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1. Environ Microbiol 77 8784 8786 Schilling AK Hotzel H Methner U Sprague LD Schmoock G et al 2012 Zoonotic agents in small ruminants kept on city farms in southern Germany Appl Environ Microbiol 78 3785 3793 Wu G Ehricht R Mafura M Stokes M Smith N et al 2012 Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages Vet Microbiol 160 197 206 For further literature please refer to http alere technologies com en science technologies publications downloads html E coli SeroGenoTyping AS 1 Kit 34 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com UPDATES AND SOFTWARE Notifications on database software updates and freeware tools can be found at 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 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
2. Take an inoculating loop of 1mm diameter filled with bacteria as shown in the right picture Optional cell lysis with A1 A2 reagent instead of 1x PBS e Centrifuge A2 tube shortly open it add 0 2 ml of Lysis Buffer A1 to Lysis Enhancer A2 and dissolve e Add an inoculating loop full of monoclonal culture material of the E coli isolate to this A1 A2 reagent and vortex thoroughly e Incubate the culture material of the E coli isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker e Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit it is as follows e Add 20 ul proteinase K from 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 coli SeroGenoTyping AS 1 Kit 9 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Important If A1 A2 reagent is not used add now 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing e Add 200 ul ethanol 96 100 e Vortex the sample and centrifuge quick spin e Transfer the complete tube content including any precipitate into a spin column that is placed in a 2 ml collection tube e Centrifuge 8 000 rpm 1 min at room temperature Time and speed need to be determin
3. coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Protocol for Quantifoil s BioShake iQ BioShake iQ by Qlnstruments equipped with a customised heating block designed to fit ArrayStrips http www ginstruments com Switch on the thermoshaker and let it pre heat to 50 C Remove the amount of ArrayStrip s needed from the pouch Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode close to row A and proper fit Pre wash the array s in two steps e First PCR grade distilled water 200 ul per well at 50 C 5 min at 550 rpm Remove the water from the well e Second C1 Hybridisation Buffer 150 ul per well at 50 C 5 min at 550 rpm Add 90 ul of C1 buffer to each tube with 10 ul labelled amplification product mix gently Remove the buffer from the well and add the mixture of C1 and labelled amplification product Incubate at 50 C 60 min at 550 rpm Meanwhile login to the ArrayMate device and prepare your worklist see section Data Analysis p 22 Remove the liquid and add 200 ul C2 Washing Buffer Incubate at 45 C 10 min at 550 rpm remove and discard E coli SeroGenoTyping AS 1 Kit 16 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Add another 200 ul C2 Washing Buffer Incubate at 45 C 10 min at 550 rpm Meanwhile prepare conjugate For each experiment add 1 ul
4. 0168 hp1148 wzy 0168 wzx 0172 11 wzy O172 11 hp1085 wzx 0174 hp1149 wzy 0174 hp1086 wzx 0177 hp1150 wzy_0177 O119 O121 O123 O124 O164 0126 0127 0128 0130 0138 0139 0141 0143 0145 0146 0147 0148 0149 0159 0167 0168 0172 0174 0177 O antigen 0118 0151 O antigen 0124 0164 O antigen 0149 DQ091854 2 O antigen O150 EU294168 1 O antigen O152 EU294170 1 O antigen 0157 AB008676 1 O antigen O147 DQ868766 1 O antigen O148 DQ167407 1 O antigen O177 DQ008593 1 H serotyping HO1 fliC HO1 11 fliC HO1 12 HO2 fliC HO2 11 HO3 fIKA HO3 11 HO4 fliC HO4 11 HO5 fliC HO5 11 HO6 fliC HO6 11 E coli SeroGenoTyping AS 1 Kit 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit AE014075 1 AF543692 1 flagellin A H antigen H03 AB128916 1 Note If repressor FIjA positive the gene fliC H16 is repressed H phenotype is H03 AY249989 1 AY249990 1 AY249991 1 www alere technologies com www alere com AB028474 1 AJ884571 1 AY249994 1 AY249995 1 AY337465 1 AY249997 1 AY249998 1 AY249999 1 H16 fliC H16 11 flagellin C H antigen H16 AB128919 1 Note If repressor fljA positive fliC H16 might be repressed H phenotype is than determined by flkA HO3 AY250001 1 AY250002 1 AY250003 1 H21 fliC H21 11 flagellin C H antigen H21 DQ862122 1 Note If repressor fljA positive fliC H21 might be repressed H pheno
5. C3 conjugate 100 x HRP to 100 ul C4 Conjugation Buffer This mixture is stable for one working day at room temperature C3 is delivered with a surplus of 100 C4 with a surplus of 200 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells Remove the Washing Buffer and add 100 ul diluted conjugate to each well incubate at 30 C 10 min at 550 rpm Remove the conjugate C3 CA add 200 ul C5 Washing Buffer Incubate at 30 C 5 min at 550 rpm Remove the Washing Buffer add 100 ul of D1 HRP substrate precipitating dye at 25 C see above per well Incubate at 25 C for 10 min but do not shake Remove liquid completely The bottom of the ArrayStrips outside surface may be cleaned cautiously with wipes Bubbles may be removed by removing and adding D1 Scan and process ArrayMate see below Please note 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 For Troubleshooting see p 29 and 38 E coli SeroGenoTyping AS 1 Kit 17 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Protocol for Eppendorf s Thermomixer Comfort with Microtiter Plate Adapter Epp
6. H 02 H 03 H 04 H 05 H 06 H 07 H 08 H 09 H 10 H 11 H 12 H 14 H 15 H 16 H 18 H 19 H 20 H 21 H 23 H 24 H 25 H 26 H 27 H 28 H 29 H 30 H 31 H 32 H 33 H 34 H 37 H 38 H 39 H 40 H 41 H 42 H 43 H 45 H 46 H 48 H 49 H 51 H 52 H 53 H 54 H 56 e The mix culture control is based on 5 probes located in consensus sequences on the 5 end of the fliC genes These probes were divided into two groups which are correlated with two groups of fliC genes fliC 5p 11 fliC 5p_12 and fliC 5p 13 fliC bp 14 fliC 5p 15 Only one group should be positive if not an E coli mix culture was probed e The gene fljA encodes for a repressor protein which repressed some of fliC genes If the fljA probe positive may two H serotype probes are positive In this case the genes fIkA and flmA encodes for the H serotype Such strains are described as biphasic Wang et al 2003 Tominaga 2004 e Probes specifying gapA gad and dnaE that were introduced to confirm the identity of E coli and to serve as genus specific controls E coli SeroGenoTyping AS 1 Kit 46 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit
7. Ilo dete fre TOES LAP Jelle b PEPE de 1 Plelel Id dale Kalle Click on flag image and the image file bmp is shown in the window on the right z VD az results results b raw data segmentation image Image Browse Search r ARCHIVE 3 2013 12 06 13 25 51 New Ru E coli Bio KDNA sero 13 0172 NM 061213 oli Bio kDNA sero CB12533 061213 B9 Bic EE E coli Bio w w CB5805 061213 18E2 E coli Bio k sero 933 ATCC7009 Etc Big E c oli Bio Egg DSM15856 061213 43 oli Bio kDNA sero FLI WH ZW O2 H25 06 oli Bio kDNA sero FLI WH ZW 02 H25 vi E coli Bio kDNA sero FLI WH zV O2 H25 we 2015 01 26 09 59 25 2015 01 26 13 47 41 2015 01 26 14 09 46 amp 2015 01 26 14 18 28 26 E coli SeroGenoTyping AS 1 Kit 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com This image of the reference isolate E coli EDL 933 shows an example for a valid test without any dust particles or non specific background The image file is automatically analysed be the ArrayMate software and a HTML report is provided that lists all markers that have been analysed Export of E coli SeroGenoTyping AS 1 Kit Test Reports 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 Othe
