abe RR aeu Ras 32 LEGAL 32 32 Rudi uie T 33 UPDATES amp SOFTWARE 2 e ene seen osea 34 APPENDIX Rel ipsc 35 APPENDIX 2 IMAGES FOR TROUBLESHOOTING c ceeeeeee eene nennen nnne nnne nt nthssssssn stt sess assassins 36 APPENDIX 38 AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit www alere technologies com BACKGROUND The AMR ve Genotyping Kit allows a quick and simple method for the detection of the most common resistance genes of Gram negative organisms using an ArrayTube based assay RNA free un fragmented genomic DNA from pure and monoclonal Gram negative bacteria e g Escherichia coli or Salmonella enterica is amplified approximately 45 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
20. ate the tab image and the picture of this particular array will appear on the right side of the window Browse Q Search a 2015 03 23 17 59 20 2015 03 27 09 37 26 Y rc results results b raw data segmentation image image SS M 8 NewRun 2015 03 27 10 00 41 2015 03 27 10 04 06 51 2015 03 27 11 16 35 2015 03 27 12 07 29 Archive 2015 03 30 15 56 45 2015 03 30 16 51 19 2015 03 31 16 01 50 2015 03 31 17 06 11 84 2015 04 02 11 07 29 2015 04 02 11 19 28 45 11 E coli Bio DNA_AMR_102_VLW 81_L50_01 E coli Bio DNA_AMR_DD_EPEC E2348 69 E coli Bio DNA DD EPEC E2348 69 2015 04 02 11 24 15 2015 04 08 17 20 48 2015 04 10 15 32 02 2015 04 10 16 37 25 2015 04 10 16 45 41 5 2015 04 13 14 46 24 2015 04 15 15 19 21 5 2015 04 17 10 04 08 5 2015 04 17 17 00 38 2015 04 20 16 18 37 anir na antar an nai oe AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit 26 www alere technologies com Export of Test Results The generated result files in an html format will show information of all target genes Possible invalid controls that might display in this report will be explained below see Troubleshooting Other files that are generated and that can be exported include e Atext file txt with the raw measurements raw data e An image file bmp
21. be stable six months post expiry DNA Labelling and Amplification e B1 V Labelling Buffer Store at 2 C to 8 C Surplus 45 96 e B2 Labelling Enzyme Store at 2 C to 8 C Surplus 300 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 tubes are to be used within two weeks Close unused ArrayTubes protect them against humidity and dust and store in the dark Avoid ANY touching or scratching the microarray on the bottom of the vial Please note Do not store or handle unused wells above 60 relative humidity since this may irreversibly corrode the spots e 1 Hybridisation Buffer Store at 18 C to 28 C Protect against sunlight Surplus 100 96 e C2 Washing Buffer 1 Store at 18 C to 28 C Surplus 140 96 AMR ve Genotyping Kit 05 16 04 0005 07 Manual AMR ve Genotyping Kit 7 www alere technologies com e 3 100x HRP Conjugate Store at 2 C to 8 C Surplus 300 96 e C4 Conjugate Buffer Store at 18 C to 28 C Surplus 500 96 e C5 Washing Buffer 2 Store at 18 C to 28 C Surplus 140 96 e D1 Horseradish Peroxidase Substrate Store at 2 C to 8 C Protect against direct sunlight Surplus 200 96 e Optional CM Reference DNA from E coli EDL933 GenBank accession number NC 002655 2 Cona 0 1 0 4 ug uL Store at 2 C to 8 C Sufficient for 5 6 tests Components required but not provid
22. blems originating 2015 04 10 16 45 41 staining control passed from the hybridization or the staining procedure 2015 04 13 14 46 24 see troubleshooting section in the manual 2015 04 15 15 19 21 4 Failed indicates problems possibly originating 2015 04 17 10 04 08 negative control passed from the staining procedure see 2015 04 17 17 00 38 troubleshooting section in the manual 2015 04 20 16 18 37 Please Note the flag results b is not active with this assay AMR ve Genotyping Kit 05 16 04 0005 07 Manual AMR ve Genotyping Kit 24 www alere technologies com Activate the tab raw data top left and the raw signal results of this particular array will appear on the right side of the window Browse l Search results results b raw data segmentation image image SpotID Substance Background Confidence Mean Signal Valid ia 2015 03 23 17 59 20 10 prob tetD 1 0 871216 1 000000 0 843380 0 030079 0 2015 03 27 09 37 26 100 hp hemL 613 0 876399 1 000000 0 863634 0 013711 0 Newnes 2015 03 27 10 00 41 101 hp hemL 614 0 872782 1 000000 0 634337 0 257193 0 2015 03 27 10 04 06 102 hp_vatE_611 0 874328 1 000000 0 649763 0 241793 0 F 6 2015 03 27 11 16 35 103 hp vatE 612 0 870589 0 532051 0 873768 0 003437 EE i HA e 104 DIM NaPP 0 869767 0 750000 0 669282 0 000525 0O Le 105 biotin 0 864314 1 000000 0 061934 0 873948 0 301503 31 18 D1 S
