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Manual Salm-SeroGenoTyping AS

1. AAA 29 Soy sae 30 LITERATURE cc M 30 UPDATES AND SOFTWARE debet tie ha entgegen Diae On acp tune beg helium aa vd M RR 30 APPENDIX L FLOW CHART nacz 31 APPENDIX 2 PROBE TO TARGET TABLE negen bi 33 APPENDIX 3 TYPING INFORMATION css RA 41 Definitions and EXplanatONS anna boa 41 List of Currently Recognised Strains ci toe ca 42 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com BACKGROUND The Alere SeroGenoTyping AS 1 Kit for Salmonella allows DNA based serogenotyping according to the Kauffmann White scheme and an assignment of unknown Salmonella isolates to known strains as well as simultaneously the detection of resistance genes of Salmonella RNA free unfragmented genomic DNA from pure and monoclonal Salmonella 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 single stranded ss DNA reaction products 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 undesired amplicons is nearly impossible and for t
2. Thermoshakers The correct temperature within the vessels is essential therefore always use appropriate equipment for heating Because of the possibility of inhomogeneous distribution of temperature within the heating block as well as possible differences between displayed and actual temperatures the use of different brands of thermoshakers might affect test performance We tested the kit with BioShake Q by Quantifoil Instruments see figure below http www qinstruments com equipped with a customised heating block designed to fit ArrayStrips recommended and Eppendorf s Thermomixer Comfort equipped with a heating block for microtitre plates When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference LT2 strains or the control DNA CM S e e serovar Typhimurium LT2 Salm SeroGenoTyping AS 1 Kit 16 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com BioShake Q by Quantifoil Instruments equipped with a customised heating block designed to fit ArrayStrips http www qinstruments com Protocol for Quantifoil s Bioshake iQ and Eppendorf s Thermomixer Comfort with microtitre plate adapter Switch on the thermoshaker and pre heat it to 55 C Remove the amount of ArrayStrip s needed from the pouch Insert the ArrayStrip s into the white frame Ensure the correct orientation
3. 1 2013 12345 10624 2 2013 12346 10624 3 2013 12347 10624 4 2013 12348 10624 5 2013 12349 10624 Salm SeroGenoTyping AS 1 Kit 20 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com 6 2013 12350 10624 7 987654 10624 Isolate referred from Dr J Doe 8 cM 10624 control strain Table 2 Positions in the 96 well format 10 18 26 34 42 50 58 66 74 82 90 11 19 27 35 43 51 59 67 75 83 91 12 20 28 36 44 52 60 68 76 84 92 13 21 29 37 45 53 61 69 77 85 93 14 22 30 38 46 54 62 70 78 86 94 15 23 31 39 47 55 63 71 79 87 95 IJOJ I m JJO old lol lp lw Inele 16 24 32 40 48 56 64 72 80 88 96 Data Acquisition in the ArrayMate Reader Insert your memory stick containing the worklist into any of the USB ports down to the right hand side of the ArrayMate Press the button a folder selection dialogue will open Select your worklist path My Computer Removable Disk Open your selected worklist by pressing Enter or Open Press the button 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 Press the button OK the worklist window will close Leave the memory stick in the ArrayMate if you intend to export Salm SeroGenotyping AS 1 Test Reports afterwards check the memory stick for co