8. Order ana ee een 33 BEE AV NAC EOE ee ee E 33 COPTIC REE EE ee 33 MERE ann ne ee nee een 34 UPDATES AND SORT WY ARE sarri iraran a ee een 35 APPENDIX L NR 36 APPENDIX 2 IMAGES FOR TROUBLESHOOTING rammnnnnnrnnenernnnnernnnnsvnnnnnernnnnevnnnnsvnnnnnsnnnnnsvennnssesnn 38 APPENDIX 3 PROBE TO TARGET TABLE sccccssecccossseccnssececsescccusseseussececssseneussencuesessuseenens 40 APPENDIX 4 TYPING INFORMATION asza s d r en 46 Dens and EXPIARAL ON JA 46 E coli SeroGenoTyping AS 1 Kit 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com BACKGROUND The Alere E coli SeroGenoTyping AS 1 Kit allows DNA based serogenotyping of most known O and H antigens RNA free unfragmented genomic DNA from pure and monoclonal E coli culture material is amplified approximately 50 fold and internally labelled with biotin 11 dUTP using a linear amplification protocol In contrast to standard PCR only one antisense primer per target is used resulting in producing single stranded ss DNA reaction products This allows 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 culture material and cannot be performed on samples such as swabs or other patient samples e g faec
9. alere technologies com www alere com Hybridisation General Remarks Handling of Arrays e Never touch the array surface e Avoid complete drying of the array surface during processing e Do not allow it to stay without liquid for more than two minutes e Never rinse the wells with distilled water after the hybridisation step only use C2 Washing Buffer Unused wells should be capped during the whole procedure The strips may be processed up to three times without a loss of quality of properly capped unused arrays Close all wells that will not be used with a cap und leave them there until you use these wells for storage conditions after use see section Kit components Storage and Stability Hybridisation and Detection Always label your ArrayStrips with a laboratory marker at the recommended position Never label them on the bottom or across the data matrix barcode This may cause errors Data Matrix code keep it clean label here 4 Avoid contact of data matrix barcode with organic solvents The ArrayMate needs the information encoded in the data matrix to perform the assay and the analysis afterwards Avoid touching the bottom of the microarray strip and keep it clean E coli SeroGenoTyping AS 1 Kit 13 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent r
10. as tab separated txt file on the memory stick provided together with the ArrayMate e To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted e You may use the software tool Worklist Generator to create a worklist easily http alere technologies com en products lab solutions software tools worklist generator html E coli SeroGenoTyping AS 1 Kit 21 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Table 1 Example worklist Please note Table header must be written exactly as shown a 201512348 10630 6 1 2015 12350 10630 7 9876504 1060 8 EcoliEDL933 10630 Table 2 Positions in the 96 well format Data Acquisition in the ArrayMate Reader e Insert your flash drive containing the worklist into any of the USB ports on the lower right hand side of the ArrayMate e Press Bir 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 E coli SeroGenoTyping AS 1 Kit 22 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Lea
11. coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com LITERATURE Anjum MF Mafura M Slickers P Ballmer K Kuhnert P et al 2007 Pathotyping Escherichia coli by using miniaturized DNA microarrays Appl Environ Microbiol 73 5692 5697 Anjum MF Tucker JD Sprigings KA Woodward MJ Ehricht R 2006 Use of miniaturized protein arrays for Escherichia coli O serotyping Clin Vaccine Immunol 13 561 567 Ballmer K Korczak BM Kuhnert P Slickers P Ehricht R et al 2007 Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray J Clin Microbiol 45 370 379 Geue L Monecke S Engelmann I Braun S Slickers P et al 2013 Rapid Microarray Based DNA Genoserotyping of Escherichia coli Microbiol Immunol 2013 Dec 3 doi 10 1111 1348 0421 12120 Geue L Schares S Mintel B Conraths FJ Muller E et al 2010 Rapid microarray based genotyping of enterohemorrhagic Escherichia coli serotype 0156 H25 H Hnt isolates from cattle and clonal relationship analysis Appl Environ Microbiol 76 5510 5519 Korczak B Frey J Schrenzel J Pluschke G Pfister R et al 2005 Use of diagnostic microarrays for determination of virulence gene patterns of Escherichia coli K1 a major cause of neonatal meningitis J Clin Microbiol 43 1024 1031 Monecke S Mariani Kurkdjian P Bingen E Weill FX Baliere C et al 2011 Presence of enterohemorrhagic Escherichia coli ST678 0104 H4 in France prior to 2011 Appl
12. genes if positive the genes fIkA HO3 or H53 or flmA H54 represent the H serotype AB128916 1 fliC non motile fl H NM 11 flagellin C from non motile isolates AY337480 1 E coli SeroGenoTyping AS 1 Kit 45 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com APPENDIX 4 TYPING INFORMATION Definitions and Explanations The displayed result will yield the following typing information e Discrimination of the 93 described O serotypes is mainly determined by the genes wzy polymerase and wzx flippase The 47 known H antigens are encoded by the gene flic e The probes immobilized on the current array version can discriminate 93 O antigens specific probes O typing O 1 O 2 O 3 O 4 0 6 O 7 0 8 O 9 O 11 0 15 0 18 O 21 O 22 0 24 0 25 0 26 0 27 0 29 0 32 0 35 0 40 0 45 0 52 0 53 0 55 0 56 0 58 0 63 0 66 0 70 O 71 0 75 0 78 0 79 0 81 0 83 0 85 0 86 0 87 0 91 0 98 0 99 O 101 0 103 0 104 0 105 0 109 O 111 O 112 O 113 O 114 O 115 O 119 O 121 O 123 O 126 O 127 0 128 O 130 O 138 O 139 O 141 0 143 0 145 O 146 O 147 O 148 O 149 O 150 O 152 0 157 0 159 O 167 O 168 O 172 0 174 O 177 grouped consensus probes O typing 10 13 O 129 O 135 10 17 0 44 0 73 0 77 O 106 10 28 0 42 10 107 O 117 0 118 0 151 10 124 0 164 e The following flagellar antigens can be identified on the array H 01
13. problem or question please contact the technical service Disclaimer This system is for research use only We do not accept any liability for damages caused by misuse The assay must not be used as a substitute for phenotypic susceptibility tests We shall not be held liable for damages caused by an 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 from whom E coli was isolated 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 for good performance and correctness of results using standard E coli DNA preparations E coli SeroGenoTyping AS 1 Kit 32 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com List of Components for Separate Order If required these reagents for the E coli SeroGenoTyping AS 1 Kit may be ordered separately 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 e mail to cct home clondiag com E coli SeroGenoTyping AS 1 Kit 33 05 16 04 0016 VO1 Manual E