23. eate a list with at least three columns that have headers written in the first line The following headers are obligatory in this order position samplelD assayID Table 1 e Positions are consecutively numbered from 1 to a maximum of 6 Do not leave empty lines in the worklist If you use EXCEL position numbers should be entered into column A Sample IDs are strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patients names should not be used as sample IDs e The Assay ID allows the system to identify the current test and to correctly use information on layout spot number and identity etc The AMR ve Genotyping Kit has the Assay ID 050103 Please note When entering assay IDs manually make sure to enter the correct number as entering wrong numbers could lead to errors or loss of data AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 20 www alere technologies com Werecommend using a printout of the worklist as a template for pipetting e Save the worklist as tab separated txt file on the memory stick provided together with the ArrayMate e To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted e You may use the software tool Worklist Generator to create a worklist easily http alere technologies com en products lab solutions software tools worklist generator
24. ecscsesesseascessceeessaaesessceseesaaasseseeeses 14 General Remarks the Substrate Precipitating Dye D1 ccccccccsscccsssseceessscecsessssecssseceessssecsessececsesseceessseeceeas 15 General Remarks Thermoshakers esie eeeess eise eene enean nn ss aa aaa nasi tese aaraa tn sein eaaa 15 intestate astaaueanace cn 16 DATASANAIYSIS 19 Starting the ArrayMate Reader sssssssssssssseee esses ene nn nnns essa than assa sies da sa assa sa 19 eriam 20 Data Acquisition in the ArrayMate Reader esses esses enean nennen rh anas sesenta sa daas assess sa sa ada asas ena 21 c 23 Export Of Test Results TETTE 27 TROUBLESHOOTING 28 28 IMAGE QUALITY cr 29 AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit www alere technologies com DNA QUALITY 29 PHYSICAL DAMAGE rond A Ln 30 AMBIGUOUS EER 30 ADDITIONAL INFORMATION 30 WARRANTY ER
25. ed e Growth media for the cultivation of bacterial isolates The test should be performed with colonies harvested from 2xTY Other rich media e g Columbia Blood Agar Standard 1 or LB also will suffice but have not systematically been tested Liquid media should not been used because contaminations or mixed cultures cannot be ruled out easily e Equipment and consumables needed for the cultivation of bacteria incubator inoculation loops Petri dishes e DNA preparation kits The assay has been tested with the DNeasy Blood Tissue Kit from Qiagen cat 69504 and the DNA preparation kit for Qiagen s EZ1 automated device cat 953034 e Equipment needed for DNA isolation e g pipettes centrifuge thermoshaker or automated device see above e 1x PBS e RNAse A we recommend Qiagen s RNase A solution 100 mg ml Qiagen Cat 19101 e Photometer OD 260 nm for measuring the concentration of DNA e Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA e Thermocycler for PCR e Thermoshaker Please note We recommend Eppendorf s Thermomixer Comfort equipped with a heating block for 1 5ml tubes AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 8 www alere technologies com e Pipettes suitable for 1uL 5uL volumes 90 100 200 1000uL e Multichannel Pipettes for 100 200 uL e Sterile reaction vials suitable for PCR VWR Cat 732 0098 e Ultrapure PCR g
26. es E My Documents c Shared Documents STICK E Removable Disk E Control Panel Devices with Removable Storage 4 Start installation double click on setup file of the AssayPlugin File Edit View Favorites Tools Help Qe 22 Search le Folders Address Conanttemp_public InesE AMR ve Folders x Name Size Type Date Modified B Desktop ArrayMate SDKs exe 468KB Application 7 17 2015 11 20 AM 5 amp My Documents AssaySoftware 050103 AMR ve 05m 2014 04 08 exe 319KB Application 3 23 2015 12 41 PM 9 My Computer B we System C 5 C3 Documents and Settings E C3 Admin AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 5 www alere technologies com 5 The welcome screen of the setup appears 15 Setup ArrayMateAssay_R_D 050103 AMR ve 05m Welcome to the ArrayMateAssay_R_D 050103 AMR ve 05m Setup Wizard This will install ArrayMateAssay_R_D 050103 AMR ve 05m 2014 04 08 on your computer It is recommended that you close all other applications before continuing Click Next to continue or Cancel to exit Setup 6 Follow the instructions and press Finish to complete the installation 7 Repeat this process for the SDK Setup 8 Log off and log in again as User R amp D default password abcde Test the AssayPlugin The Software installation can be tested with the unprocessed Array by the following steps 1 Logon to the A
27. esistance acc prob acc1 11 blaACC 1 ACC 1a4 class C beta lactamase EF554600 1 ACC 2 ACC 4 beta lactam resistance act1 prob_act1_11 blaACT 1 ACT 2 class C beta lactamase U58495 2 ACT 3 MIR 8 Mir2 Mir4 AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 38 www alere technologies com prob cmy 11 a UT hp blaCcMY 611 consensus sequence to blaCMY 13 blaCMY 2 blaCMY 24 blaCMY 35 blaCMY CFE1 blaCMY CFE2 Citrobacter spp blaCMY Cmur Citrobacter murliniae blaCMY Cwer Citrobacter werkmanii blaCMY Cyou Citrobacter youngae blaCMY HG3 blaCMY HG4 B E consensus sequence to blaCMY 13 blaCMY 2 prob ctxM1 11 ctxM15 ctxM28 extended spectrum beta lactamase class A beta ctxM29 ctxM3 lactamase X92506 1 including ctxM15 ctxM32 ctxM33 HQ202266 1 men 1 prob_ctxM1_12 ctxM15 ctxM28 extended spectrum beta lactamase class A beta ctxM29 ctxM3 lactamase X92506 1 including ctxM15 ctxM32 ctxM33 HQ202266 1 men 1 prob ctxM9 11 ctxM13 ctxM14 class A beta lactamase AF174129 3 ctxM16 ctxM17 CtxM18 ctxM21 ctxM24 ctxM27 ctxM51 ctxM9a ctxM9b KLUY 2 KLUY 3 KLUY A consensus sequences to blaCMY 13 blaCMY 2 blaCMY 24 blaCMY 35 blaCMY CFE1 blaCMY CFE2 Citrobacter spp blaCMY Cmur Citrobacter murliniae blaCMY Cwer Citrobacter werkmanii blaCMY Cyou Citrobacter youngae blaCMY HG3 beta lactam resistance beta lactam resistance b