14. the ArrayMate It uses an image taken by the ArrayMate or a txt file raw signal data file and generates a report from the raw signal data E coli SeroGenoTyping AS 1 Kit 35 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com APPENDIX 1 FLOW CHARTS The figure summarise the test procedure for the thermoshaker BioShake iQ by Quantifoil Please always refer to the text section of this manual for further important details Protocol Quantifoil BioShake iQ pro hands prepare ArraysStrips prepare DNA cessing on time time grow CLONAL E coli isolate over 5 min not part of the kit night isolate genomic DNA 3h 40 min not part of the kit label RNA free DNA in thermocycler Cx D 3h 5 min 5 ul DNA cona 0 1 0 4 ug ul rinse ArrayStrips p Mester 3 9 ul B1 0 1 ul B2 1 ul B3E1 200 ul water 50 C 550 rpm 5 min da s discard water prepare labeled DNA 150 ul Buffer C1 50 C 550 rpm 5 min to 10 ul of labeled DNA add 90 ul of 15min 2 min discard C1 process promptly Buffer C1 transfer 100 ul labeled DNA to ArrayStrips 2 min 2 min Barcodes wo here hybridise 50 C 550 rpm 60 min Meanwhile Login to the ArrayMate device and prepare your worklist 60min 10min discard labeled DNA 20min 5min incubate twice in 200 ul Buffer C2 45 C 550 rpm 10 min prepare C3 C4 Conjugate C3 C4 1 100 preheat Substrate D1 25 C discard Buffer C2 15 mi
15. 000001 1 wbdA AB031867 1 0103 0104 0105 0107 0117 hp1056 wzx_0107 0117 O antigen 0107 0117 0109 0111 wbdH O111 11 wbdM O111 11 wzx O antigen 0111 AF078736 1 O111 11 wzy O111 11 0112 hp1059 wzx 0112 O antigen 0112 EU296413 1 hp1123 wzy O112 hp1058 wzx O112ac hp1122 wzy O112ac O113 hp1060 wzx 0113 wzy O113 11 O antigen O113 AF172324 1 O114 wzx 0114 11 wzy 0114 11 O antigen O114 AY573377 1 E coli SeroGenoTyping AS 1 Kit 41 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com 0115 0118 0151 hp1061 wzx O115 hp1124 wzy 0115 hp1062 wzx_0118 0151 hp1125 wzy 0118 0151 hp1063 wzx 0119 hp1126 wzy 0119 Wzx O121 11 wzy O121 11 hp1064 wzx 0123 hp1127 wzy 0123 hp1065 wzx_0124 0164 hp1128 wzy 0124 0164 hp1066 wzx 0126 hp1129 wzy 0126 hp1067 wzx 0127 hp1130 wzy 0127 wzx 0128 11 wzy O128 11 hp1068 wzx 0130 hp1131 wzy 0130 hp1069 wzx 0138 hp1132 wzy 0138 hp1070 wzx 0139 hp1133 wzy 0139 hp1071 wzx 0141 hp1134 wzy O141 hp1072 wzx 0143 hp1135 wzy 0143 hp1073 wzx 0145 hp1136 wzy O145 hp1074 wzx 0146 hp1166 wzy 0146 hp1075 wzx O147 hp1138 wzy O147 hp1076 wzx 0148 hp1139 wzy 0148 hp1077 wzx 0149 hp1140 wzy 0149 O150 hp1155 wzx 0150 hp1142 wzy 0150 0152 hp1081 wzx O152 hp1144 wzy 0152 0157 rfbE O157 11 wzx 0157 11 hp1145 wzy 0157 hp1082 wzx 0159 hp1146 wzy 0159 hp1083 wzx 0167 hp1147 wzy 0167 hp1084 wzx
16. 49 0 36 41 3111 120 For up to date information regarding the kit please visit our website at http www alere technologies com Safety Precautions e The assay is intended for use by personnel trained in microbiological and molecular methods Preparation of DNA from pure E coli colonies clones requires expertise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed e Isolated cell free E coli DNA may be processed without further biosafety precautions although contamination with E coli or other bacteria needs to be ruled out E coli SeroGenoTyping AS 1 Kit 2 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Always wear protective clothing as required for laboratory work according to your specific regulations on laboratory safety Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest amendments to the European Union Directives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet MSDS They do not contain more than 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 swall
17. 83 0 750000 0 884618 0 009758 a 2015 01 26 09 59 25 107 wzy O8_11 0 896543 0 782051 0 891362 0 005440 o 2015 01 26 13 47 41 108 wzy O91 11 0 899453 0 750000 0 894491 0 005194 0 2015 01 26 14 09 46 109 wzy O9 11 0 898260 0 788335 0 893795 0 004680 a 2015 01 26 14 18 28 11 Flic HO2_11 0 882257 0 765126 0 886245 0 004256 0 E coli SeroGenoTyping AS 1 Kit 25 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Click on flag segmentation image and the grid alignment accuracy is shown in the window on the right Browse E results results b raw data segmentation image image NewRun E coli Bio kDNA sero 13 0172 NM 061213 E coli Bio kDNA sero CB12533 061213 B9 p E coli Bio kDNA sero CB5805 061213 8E2 E coli Bio kDNA sero DD EDL933 ATCC7009 y Archive E coli Bio kDNA_sero_DSM15856_061213 3 coli Bio kD JER PIRI BEP KPIDL POLE EEE VE M Pals CEEPICLEC 2015 01 26 13 47 41 PIP o SS Legal Pole DIE GEO ABBA MERC EEE 01 18 C ie IP Ibe Wolfe Lae OLOO m Ei bila SEE C ELPOOPLLIE ordo M scorio B CO olaleld OELE gor Pl F ler a HE obl Tool SP EISELE LLAP fe TO bd deletae e e eje ldo I LSP BIG op Ble SSO LIP b Pe JOLY Tafel STE 9 SERE pe OB Lear sf ele el IA HSC JOTSe TS Te loot d Ge dle SOL Se Gla le II Talis PC ENE Og TT Tolol Iole eio el el e Te NPE olo PIE IO Seer ae
18. 96 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells e Remove the Washing Buffer and add 100 ul diluted conjugate to each well incubate at 30 C 10 min at 550 rpm e Remove the conjugate C3 C4 add 200 ul C5 Washing Buffer Incubate at 30 C 5 min at 550 rpm e Remove the Washing Buffer add 100 ul of D1 HRP substrate precipitating dye at 25 C see above per well e Incubate at 25 C for 10 min but do not shake e Remove liquid completely e The bottom of the ArrayStrips outside surface may be cleaned cautiously with wipes Bubbles may be removed by removing and adding D1 e Scan and process ArrayMate see below Please note 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 For Troubleshooging see p 29 and 38 E coli SeroGenoTyping AS 1 Kit 19 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Data Analysis Starting the ArrayMate Reader We recommend starting the ArrayMate Reader after starting the hybridisation this allows the convenience of starting the device and to importing the worklist file Please note that this is a short instructio
19. AS 1 Kit 20 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com must be saved in the txt file format that can be imported into the test specific ArrayMate software Do not use special characters such as V etc e Create a list with at least three columns that have headers written in the first line The following headers are obligatory in this order position samplelD assayID Table 1 e Positions are consecutively numbered from 1 to a maximum of 96 Position 1 would correspond to A1 8 to H1 9 to A2 and 96 to H12 Table 2 Do not leave empty lines in the worklist If you use EXCEL position numbers should be entered into column A e Sample IDs are strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patients names should not be used as sample IDs e The Assay ID allows the system to identify the current test and to correctly use information on layout spot number and identity etc The E coli SeroGenoTyping AS 1 Kit has the Assay ID 10630 Please note Assay ID numbers must not be confused as this could lead to errors or loss of data e You may add further columns and headers with notes and comments at your convenience Information from these columns will not appear on the result screen or in the Test Report e We recommend using a printout of the worklist as a template for pipetting e Save the worklist
20. H25 we 2015 01 26 09 59 251 2015 01 26 13 47 41 2015 01 26 14 09 46 2015 01 26 14 15 28 E coli SeroGenoTyping AS 1 Kit 24 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Click on a Sample ID and the E coli SeroGenoTyping AS 1 Kit Test Report for this array is shown in the window on the right Browse 4 Search results results b raw data segmentation image image FI E ARCHIVE e 2013 12 06 13 25 51 For Research Use Only Not For Use In Diagnostic Procedures New Run E coli Bio kDNA sero 13 0172 NM 061213 Operator E coli Bio kDNA sero CB12533 061213 B9t E coli Bio KDNA_sero_CB5805_061213 _ 18E2 Sample ID E coli Bio DNA sero EDL933 ATCC700927 061 21 3 E coli Bio kDNA sero DD EDL933 ATCC7009 Experiment ID E coli Bio DNA sero EDL933 ATCC700927 061213 am aa DateotResut muna 1201302015 E coli Bio kDNA_sero_FLI WH_ZY_O2 H25_vie Assay Name Ecoli SeroType AS 1 E coli Bio kDNA sero FLI WH zV O2 H25 we Assay ID 10630 2015 01 26 09 59 25 2015 01 26 13 47 41 Well Position E 2015 01 26 14 09 46 Software Version 2015 01 15 2015 01 26 14 18 28 Device f print Results Result Positive Marker s O serotyping 0157 H serotyping HO Controls control result explanation Failed indicates possible problems originating from the hybridization or the staining procedure see tro
21. Surplus 50 e A2 Lysis Enhancer lyophilised Cat 245102000 Store at 18 to 28 C ambient temperature Centrifuge A2 tubes shortly prior to opening Add 200 ul Buffer Al to Lysis Enhancer before use Mix well and store for less than 1 week at 2 8 C Sufficient for 96 isolations DNA Labelling and Amplification e 81 Labelling Buffer Store at 2 8 C Surplus 40 96 e B2 Labelling Enzyme Store at 2 8 C Surplus 100 96 e B3 yophilised Labelling Primermix two tubes dilute each in 70 ul molecular grade water Store at 20 C Surplus 5096 E coli SeroGenoTyping AS 1 Kit 4 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Hybridisation and Detection e ArrayStrips 12 x 8 samples Protected against light and sealed under inert gas Store at 18 to 28 C After opening to be used within two weeks Close the unused wells with caps to protect against humidity and dust and store in the dark Avoid any touching or scratching of the microarray surface at the bottom of the well Do not store or handle unused wells at an air humidity of more than 60 since this may irreversibly corrode the spots e StripCaps 24 units e C1 Hybridisation Buffer Store at 18 to 28 C protect against sunlight Surplus 150 96 e C2 Washing Buffer 1 Store at 18 to 28 C protect against direct sunlight Surplus 200 e C3 HRP Conjugate 100 x Store at 2 to 8 C pro
22. WS section of our website http alere technologies com Support is available via cct home clondiag com Components Required but not Provided e Growth media for the cultivation of E coli The test should be performed with colonies harvested from 2 x TY or Columbia Blood Agar Other rich media e g Standard 1 or LB may also suffice but have not been tested systematically Liquid media should not be used because contaminations or mixed cultures cannot be ruled out easily e Equipment and consumables needed for the cultivation of E coli incubator inoculation loops Petri dishes e DNA preparation kits The assay has been tested using the DNeasy Blood amp Tissue Kit by Qiagen Cat 69504 and High Pure DNA Isolations Kit from Roche Cat 11796828001 Please note The DNA specimen needs to be free of RNA Recommendation a pre treatment using the cell lysis components A1 A2 see below or a standard RNase A treatment during DNA preparation E coli SeroGenoTyping AS 1 Kit 6 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Equipment needed for DNA isolation e g pipettes centrifuge thermoshaker or automated device see above e Photometer OD 260 nm for measuring the DNA concentration e Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA e Thermocycler for PCR e Thermoshaker Please note We strongly recommend the B
23. carefully or try to manually identify the test If no obvious reason for the fault can be discovered please contact the technical service E coli SeroGenoTyping AS 1 Kit 30 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Ambiguous Results Apart from a positive or negative result for the individual markers on E coli SeroGenoTyping AS 1 Test Report the result can also be ambiguous This can be caused by poor sample quality poor signal quality or less then optimal stringency of the hybridisation process temperatures in hybridisation process are too low or too high E coli SeroGenoTyping AS 1 Kit 31 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com ADDITIONAL INFORMATION Warranty Alere Technologies GmbH 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 28 C Kit components comprise the arrays and their caps the Lysis Enhancer the reagents for DNA labelling and for detection of labelled DNA products on the array the ArrayMate reader and its software In case one of these components fails within the expiry date due to other reason than misuse contact Alere Technologies GmbH for replacement or refund Terms and conditions apply If you have any
24. ed depending on the sample viscosity and the type of centrifuge used All liquid should be collected in the collection tube afterwards e Discard collection tube with liquid e Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 ul Buffer AW1 e Centrifuge 8 000 rpm 1 min at room temperature e Discard collection tube with liquid e Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 ul Buffer AW2 e Centrifuge 14 000 rpm 3 min 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 not provided with the kit e Add 100 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 8000 rpm 1 min at room temperature E coli SeroGenoTyping AS 1 Kit 10 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Optional Add another 100 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 Contamination with Washing Buffer might occur during the elution of prepared DNA by drop
25. emove and discard reagent D1 as completely as possible and scan immediately ArrayMate The dye precipitate fades slowly in presence of liquids General Remarks Thermoshakers The correct temperature within the vessels is essential therefore always use the appropriate equipment for heating Because of a possibly inhomogeneous distribution of the temperature within the heating block and because of possible differences between displayed and actual temperatures the use of different brands of thermoshakers might affect test performance We tested the assay with BioShake iQ by Qlnstruments see picture below http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips and Eppendorfs Thermomixer Comfort equipped with a heating block for microtiter plates When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference strains or the control DNA CM E coli EDL933 ATCC700927 The difference between the protocols for Qlnstrument s BioShake iQ and Eppendorf s Thermomixer Comfort with microtiter plate adapter is only the washing temperature after the hybridisation step Please note The Quantifoil s BioShake iQ has no active cooling function Please use the second pre temperatured passive cooling block to reduce the incubation temperature quickly E coli SeroGenoTyping AS 1 Kit 15 05 16 04 0016 VO1 Manual E