28. eta lactam resistance blaCMY 24 blaCMY 35 blaCMY CFE1 blaCMY CFE2 Citrobacter spp blaCMY Cmur Citrobacter murliniae blaCMY Cwer Citrobacter werkmanii blaCMY Cyou Citrobacter youngae blaCMY HG3 blaCMY HG4 beta lactam resistance beta lactam resistance beta lactam resistance beta lactam resistance prob ctxM2 11 ctxM20 ctxM3 extended spectrum beta lactamase CTX M2 class A ctxM35 ctxM5 beta lactamase 4M040709 1 ctxM6 beta lactam resistance prob ctxM26 11 ctxM25 ctxM39 extended spectrum beta lactamase class A beta ctx M41 lactamase AF518567 2 beta lactam resistance prob ctxM8 11 blaKLUG 1 ctxM40 extended spectrum beta lactamase class A beta ctxM63 lactamase AY750914 2 consensus sequence for blaFOX genes AJ703795 1 fox consensus sequence for blaFOX genes AJ703795 1 beta lactam resistance mox hp blaMOX ampC blaCMY 10 class C beta lactamase extended spectrum beta CMY9 611 blaCMY 9 MOX 4 lactamase precursor associated with resistance to cephalosporins AF381617 1 AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 39 www alere technologies com beta lactam resistance hp blaMOX ampC blaCMY 10 class C beta lactamase extended spectrum beta CMY9 612 blaCMY 9 MOX 4 lactamase precursor associated with resistance to cephalosporins AF381617 1 beta lactam resistance hp blaMOX ampC blaCMY 10 class C beta lactamase extended spectrum beta CMY9 613
29. guided by phenotypic susceptibility tests Furthermore we do not accept any liability for damages caused by inappropriate use of the device as a personal computer for instance related to the use of additional software to network connections or to a breach of privacy related to the storage of confidential information such as names of patients on its hard disk and or to the use of external storage devices that might be contaminated with spyware Quality Control Each batch is stringently tested with the use of standard DNA preparations for good performance and correctness of results AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 31 www alere technologies com List of Components for Separate Order If required these reagents may be ordered separately compost mme amou uw sere p labeling rayme 2st 248102000 ci mbrdsetonSufer pomi 1828 C cz WssmgBuferi Xm 245106000 1828 Conjugate 100 a00ut_ 248107000_ 2 8 c pomi 245108000 1828 C cs WasmgBuferz Xm 24510000 1828 pi RP substrate smi 5009 feere B1 B2 Ci C2 C3 C4 C5 D1 M 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 cct h
30. he archive by default they are named by date and time of day of creation which you may have changed see section setup of the ArrayMate reader Browse Q Search TotalSize of D 107 GB FreeSapce on D 96 GB Ea ARCHIVE 2013 12 06 13 25 51 New Run 5 2015 01 26 09 59 25 2015 01 26 13 47 41 2015 01 26 14 09 46 2015 01 26 14 18 28 Archive 2015 01 30 13 03 40 2015 01 30 13 24 09 2015 01 30 13 44 58 2015 01 30 13 46 29 7 9 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 l Search experiment sample ID position assay image raw data results E coli Bio E coli Bio D 1 050103 x x a amp 2015 04 02 11 07 29 E coli Bio E coli Bio D 2 050103 x x x 2015 04 02 11 19 28 P E coli Bio E coli Bio D 3 050103 x x x New Run C freu Bio DNA CH Nord49 R436 P E coli Bio E col Bio D 4 050103 x x x C freu Bio DN4 amp _AMR_CH_Nord49_R436_f C freu Bi C freu Bio 5 050103 x x 5 E coli Bio DN AMR 102 VLW 81 45 01 C freu Bi C freu Bio 6 050103 x x x E coli Bio DM AMR 102 VLW 81 150 01 Archive E coli Bio DNA DD EPEC E2348 69 E coli Bio DNA DD EPEC E2348 69 2015 04 02 11 24 15 AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit 23 www alere technologies com
31. he final image of the DNA microarray on the ArrayMate a specific software plug in is required This software plugin can be downloaded from our website www alere technologies com under Downloads plug ins Please install it on your reader according to the following instructions AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 4 www alere technologies com Assay Plugin and SDK for the ArrayMate The following instruction describes the installation of the AssayPlugin and ArrayMate installation software SDK Download the AssayPlugin and the ArrayMate SDK from http alere technologies com en products lab solutions amr ve genotyping html 1 Copy downloaded files Plugin and SDK setup to an USB Memory Stick and connect it to the ArrayMate 2 Log on as user admin to the ArrayMate default password 12345 3 Open the Windows Explorer and navigate to the downloaded setup files 5 My Computer File Edit View Favorites Tools Help 5 93 pa Search Folders Address 3 My Computer Name Type System Tasks Files Stored on This Computer View system E Shared Documents File Folder information E Admin s Documents File Folder programs user s Documents File Folder g Change a setting user R amp D s Docu File Folder Eject this disk 3 Hard Disk Drives System C Local Disk Se Data D Local Disk Other Places My Network Plac