26. endorf Thermomixer with thermoblock for MTPs and deepwell plates e Switch on the thermoshaker and let it pre heat to 50 C e Remove the amount of ArrayStrip s needed from the pouch e Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode close to row A and proper fit e First add PCR grade distilled water 200 ul per well at 50 C 5 min at 550 rpm Remove the water from the well e Second add C1 Hybridisation Buffer 150 ul per well at 50 C 5 min at 550 rpm e Add 90 ul of C1 buffer to each PCR tube with 10 ul labelled amplification product mix gently e Remove the buffer from the well and add the mixture of C1 and labelled amplification product e Incubate at 50 C 60 min at 550 rpm e Meanwhile login to the ArrayMate device and prepare your worklist see section Data Analysis p 22 e Remove the liquid and add 200 ul C2 Washing Buffer Incubate at 50 C 10 min at 550 rpm remove and discard e Add another 200 ul C2 Washing Buffer Incubate at 50 C 10 min at 550 rpm E coli SeroGenoTyping AS 1 Kit 18 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Meanwhile prepare conjugate For each experiment add 1 ul C3 conjugate 100 x HRP to 100 ul C4 Conjugation Buffer This mixture is stable for one working day at room temperature C3 is delivered with a surplus of 100 96 C4 with a surplus of 200
27. es Resulting biotin labelled ssDNA is transferred and hybridised to DNA oligonucleotide microarrays with 265 probes for different genetic markers and a biotin staining positive and buffer negative control All probes for serogenotyping are printed in two duplicate spots Targets include a variety of species and serotyping markers including genes encoding 93 different O antigens and 47 H antigens see also PROBE TO TARGET TABLE page 40 Based on a digital image of the arrays spot recognition is performed automatically and results are given as an HTML file with a description of each analysed target E coli SeroGenoTyping AS 1 Kit 1 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not Intended for Use in Clinical Diagnostics This kit allows genotypic characterisation of E coli isolates for research and epidemiological applications It must not be used as a substitute for phenotypic susceptibility tests and for the guidance of antibiotic therapy Specifications Upon receipt the kit components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 C to 28 C Technical Support If you require any further information on this product please contact Email cct home clondiag com Phone 49 0 36 41 3111 155 Fax
28. eservoirs We strongly recommend that the liquid is removed by pipetting rather than by inverting the strips and flicking the liquids out Fine tipped soft disposable Pasteur pipettes are suited best such as VWR Cattt 612 2856 Always place the pipette tip at the cavity between the array and the wall of the reagent well If you touch the array surface probes may be scratched off and this may cause errors Pasteur pipette plastic with a flexible tip m 1 flexible tip I Pipette tip Use the cavity between array and the wall of the tube Do never touch the array bh Array General Remarks The Substrate Precipitating Dye D1 An appropriate amount of D1 substrate precipitating dye should be transferred into an Eppendorf tube and taken out of the refrigerator when starting the procedure allowing it to acclimatise to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged prior to use to remove bubbles as well as possible precipitates quick spin E coli SeroGenoTyping AS 1 Kit 14 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Triggered by peroxidase the dye precipitates in case of positive reactions 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 or thereafter After completion of staining r
29. he DNA specimen needs to be RNA free and it should not be fragmented This can be determined by agarose gel electrophoresis Additionally the microarray includes probes to internally check for RNA contamination The automatic software analysis will give an invalid if high RNA contamination occurred during DNA isolation DNA should not be prepared by disrupting F coli cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the E coli SeroGenoTyping AS 1 Kit are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend to obey the protocols outlined below Please note To yield more genomic DNA we recommend the optional cell lysis step with A1 A2 reagent E coli SeroGenoTyping AS 1 Kit 8 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com DNA extraction using spin columns produce higher amounts of pure DNA which can be stored 4 8 C whereas DNA extraction with the fast and economic boiling procedure results in DNA for direct subsequent use without storage DNA Extraction by Spin Columns e g Qiagen e Add an inoculating loop full of monoclonal culture material of the E coli isolate to 0 2 ml 1 x PBS 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
30. he well is contaminated with a liquid e g buffer Substrate D1 residue in the array edges Signal intensity is too low Chip was not in focus during image acquisition E coli SeroGenoTyping AS 1 Kit 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit Please clean the bottom surface with a cleanroom wipe scan and process again Remove substrate D1 completely with a Pasteur pipette 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 Repeat image acquisition after fitting the ArrayStrip in the frame 39 www alere technologies com www alere com APPENDIX 3 PROBE TO TARGET TABLE Target genes Proes jFuxton O Family and Species Marker gad gad 10 glutamate decarboxylase AE014075 1 locus tag c4328 genus specific marker for Escherichia Shigella dnaE hp dnaE 612 hp dnaE 613 DNA polymerase III subunit alpha genus specific marker for Escherichia Shigella U00096 3 rrs hp rrs 611 hp rrs 612 16S rRNA species specific marker for Escherichia coli U00096 3 glyceraldehyde 3 phosphate dehydrogenase A CP000468 1 locus gapA prob gapA 611 tag APECO1 847 family specific marker for Enterobacteriaceae hp1021 wzx 0001 hp1087 wzy O001 O antige
31. ioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips Alternatively you may use Eppendorf s Thermomixer Comfort equipped with a heating block for microtiter plates e Pipettes Suitable for 1 ul 5 ul volumes 90 ul 100 ul 200 ul 1000 ul e Multichannel pipettes for 100 200 ul e Sterile reaction vials suitable for PCR e Ultrapure PCR grade water e RNase A we recommend Qiagen s RNase A solution 100 mg ml Qiagen Cat 19101 e Pasteur pipettes VWR Cat 612 2856 E coli SeroGenoTyping AS 1 Kit 7 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com PROTOCOLS Culturing and Harvesting Bacterial Cells Members of the genus Escherichia are potential pathogens All procedures for cultivation of the bacterium and DNA preparation need to be performed by properly trained staff in a biosafety level 2 facility Grow E coli on 2 x TY or Columbia Blood agar overnight at 37 C or 48 h at room temperature Obtain confirmation of the identification as EF coli API VITEK MALDI and make sure that you have a pure monoclonal E coli culture Contamination by other bacteria especially other Enterobacteriaceae must be strictly avoided DNA Extraction The required sample type for the E coli SeroGenoTyping AS 1 Kit is 0 5 2 ug Cona 0 1 0 4 ug ul of intact genomic DNA from a single clone T