32. image quality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel In order to determine whether any problems originated from the DNA preparation you might perform an experiment with the Control Material CM P This is genomic DNA from E coli EDL933 GenBank accession number NC 002655 2 It is be provided free of charge upon request If the control experiment yields a valid result and a correct identification of probes gapA ihfA dnaE and hemL there was probably an issue with DNA preparation If the control experiment also fails an error affecting later steps or a degradation of reagents from later steps is likely See also Appendix 2 Images for troubleshooting p 35 and 36 DNA Quality The amount of DNA is crucial because of the linear kinetics of amplification see Introduction DNA should be free of RNA as free RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation A260 readings will cover RNA and other contaminants as well Therefore pure DNA preparations without RNA contaminations are a prerequisite for proper DNA concentration measurement RNAse treatment prior to A260 reading therefore is necessary DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites DNA should for this reason not
33. mages may be out of focus 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 Barcodes on ArrayTubes and holder must be clean Press Next at the bottom right on the screen reader closes analysis program starts it takes about 2 10 min depending on the number of ArrayTubes the reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols in analysis ready The reader indicates the end of the entire process with an acoustic signal beep Press Next at the bottom right on the screen reader is opening Remove the adapter with the ArrayTubes Press Next at the bottom right on the screen reader is closing AMR ve Genotyping Kit 05 16 04 0005 V07_Manual_AMR ve_Genotyping Kit 22 www alere technologies com Results On the left hand side of the screen you will see a list showing all runs stored on the ArrayMate s hard disk A run contains the results from all arrays analysed together within one frame If this list is not displayed e Press Archive left hand side and activate the flag Browse at the top left e The runs are organised like folders in Windows Explorer and named by default according to the date of acquisition Example there are several readings in t
34. n your memory stick e Do NOT remove the memory stick as long as the hourglass symbol is visible e Switch off the device by clicking Power at the bottom left on the screen D e Switch off the screen There is no need to physically switch off the ArrayMate Reader TROUBLESHOOTING In case of trouble always make sure that reagents are within the recommended shelf life and stored under appropriate conditions Should you encounter a problem we will always be happy to support you Please e mail to cct home clondiag com and include a description of the problem as well as the array images bmp files in question Staining Control A staining control is included to check whether possible problems originate from the hybridisation or the staining procedure If the staining control has Failed proceed as follows Horseradish peroxidase conjugate may have degraded during storage Add 1 uL mixture 3 4 to 9 uL D1 substrate If the solution turns green within 3 5 seconds the horseradish peroxidase still has sufficient enzymatic activity Enzymatic reaction is inhibited by carryover of buffer C1 Ensure proper washing with buffer C2 of the wells to remove all of buffer C1 prior to adding horseradish peroxidase conjugate If the staining control has Passed refer to the following hints AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 28 www alere technologies com Image Quality In case of poor
35. nicol prob_catB3_11 catB4 chloramphenicol acetyltransferase group B resistance AJ009818 1 chloramphenicol prob catB8 12 chloramphenicol acetyltransferase AF227506 1 resistance chloramphenicol cml prob_cmlA1_11 cmlAcmlA1 cmlA4 chloramphenicol transporter EF113389 1 resistance cmlAS cmlA6 cmlA7 chloramphenicol flo prob_floR_11 florfenicol export protein AF252855 1 resistance erythromycin ereA hp_ereA_611 ereA2 type erythromycin resistance AY183453 1 resistance erythromycin ereA hp_ereA_612 ereA2 type erythromycin resistance AY183453 1 resistance erythromycin ereB hp_ereB_611 ereB type Il erythromycin resistance AB207867 1 resistance erythromycin ereB hp ereB 612 ereB type Il erythromycin resistance AB207867 1 resistance family marker gapA prob gapA 611 glyceraldehyde 3 phosphate dehydrogenase A marker for Enterobacteriaceae AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 40 S chloramphenicol prob catlll 1 resistance www alere technologies com famly marker ihfA prob ihfA 611 integration host factor subunit alpha marker for Enterobacteriaceae genus marker dnaE hp dnaE 611 DNA polymerase subunit alpha Species marker for Salmonella spec Escherichia spec and Shigella spec genus marker dnaE hp dnaE 612 DNA polymerase subunit alpha Species marker for Salmonella spec Escherichia spec and Shigella spec genus marker dnaE hp dnaE 613 DNA p
36. noglycoside acetyltransferase associated with resistance to apramycin dibekacin gentamicin netilmicin sisomicin tobramycin EU784152 1 prob aac6lb 1 aacA4 aminoglycoside 6 N acetyltransferase associated with resistance to streptomycin spectinomycin M21682 1 aminoaminoglycoside aadA prob_aadA1_1 aadA1 aminoglycoside adenyltransferase associated with resistance resistance to streptomycin spectinomycin EU704128 1 aminoaminoglycoside aadA prob aadA2 1 aadA2a aadA2b aminoglycoside adenyltransferase associated with resistance aadA2c aadA3 resistance to streptomycin spectinomycin aadA8 EU704128 1 aminoaminoglycoside resistance aminoaminoglycoside aadA prob aadA4 1 aadA5 aminoglycoside adenyltransferase associated with resistance resistance to streptomycin spectinomycin EU704128 1 aminoaminoglycoside ant2 prob ant2la 1 aadB aminoglycoside 2 adenylyltransferase associated resistance with resistance to dibekacin gentamicin kanamycin sisomicin tobramycin L06418 4 aminoaminoglycoside strA prob strA 611 aph3 aminoglycoside 3 phosphotransferase locus A resistance associated with resistance to streptomycin EF090911 1 aminoaminoglycoside strB prob strB 611 aph6 aminoglycoside 6 phosphotransferase associated resistance with resistance to streptomycin EF090911 1 beta lactam resistance acc prob acc2 11 blaACC 1 ACC 1a4 class C beta lactamase EF554600 1 ACC 2 ACC 4 beta lactam r
37. ntensity of the signal spot where 1 maximum brightness and 0 maximum darkness Please note 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 AMR ve Genotyping Kit 05 16 04 0005 07 Manual AMR ve Genotyping Kit 25 www alere technologies com Activate the tab segmentation image and the analysed picture of this particular array will appear on the right side of the window Browse S Search results results b raw data segmentation mage image d 2015 03 23 17 59 20 2015 03 27 09 37 26 New Run 2015 03 27 10 00 41 2015 03 27 10 04 06 5 2015 03 27 11 16 35 2015 03 27 12 07 29 Archive 2015 03 30 15 56 45 5 2015 03 30 16 51 19 2015 03 31 16 01 50 2015 03 31 17 06 11 2015 04 02 11 07 29 S 2015 04 02 11 19 28 C freu Bio DNA CH Nord49 R436 t E col Bio DNA 102 VLW 81 145 01 E col Bio DNA AMR 102 VLW 81 150 01 E coli Bio DNA AMR DD EPEC E2348 69 E coli Bio DNA DD EPEC E2348 69 5 2015 04 02 11 24 15 2015 04 08 17 20 48 2015 04 10 15 32 02 2015 04 10 16 37 25 2015 04 10 16 45 41 2015 04 13 14 46 24 2015 04 15 15 19 21 2015 04 17 10 04 08 2015 04 17 17 00 38 2015 04 20 16 18 37 2015 04 20 16 23 21 Activ
38. ologies com DEVICES SOFTWARE AND REAGENTS Devices e ArrayMate Reader to be ordered separately for details see below e Alternatively Reader ATRO3 to be ordered separately for details see below e conoclust software provided with the reader e Report Generator optional Whilst the AMR ve Genotyping assay 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 us Assay specific software plug in is delivered with the reader or can be downloaded from our website where it will occasionally be updated The ArrayMate Reader by default has all software on board However the AMR ve Genotyping assay specific package might be missing e g if you obtained the device for the use with another assay Then you may need to install it separately It will be provided upon kit order and can also be downloaded from our website as discussed above No issues regarding compatibility of software have been observed with the ArrayMate device The ATROS3 reader requires several pieces of software to be installed on an external PC please refer to the latest version of the ATRO3 manual for details The AMR ve Genotyping assay specific software is not compatible with iconoclust versions older than version 4 4 Software Installation For analysis of t
39. olymerase subunit alpha Species marker for Salmonella spec Escherichia spec and Shigella spec hp hemL 611 gsa popC glutamate 1 semialdehyde aminotransferase genus marker hemL hp hemL 612 gsa popC glutamate 1 semialdehyde aminotransferase Species marker for Salmonella spec Escherichia spec and Shigella spec Species marker for Salmonella spec Escherichia spec and Shigella spec genus marker hemL hp hemL 613 gsa popC glutamate 1 semialdehyde aminotransferase Species marker for Salmonella spec Escherichia spec and Shigella spec genus marker hemL hp hemL 614 gsa popC glutamate 1 semialdehyde aminotransferase Species marker for Salmonella spec Escherichia spec and Shigella spec integras imt prob intl1 1 class 1 integrase AY260546 3 integrases int prob intl2 11 class 2 integrase AY183453 1 macrolide rmB hp_ermB_611 rRNA adenine N 6 methyltransferase lincosamide and streptogramin B resistance protein 89505 1 and streptogramin B resistance protein 89505 1 qnrA1 quinolone or fluoroquinolone resistance protein AY931018 1 hp_ermB_612 rRNA adenine N 6 methyltransferase lincosamide t t quinolione resistance nr prob qnrB 11 qnrB2 qnrB3 qnrB4 quinolone or fluoroquinolone resistance protein qnrB5 qnrB6 AB281054 1 quinolione resistance nr prob qnrB 12 qnrB2 qnrB3 qnrB4 quinolone or fluoroquinolone resistance protein qnrB5 qnrB6 AB2810