32. ions without RNA contaminations are a prerequisite for proper DNA concentration measurement Therefore RNase treatment is necessary prior to A go reading component A2 contains RNAse DNA must be unfragmented as fragmentation reduces the amplification and labelling efficiency due to the distance between primer and probe binding sites For this reason DNA should not be prepared by disrupting F coli cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols We made good experiences with positive experiences when using the manual QIAGEN DNeasy Kit and the Roche High Pure Kit DNA must be free of any trace of ethanol as ethanol has a strong impact on the amplification It is possible to heat the sample prior to adding it to the labelling mix 5 10 minutes at 70 C in order to evaporate any traces of ethanol Physical Damage to the Array Scratching the array surface with a pipette tip may damage array spots which may lead to the impairment or absence of a valid signal In this case the respective marker will not be assigned as negative but instead the message none appears next to the marker name Report Unavailable If the ArrayMate indicates that no report is available for an array or multiple arrays on one strip please check that the strip was positioned properly into the frame Scratches or drops of condensed water might render the Data Matrix code identifier unreadable please wipe it
33. lick Make New Folder on the bottom a new folder icon appears e Rename the new folder e g with the experiment name or date e Click Ok data are exported into the new folder on your flash drive e Do NOT remove the flash drive 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 E coli SeroGenoTyping AS 1 Kit 28 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com TROUBLESHOOTING When troubleshooting always make sure that reagents are within the recommended shelf life and stored appropriately Should you encounter a problem we will always be happy to support you Please e mail to cct homeQclondiag 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 buffer C3 C4 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 of the we
34. lls with C2 buffer to remove all C1 buffer prior to adding horseradish peroxidase conjugate If the staining control has Passed refer to the following hints Image Quality In case of poor image quality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel In order to determine whether any problems originated from the DNA preparation perform an experiment with the Control material CM This is DNA from the E coli EDL933 reference strain GenBank accession number NC 002655 2 which should be identified by the assay as E coli with O157 and H7 If the control experiment yields a valid result and a correct identification 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 38 and 39 E coli SeroGenoTyping AS 1 Kit 29 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com DNA Quality and RNA Contamination Control The amount of DNA is crucial because of the linear kinetics of amplification see Introduction DNA should be RNA free as free RNA reduces the amplification and labelling efficiency 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 preparat
35. n 2 min incubate in 100 ul C3 C4 Conjugate 30 C 550 rpm 10 min U discard C3 C4 conjugate 5 min 2 min incubate once in 200 ul Buffer C5 30 C 550 rpm 5 min discard Buffer C5 15 min 2 min incubate with 100 ul Substrate D1 25 C 10 min U discard Substrate D1 analyse ArrayMate 10 min 5min total time requirement overnight app 120 min app 8h E coli SeroGenoTyping AS 1 Kit 36 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com The figure summarise the test procedure for the thermoshaker Thermomixer Comfort by Eppendorf with heating block for microtiter plates Please always refer to the text section of this manual for further important details Protocol Eppendorf Themoshaker comfort pro hands prepare ArraysStrips prepare DNA cessing on time time grow CLONAL E coli isolate over 5 min not part of the kit night isolate genomic DNA 3h 40 min not part of the kit label RNA free DNA in thermocycler 5 mi 5 ul DNA cpya 0 1 0 4 g l an rinse ArrayStrips PIE Sn 3 9 ul B1 0 1 ul B2 1 ul B3 c01 200 ul water 50 C 550rpm smin gt discard water prepare labeled DNA 150 ul Buffer C1 50 C 550 rpm 5 min to 10 ul of labeled DNA add 90 ul of 15min 2 min discard C1 process promptly Buffer C1 transfer 100 ul labeled DNA to ArrayStrips 2 min 2 min aian Pais here hybridise 50 C 550 rpm 60 min Meanwhile Login t
36. n O1 GU299791 1 hp1022 wzx 0002 hp1088 wzy 0002 O antigen O2 GU299792 1 hp1023 wzx 0003 hp1089 wzy 0003 O antigen O3 EU694097 1 hp1024 wzx 0004 wzy 04 11 AY568960 1 ere ee S i CP000247 1 hp1012 wzm OO008 O antigen O8 AF013583 1 hp1015 wzt 0008 wzx O8 11 wzy O8 11 hp1013 wzm 0009 O antigen 09 wzm wzt hp1016 wzt 0009 wzx O9 11 wzy D43637 1 wzx AF104912 2 wzy O9 11 AB031867 1 O13 O129 O135 hp1026 wzx_0013 0129 0135 O antigen 013 0129 0135 hp1092 wzy_0013 0129 0135 EU296422 1 EU296423 1 EU296424 1 015 wzx 015 11 wzy 015 11 O antigen 015 CP002291 1 CP002291 1 O17 O44 O73 077 0106 hp1027 wzx 0017 0044 0073 0077 O antigen 017 044 073 O77 or 0106 CU928163 2 hp1093 wzy 0017 0044 0073 0077 FN554766 1 DQ000313 1 DQ000314 1 DQ000315 1 O serotyping O O O E coli SeroGenoTyping AS 1 Kit 40 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com 027 028 042 hp1034 wzx 0028 0042 O antigen 028 042 029 035 040 hp1038 wzx 0040 hp1104 wzy 0040 O antigen 040 EU296417 1 045 055 058 066 hp1044 wzx 0066 hp1109 wzy 0066 O antigen 066 DQ069297 1 075 079 wbdU 079 11 wzx 079 11 O antigen 079 EU294162 1 er et 081 086 091 098 hp1014 wzm 0099 O antigen 099 FJ940773 1 hp1018 wzt 0099 wz O101 11 wbdA 09 11 O antigen 0101 wzt 0101 AFAH02
37. n only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate 1 main switch on the rear below the electric cable plug m operating switch on the bottom left corner of the front side e Switch on the screen switch is on the right hand side below the screen e Log in as R amp D User Research and Development User for full access to test specific software a default password will be provided together with the ArrayMate device If you log in as User you will obtain only raw values but neither positives negatives interpretation nor strain assignment The Administrator log in will allow the installation of a new assay specific plug in which can be downloaded at http alere technologies com see p 31 e The user interface will be loaded the ArrayMate performs internal testing It requires slightly less than a minute e Click New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience e Type in your operator ID optional Worklist A Worklist file allows linking an identifier such as a laboratory or sample number to a position of an array within 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 E coli SeroGenoTyping