40. ome clondiag com AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 32 www alere technologies com LITERATURE 1 Development of a miniaturised microarray based assay for the rapid identification of antimicrobial resistance genes in Gram negative bacteria Batchelor M Hopkins KL Liebana E Slickers P Ehricht R Mafura M Aarestrup F Mevius D Clifton Hadley FA Woodward MJ Davies RH Threlfall EJ Anjum MF Int J Antimicrob Agents 2008 Feb 1 2 Identifying antimicrobial resistance genes of human clinical relevance within Salmonella isolated from food animals in Great Britain Muna F Anjum Suman Choudhary Victoria Morrison Lucy C Snow Muriel Mafura Peter Slickers Ralf Ehricht and Martin J Woodward J Antimicrob Chemother 2011 For further literature please refer to http alere technologies com en science technologies publications AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 33 www alere technologies com UPDATES amp SOFTWARE Notifications on database software updates and freeware tools can be found at http alere technologies com en products lab solutions e coli e coli genotyping kit html http alere technologies com en products lab solutions software tools html and or http alere technologies com en news html Currently available freeware programs are e Alere Result Collector for the conversion of multiple result xml files from the ArrayMate
41. rade water e Pasteur pipettes VWR Cat 612 2856 PROTOCOLS Culturing and Harvesting Bacterial Cells Enterobacteriacae are potential pathogens All procedures for cultivation of the bacteria and DNA preparation need to be performed by properly trained staff in a biosafety level 2 facility Grow bacteria on suitable agar see above overnight at 37 C or 48 hrs at room temperature Make sure that you have a pure monoclonal culture Contamination with other bacteria needs to be strictly avoided as this can introduce false positive signals and patterns Extraction of DNA The required sample type for the AMR ve Genotyping test is 0 5 2 ug cDNA 0 1 0 4 ug uL of intact DNA from a single clone of Gram negative bacteria This is much more DNA than for standard PCR applications see Introduction 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 cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend following the protocols outlined below The use of automated systems for DNA preparation EZ1 Qiacube Magnapure etc has not yet been systematically evaluated with this assay While there are positive experiences with some of our other assays
42. rotocol with a set sample volume of 200 uL and an elution volume of 50 e Concentrate DNA and evaporate traces of solvents by heating the sample at 70 C for 5 10 minutes AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 12 www alere technologies com Linear Amplification and Internal Biotin Labelling AMR ve Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 labelling reagent is 4596 e Prepare a Master Mix by combining 4 9 uL of B1 V labelling reagent and 0 1 uL of B2 DNA polymerase per sample e Add 5 uL of E coli DNA cona 0 1 0 4 ug uL prepared as described above to 5 uL of the Master Mix B1 V B2 Do not forget to label the vial e Perform amplification in a pre programmed thermocycler such as Mastercycler gradient with heated lid according to following protocol Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 50 C 45 cycles with 30 sec at 72 C 20 sec at 96 C Cool down to 4 C hold e The amplification products can be stored frozen until usage Please note When using another device some adaptations might be necessary Before starting routine use please test the protocol with a few known reference strains and the control DNA CM supplied with the kit Hybridisation procedure General Remarks Handling of Arrays e Never touch the array surface AMR ve Genotyping Kit 05
43. rrayMate in User R amp D Mode default password abcde and start a New Run 2 Choose automatic detection in Experiment Infos and press Next Place the ArrayTube rack with an unprocessed ArrayTube into the ArrayMate than press Next After the Experiment Run the ArrayMate automatically enters the Archive mode and displays the results of the last experiment 3 Each cell of the columns image raw data and results must contain an X Otherwise please retry the installation process of the AssayPlugin and the installation software SDK AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 6 www alere technologies com Exper gt sample ID position assay image raw data results 01 4 1 050103 x X 01 B 2 050103 X X X D1 C 3 050103 X X x 1 4 050103 X X x 01 E 5 050103 x x x 01 6 050103 X X x Kit Components Storage and Stability All reagents are provided in surplus see below If necessary all components may be ordered separately please refer to the catalogue reference numbers Cat at the end of this manual For pricing please contact your local representative or our customer service respectively The expiry date can be found on each bottle and on the outer packaging All components have been stability tested for short term shipment lt 1 week at ambient temperature lt 37 C The assay components with limited stability are D1 and C3 The other kit components have proven to
44. scsseecececuecnsssseceseceeeasessecesseueguneesseessegagaseeeeees 7 DNA Labelling and Amplification ii rii dha cadessnasbedaagiadssaacaiee lt assaceveas 7 Hybridisation and Detection sess eese sensn nsns nh annn nasse sea aaa assa sa sa dadas sss sss s da sa dass assa essa sa assa dan 7 COMPONENTS REQUIRED BUT NOT PROVIDED us csenctencenstesschsesersonnchasesaeedeesneveadeshtevecesheekucedtendtentacutersahtakeesobesteenchanebentest gt 8 PROTOCOLS ea 9 CULTURING AND HARVESTING BACTERIAL CELUS cicssncessvactecssnsecveianssnscssnieesesocacncstepseosatentsosocenaacesssonecedcesnneedosattesvsbsnacevanses 9 EXTRACTION OF IDNA 9 Extraction of DNA by Spin Columns e g Qiagen DNeasy Blood amp Tissue cesses 10 Extraction of DNA by Automated Device sccscccccceseessssscesccsssesssecesecsseessesesecseeesesseassesscseeasaassesseseessaasseseeeses 12 LINEAR AMPLIFICATION AND INTERNAL BIOTIN LABELLING essere enne nnne nnnennn tnn ne nnne th sn trennen 13 HYBRIDISATION aiai 13 General Remarks Handling of Arrays eese 13 General Remarks Handling of LiQuids ccssscccccccsseessssssescesseesesesesecsceeesseses
45. single or few nucleotides This can cause the effect that the actual allele yields a positive signal while other mismatching probes give ambiguous rather than negative results ADDITIONAL INFORMATION Warranty Alere guarantees the performance as described in this manual Usage of the 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 If you have any problem or question please contact the technical service AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 30 www alere technologies com Disclaimer This system is for research use only We do not accept any liability for damages caused by misuse Misuse comprises especially but not exclusively of a use of the system for the detection of resistance genes in order to predict phenotypic antibiotic resistances or susceptibilities for the guidance of an antibiotic chemotherapy Since resistance might be caused by genes or mutations not covered by this array or by hitherto unknown genes or mutations any antibiotic chemotherapy MUST be
46. te Please note Do not move ArrayTubes during staining The reagent D1 contains a substrate for Horseradish Peroxidase Please keep in mind the limited surplus of D1 200 26 e Set the thermoshaker to 30 C for the following steps Add 100 uL pre warmed reagent D1 to each well supernatant of centrifuged D1 without precipitate e Incubate at 25 C WITHOUT agitation for 10 min e Read out WITHOUT removing D1 removal of D1 would leave air bubbles within the tubes Please note The ArrayTubes as used in this kit do have a different geometry than the 8 well ArrayStrips that are used in other kits Therefore unlike the directive for ArrayStrips D1 is NOT to be removed from the ArrayTubes before reading Check immediately all images for cleanliness i e absence of dust particles residual liquids and for good focus Dust particles and residual fluids inside the vial can be removed by cautiously washing twice with 200 uL PCR grade distilled water If necessary scan and process again Data Analysis Starting the ArrayMate Reader e We recommend starting the ArrayMate Reader after starting the hybridisation this allows the convenience of starting the device and importing the worklist file see below e Please note that this is a short instruction only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate main switch on the rear below the electric cable plug operating switch on
47. te Meanwhile login to the ArrayMate device and prepare your worklist see section worklist S 21 Dilute Streptavidin Horseradish Peroxidase C3 C4 e Combine reagent C3 Streptavidin Horseradish Peroxidase and Buffer C4 in a ratio of 1 100 the mixture is stable for 1 day at room temperature C3 is delivered with a surplus of 300 C4 is delivered with a surplus of 500 Pipetting scheme ArrayTubes AT 1 AT 2 3 ATs 4 6 ATs 7 10ATs 11 15 ATs C3 15ygL 35ygL 7 11 16 uL C4 150gL 350gL 700uL 1100 1600 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 Washing after hybridisation e Please keep in mind the limited surplus of C2 140 96 e Remove the ArrayTubes from the thermoshaker e Set the thermoshaker to 30 C for the following steps e Carefully open the tubes and remove the hybridisation mixture as completely as possible without touching the array surface AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 17 www alere technologies com e 1 Washing step after hybridisation add 500 pL of buffer C2 and incubate in the thermoshaker at 30 C 550 rpm for 5 min remove and discard the washing solution e 2
48. the bottom left corner of the front side e Switch on the screen switch right hand below the screen AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 19 www alere technologies com e Log in as R amp D User Research and Development User for full access to test specific software default password abcde If you log in as User you will obtain only raw values but neither positives negatives interpretation nor strain assignment The Administrator log in default password 12345 will allow the installation of a new assay specific plug in which can be downloaded at http alere technologies com e The user interface will be loaded ArrayMate performs internal testing This will require slightly less than a minute Click on the icon New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience Worklist A Worklist file allows an identifier such as a laboratory or sample number to be linked to the respective array position on the ArrayStrip For privacy reasons arrays should not be identified by patient names Worklists can be generated using spreadsheet software such as EXCEL see below but must be saved in the txt file format that can be imported into the test specific ArrayMate software Do not use special characters such as V etc e Cr