38. o the ArrayMate device and prepare your worklist 60 min 10min discard labeled DNA 20 min 5 min incubate twice in 200 ul Buffer C2 50 C 550 rpm 10 min prepare C3 C4 Conjugate C3 C4 1 100 preheat Substrate D1 25 C U discard Buffer C2 15min 2min incubate in 100 ul C3 C4 Conjugate 30 C 550 rpm 10 min i discard C3 CA conjugate 5 min 2 min incubate once in 200 ul Buffer C5 30 C 550 rpm 5 min discard Buffer C5 15 min 2 min incubate with 100 ul Substrate D1 25 C 10 min i discard Substrate D1 analyse ArrayMate 10 min 5 min total time requirement overnight app 120 min app 8h a with heating block for microtiter plates E coli SeroGenoTyping AS 1 Kit 37 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com APPENDIX 2 IMAGES FOR TROUBLESHOOTING g a Valid experiment Valid results no error messages The bottom of the well is contaminated with dust Please clean the bottom of the well scan particles and process again 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 microarray surface is contaminated with dust particles E coli SeroGenoTyping AS 1 Kit 38 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com The bottom of t
39. owed 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 any liquids are spilled clean with disinfectant and or laboratory detergent and water Alere assumes no liability for damage resulting from handling or contact with these products If you have any questions please contact our Technical Support see above Shipping Precautions RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods E coli SeroGenoTyping AS 1 Kit 3 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com REAGENTS AND DEVICES 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 were 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 proved to be stable six months after post expiry Cell Lysis optional order e Al Lysis Buffer Cat 245101000 Store at 18 to 28 C ambient temperature
40. r files that are generated and that can be exported include e A txt file with the raw measurements e An image file bmp with the actual photo of the array e A second image file png in which the coordinate grid is superimposed and the recognised spots are circled and e A XML xml files that contains the same information as the html result sheets for future export into databases etc Please note Only complete runs can be exported The export of individual E coli SeroGenoTyping AS 1 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 Tr S Browse 4 Search FI ARCHIVE 2009 02 05 09 00 42 expe sample ID order37x order37x23 order37x order37x78 order37x order37x82 order 2x order42x01 Browse For Folder EE en va order42x03 order42x06 va order42x07 Order78x01 Order78x01 4 Order78x08 order42x02 Choose a directory Desktop My Documents amp Se Local Disk C Sw Local Disk D amp w Removable Disk E Folder My Computer Make New Folder lt E coli SeroGenoTyping AS 1 Kit 27 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Click My Computer then Removable Disk and choose the folder where to save or c
41. s adhering to the spin columns funnels Therefore these funnels should be gently touched and dried with sterile filter paper or wipes prior to the elution step Alternatively prepared DNA can be heated shortly 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 concentration A go method it should not be lower than 0 1 ug ul The concentration might be increased by heating and evaporating water or by using a speed vac centrifuge not recommended when the same preparation shall be used in PCR experiments DNA Extraction by Heat Lysis Please note Only a fresh overnight culture can be used After DNA extraction by boiling the linear amplification must be done immediately Storage of extracted DNA is not recommended e Adda 1 ul inoculating loop Please Note do not use too much culture material see figure below of a monoclonal isolate to 50 ul PCR grade distilled water and vortex thoroughly e Incubate at 99 C 15 min at 550 rpm in a thermoshaker e Centrifuge for 5 min at 13 600 rpm at room temperature e Carefully pipette 25 ul supernatant into a new 1 5 ml tube and discard the old tube with the pellet E coli SeroGenoTyping AS 1 Kit 11 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Add 0 25
42. t on the screen reader is opening e Remove the white frame with the ArrayStrip s E coli SeroGenoTyping AS 1 Kit 23 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com e Press Next at the bottom right on the screen reader is closing Results On the left hand side of the screen there 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 is one experiment run in this archive gp Browse Search Lug zi ARCHIVE m 2013 02 06 13 22 37 Archive If you click on the plus symbol left to the run name the folder opens and you will see a list of the individual arrays alphabetically ordered by Sample ID Browse Search i E ARCHIVE 7013 12 06 135 25 51 New Run E coli Bio kDNA sero 13 O172 MM D61213 E coli Bio kDNA sero CB12533 061213 159 p5 E coli Bio kDNA sero CB5805 061213 IBEZ E coli Bio kDMA sero DD EDL933 ATCC7009 Archive E coli Bio kEDNA sero D 5M15856 061213 3 E coli Bio kDNA sero FLI WH 7 02 H25 D6 E coli Bio kDNA sero FLI WH zw O2 Hz5 vie E coli Bio kDMA sero FLI WH zv O2
43. tect against direct sunlight Surplus 100 96 e C4 Conjugate Buffer Store at 18 to 28 C protect against direct sunlight Surplus 200 e C5 Washing Buffer 2 Store at 18 to 28 C protect against direct sunlight Surplus 500 e D1 Horseradish Peroxidase Substrate Store at 2 to 8 C protect against direct sunlight Surplus 50 e Optional CM P Reference DNA from E coli EDL933 GenBank accession number NC 002655 2 Cona 0 1 0 4 ug ul Store at 2 to 8 C Sufficient for 5 6 tests E coli SeroGenoTyping AS 1 Kit 5 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com Instrumentation and Software e ArrayMate Reader to be ordered separately for details see below The ArrayStrip based E coli SeroGenoTyping AS 1 Kit can be used on the ArrayMate reader only The alternative devices ATRO1 03 are not suitable for reading ArrayStrip based assays In case of any questions please contact us e conoclust software provided with the reader e Test specific software plug in can be downloaded from Alere Technologies GmbH website check periodically for updates for details see below Information such as spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is delivered with the reader or can be downloaded from our website These test specific plug ins will occasionally be updated Please check the NE