49. we recommend testing some known strains for evaluation prior to routine AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 9 www alere technologies com 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 Extraction of DNA by Spin Columns e g Qiagen DNeasy Blood amp Tissue e Add an inoculating loop full of monoclonal colony material of the E coli isolate to 0 2 ml 1xPBS and vortex thoroughly Loop empty Loop full It is important to harvest enough bacteria this is a prerequisite for extraction of a sufficient amount of DNA Take an inoculating loop of 1 mm diameter filled with bacteria as shown in the left picture e Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit that is as follows e Add25 ul proteinase K Qiagen Kit or equivalent and add 200 uL buffer AL Qiagen Kit e Vortex shortly or shake vigorously e Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker e Add 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing e Add 200 uL ethanol 96 10096 e Vortex the sample and centrifuge shortly e Transfer the complete content of the tube into a spin column that is placed in a 2 ml collection tube e Centrifuge at room temperature time
50. ww ginstruments com equipped with a customised heating block designed to fit ArrayTubes and Eppendorf s Thermomixer Comfort equipped with a heating block for 1 5 ml Eppendorf tubes Thus we recommend the use of either device When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference strains or the control DNA supplied with the kit Protocol 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 Remove the ArrayTube from the bag open the bag at its predetermined breaking point Add 500 of ultrapure water to each tube Incubate in the thermoshaker at 55 C 550 rpm for 2 minutes e Remove and discard the water WITHOUT TOUCHING THE ARRAY SURFACE Add 500 uL buffer C1 to each tube Incubate in the thermoshaker at 55 C 550 rpm for 4 minutes e Remove and discard buffer C1 Proceed promptly hybridisation mixtures must be ready when buffer C1 is removed AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 16 www alere technologies com Hybridisation e Transfer each hybridisation mixture 100 uL to a prepared ArrayTube avoid extensive foaming e Incubate for one hour at 55 C and 550 rpm on a thermoshaker No
51. www alere technologies com www alere com Manual AMR ve Genotyping Kit Array Hybridisation Kit for DNA based detection of the most common resistance genes of Gram negative bacteria Kit order number 205300050 50 reactions ArrayTube format For Research Use Only Not for Use in Diagnostic Procedures www alere technologies com www alere com CONTENT BACKGROUND 1 GENERAL INSTRUCTIONS FOR USE zioenean osaansa ananasen esanen tuens sat eda tn SAS uan deen E Oe EAAS SASEA s ane E rne esee 2 ipsi M 2 C 2 TECHNICAL 2 SAFETY PRECAUTIONS 2 MATERIAL SAFETY DATA SHEETS MSDS scccsssccceessececesssececsssseceesaececesssececsssecceesaesecesasececsssseceesseeecsessacecsssseceesaeseceeaaes 3 SHIPPING PRECAUTIONS PES 3 DEVICES SOFTWARE AND REAGENTS 5 2 4 4 SOFTWARE INSTALLATION misici derenn REAR E E aE APEA EA M 4 Assay Plugin and SDK for the ArrayMate cccccccccessessssscesecessessasscesecsseeensesesecsseeseasesesecseeesesseaesecscceseaseasseeseseesaea 5 KIT COMPONENTS STORAGE AND STABILITY cc ccssssssscecececeecssescecesseeeessescececsen
52. y between array and the wall of the tube do never touch the 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 warm to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged 1 min 13 000 rpm prior to use to remove bubbles as well as possible precipitates Triggered by peroxidase in case of positive reactions the dye precipitates but it is not covalently bound The precipitate can be dissolved by vigorous shaking Thus the arrays must not be shaken dropped or moved abruptly during the staining procedure and afterwards After completion of staining do not remove reagent D1 and scan immediately The dye precipitate fades slowly in presence of liquids General Remarks Thermoshakers The correct temperature within the vessels is essential therefore always use the appropriate equipment for heating Because of a possibly inhomogeneous distribution of the temperature within the heating block and because of possible differences between displayed and actual AMR ve Genotyping Kit 05 16 04 0005 VO7 Manual AMR ve Genotyping Kit 15 www alere technologies com temperatures the use of different brands of thermoshakers might affect test performance We tested the assay with BioShake iQ by Quantifoil Instruments http w

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