44. tin Labelling eese 12 g NOS tl OU T E A E LE Emm 13 General Remarks Handling of Arrays an un nk 13 General Remarks Handling of Liquids rronnrrnnnnrrnnnnrrnnnnnrrnnnnrrnnnnernnnnnernnnnernnnnsennnnnennnnsesenneee 14 General Remarks The Substrate Precipitating Dye D1 rrrrrnnnnnnrrrnnnnnrrrnnnnnernnnnnnerrnnnnneeene 14 General Remarks Tie NOEN aa ae 15 Protocol Tor Ouantitoil S BioShake IQ esas ceis cobi kun a 16 Protocol for Eppendorf s Thermomixer Comfort with Microtiter Plate Adapter 18 BELE INS NETTE T m 20 Starting the ArrayMate RE Ta ee EEE 20 NN 20 Data Acquisition in the ArrayMate Reader eese nennen nnns 22 FO SCS ssc gates ds c M H 24 Export of E coli SeroGenoTyping AS 1 Kit Test Reports rrrrrnnrrnnnnrrrnnnnrrnnnnrrnnnnnrrnnnnernnnnseennn 27 TROUBLESHOOTING zu este sa VES TOE 29 StaIBIhg CONO EE ee ee 29 MEN E 29 DNA Quality and RNA Contamination Control rrrrnnnrrnnannvnnnnnrnnnnnernnnnernnnnernnnnnennnnnsrnnnnssnnnnee 30 Physical Damage to Me Array ae 30 Re NNN 30 AMD 01000 RO JE EE ee 31 ADDITIONALINFORMATION arrene en ei 32 WALL ACY EEE 32 E coli SeroGenoTyping AS 1 Kit 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com RANT 32 UN ON 32 List f Components tor Separate
45. type is than determined by flmA H54 or flkA H53 AY250005 1 AY250006 1 ADUP01000024 1 AY250008 1 CU928162 2 AY250010 1 AY250012 1 AY250011 1 E coli SeroGenoTyping AS 1 Kit 43 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com AY250013 1 DE ir AY250014 1 DE uM AY250015 1 AY250016 1 AY250017 1 mn AY250018 1 AY250019 1 m H40 11 flagellin C H antigen H40 AJ865464 1 Note If repressor fljA positive fliC H40 might be repressed H phenotype is than determined by flkA H53 rs AY250020 1 AY250021 1 AY250022 1 AY250023 1 AY250024 1 AY250025 1 ee GN PGE AY250026 1 m em PGE AY250027 1 AY250028 1 H53 fIkA H53_11 flagellin A H antigen H53 AB128917 1 Note If repressor FIjA pm alan m nome H21 are repressed H phenotype is H53 H54 flmA H54 11 flagellin A H antigen H54 AB128918 1 Note If repressor FIjA positive the gene fliC H21 is repressed H phenotype is H54 H56 fliC H56 11 flagellin C H antigen H56 AY250029 1 E coli SeroGenoTyping AS 1 Kit 44 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www alere technologies com www alere com H serotyping additional marker fliC Marker fliC bp 11 fliC 5p 12 fliC 5p 13 fliC fliC marker consensus sequence 5p 14 fliC 5p 15 for all fliC genes fljA Repressor FliA represses the expression of fliC
46. ubleshooting section in the manual staining control passed Failed indicates possible problems originating from the staining procedure see troubleshooting section in the manual mixed culture Invalid indicates a mixed or contaminated culture passed P control comprising of two or more E coli strains Invalid indicates a contamination of the genomic DNA with ribosomal RNA which potentially affects the labeling efficiency negative control passed RNA contamination passed Click on flag raw data and the raw data is shown in the window on the right Browse Search results results b raw data segmentation image image Spot ID Substance Background Confidence Mean Signal valid FI EJ ARCHIVE 1 FI H NM 11 0 872344 0 675382 0 880746 0 009068 0 2013 12 06 13 25 51 10 FIIC HO1_13 0 888505 0 750000 0 889262 0 000803 0 nl E coli Bio kDNA sero 13 O172 NM 061213 100 wzy 0172 11 0 891934 0 796520 0 889874 0 002175 0 E E coli Bio KONA_sero_CB12533_061213 189 101 w2y 026 11 0 889577 1 000000 0 893255 0 003892 0 EJ EON wo ES GLE EA 102 wzy 04_11 0 895839 1 000000 0 893148 0 002828 0 kk ESTER ur 103 wzy O53 11 0 894118 0 822749 0 892753 0 001437 o inii eel ara 104 wzy OSS 11 0 891186 0 782051 0 896237 0 005336 0 l EMS SSA 105 wzy O6 11 0 891890 0 762481 0 893411 0 001605 0 E col fio kDNA sero FLI WH 2 02 425 we 106 wzy O86 11 0 8938
47. ul RNase A not provided see above with a stock concentration of 1 mg ml e Incubate at 37 C 5 min at 550 rpm in a thermoshaker e Use directly 5 ul of this DNA suspension for the linear amplification and internal biotin labelling process Linear Amplification and Internal Biotin Labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 labelling reagent is 4096 EcO1 e Prepare a Master Mix by combining 3 9 ul of B1 labelling reagent 1 ul B3 Labelling Primermix 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 B2 B3 Do not forget to label the vial e Perform amplification in a pre programmed thermocycler e g Eppendorf Mastercycler gradient with heated lid according to the following protocol Pre heat cover lid to 105 C 20 sec at 50 C 300 sec at 96 C 50 cycles with 40 sec at 72 C 60 sec at 96 C Cool down to 4 C hold e The samples can be stored frozen until they will be used Please note When using a different device some adaptations such as an increase of the number of cycles might be necessary Before establishing routine use please test the protocol with a few known reference strains and the control DNA CM supplied with the kit E coli SeroGenoTyping AS 1 Kit 12 05 16 04 0016 VO1 Manual E coli SeroGenoTyping AS 1 Kit www
48. ve the flash drive in the ArrayMate if you intend to export E coli SeroGenoTyping AS 1 Kit reports afterwards Check the flash drive regularly for computer viruses and malware using an appropriate program e Press Next at the bottom right on the screen reader is opening e Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply strong force Ensure proper fit otherwise the images may be out of focus e Carefully insert the white frame with the array strips into the metallic adapter Ensure the correct orientation Position A1 in the frame next to the data matrix barcode on the adapter and proper fit otherwise the images may be out of focus ArrayStrip frame with inserted strips Strips are inserted in accordance with the Worklist Please note ArrayStrips must be clean They should not contain any liquids during analysis Data matrix codes must be clean There must be no Array StripCaps on the wells to be analysed however unused wells should remain capped e 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 strips the reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analysis ready e The reader indicates the end of the entire process with an acoustic signal beep e Press Next at the bottom righ
49. www alere technologies com www alere com Manual E coli SeroGenoTyping AS 1 Kit Array Hybridisation Assay for DNA based serogenotyping of Escherichia coli Kit order number 246300096 96 reactions ArrayStrip format For Research Use Only Not Intended for Use in Clinical Diagnostics www alere technologies com www alere com CONTENT BACKOROUND ee ee 1 GENERALINSTRUCTIONS FOR USE une nnd 2 NEVE ae er ee 2 NNN 2 TFT 2 Safety PN 2 Material Safety Data Sheets IMSDS anne een 3 SIIDDIHP Prec ION EE 3 REAGENTSAND DENLEE Sirenen recs sense AA as a en se en 4 Kit Components Storage and Stability rrrarrrnnnnnrnnnnnrrnnnnrrnnnnernnnnnrrnnnnernnnnsrnrnnnennnnsernnnsssnnnneene 4 Cell Lysis optional Order enter 4 DNA Labelling and Amplification rrrrnnnnrrnnnnrrnnnnnrrnnnnernnnnernnnnernnnnnrrnnnnernnnnsvnnnnsennnunsnnnnnesnnnnee 4 Hybridisation and DCEO 00 ea 5 instrumentation and NNN 6 Components Required but not provided rrrnronnrrnnnnrrnnnnernnnnernnnnnrnrnnnernnnnernnnnsvnnnnnsnnnunenennneeee 6 PROTOCOL c 8 Culturing and Harvesting Bacterial Cells rrrrrannrrnnnnrrnnnnrrnnnnernnnnnrrnnnnernnnnernnnnernnnnevnnnnsevnnnnssesne 8 NMF 8 DNA Extraction by Spin Columns e g Qiagen rrrrronrrrnnrnnnrrrnnnnrrrnnnnnnrrnnnnnnernnnnnnernnnnnverensnneee 9 DNA Extraction Dy Heat L SS osos inia rant een 11 Linear Amplification and Internal Bio

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