4. MM MasterMix 7 8h 3 with heating block for microtitre plates Hands on time 5 min 40 min 5 min 2 min 2 min 0 min 5 min 2 min 2 min 6 min 10 min app 120 min Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 32 www alere technologies com www alere com APPENDIX 2 PROBE TO TARGET TABLE Genoserotyping No Probes Targets 1 hp 3001 FL 1 e n x e n x z6 e n x z15 2 hp 3003 FL 1 e n x e n x z6 e n x z15 1 5 1 6 3 hp 3004 FL 1 e n x 1 2 1 5 1 2 7 1 5 7 1 6 1 7 z 1 11 16 1 12 4 hp 3005 FL 1 e n x 1 11 16 1 2 1 5 1 12 1 2 7 1 5 7 1 6 1 7 z 5 hp 3006 FL 1 e n x z 1 5 1 2 1 2 7 1 5 7 1 6 1 7 1 11 16 1 12 6 hp 3007 FL 1 e n x 1 5 1 6 7 hp 3008 FL 1 e n x 1 5 1 6 e n x 26 e n x z15 1 2 8 hp 3009 FL 1 e n x 1 5 1 6 e n x 26 e n x z15 1 2 9 hp 3012 FL 1 e n x 1 2 e n x 26 e n x z15 z 1 11 16 1 12 1 5 1 2 7 1 7 1 5 7 1 6 10 hp 3013 FL 1 e n x 1 2 e n x z15 e n x z6 z 1 11 16 1 12 1 2 1 2 7 1 5 7 1 5 1 6 1 7 11 hp 3014 FL 1 e n x e n x z15 1 2 e n x z6 z 1 11 16 1 12 1 5 1 2 7 1 7 1 5 7 1 6 12 hp 3015 FL 1 e n x 1 5 1 6 1 2 e n x z6 e n x z15 z 1 11 16 1 12 1 2 7 1 7 e n x z15 13 hp 3016 FL c C 14 hp 3017 FL c c 15 hp 3018 FL d
5. data matrix code close to row A and proper fit Pre wash the array in two steps e First PCR grade distilled water 200 ul per well at 55 C 5 min and 550 rpm Remove the water from the well e Second C1 Hybridisation Buffer 150 ul per well at 55 C 5 min and 550 rpm Add 90 ul of buffer C1 to each tube with 10 ul labelled amplification product mix gently Remove the buffer from the well with the array and add the mixture of C1 and labelled amplification product Incubate at 55 C 60 min and 550 rpm Salm SeroGenoTyping AS 1 Kit 17 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com TM e Meanwhile log on to the ArrayMate device and prepare your worklist see section Data Analysis p 17 e Remove liquid and add 200 ul C2 Washing Buffer Incubate at 45 C 5 min and 550 rpm remove and discard e Add another 200 ul C2 Washing Buffer Incubate at 45 C 5 min and 550 rpm remove and discard e Meanwhile prepare the conjugate For each experiment add 1 ul C3 HRP conjugate to 99 ul of C4 Conjugation Buffer This mixture is stable for around one working day at room temperature C3 is delivered with a surplus of 100 26 C4 with a surplus of 200 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 C3 15ul 3 5 ul 7 ul 11 16 ul 21 32 ul 42 ul 4 150 u
6. B 0 4 Yes S e enterica Duisburg SGSC2472 B 0 4 1 4 12 27 d e n z15 B 0 4 Yes S e enterica Schwarzengrund CDC1629 B 0 4 1 4 12 27 d 1 7 B 0 4 Yes SGSC2514 5 6 enterica Stanley CDC000477 B 0 4 1 4 5 12 27 d 1 2 B 0 4 Yes SGSC2517 S e enterica Chester CDC17 B 0 4 1 4 5 12 e h e n x B 0 4 Yes S e enterica Reading CDC19 B 0 4 1 4 5 12 e h 1 5 B 0 4 Yes SGSC2510 S e enterica Saintpaul CDC108 B 0 4 1 4 5 12 e h 1 2 B 0 4 Yes S e enterica Sandiego CDC18 B 0 4 1 4 5 12 e h e n z15 B 0 4 Yes S e enterica Derby CDC20 B 0 4 1 4 5 12 f g 1 2 B 0 4 Yes S e enterica Agona CDC1636 B 0 4 1 4 5 12 f g s 1 2 B 0 4 Yes 5 6 enterica California CDC1109 B 0 4 4 12 g m t 267 B 0 4 Yes S e enterica Budapest CDC23 B 0 4 1 4 12 27 g t B 0 4 Yes 5 6 enterica Travis CDC990318 B 0 4 4 5 12 g 251 1 7 B 0 4 Yes S e enterica 1 4 5 12 i CDCQA126 B 0 4 1 4 5 12 i B 0 4 Yes NRL688 NRL813 S e enterica Agama CDC513 B 0 4 4 12 i 1 6 B 0 4 Yes S e enterica Gloucester CDC443 B 0 4 1 4 12 27 i l w B 0 4 Yes 5 6 enterica Typhimurium CDC14 B 0 4 1 4 5 12 i 1 2 B 0 4 Yes DSM10506 DSM17058 DSM17058 DSM19587 DSM554 LT2 S e enterica Brandenburg CDC2519 B 0 4 4 5 12 l v e n z15 B 0 4 Yes SGSC2460 S e enterica Bredeney C
7. bottle and on the outer packaging All components were tested for stability for short term shipment lt 1 week at ambient temperature lt 37 C The kit components with limited stability are D1 and C3 The other components proved to be stable even six months after passing the kit expiry date Cell Lysis optional order e A1 Lysis Buffer cat 245101000 Store at 18 28 C ambient temperature Surplus 50 96 e A2 Lysis Enhancer lyophilised cat 245102000 Store at 18 28 C ambient temperature Centrifuge A2 tubes shortly prior to opening Add 200 ul Buffer A1 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 BU Labelling Buffer Store at 2 8 C Surplus 40 96 e B2 Labelling Enzyme Store at 2 8 C Surplus 100 96 e B3 lyophilised Labelling Primermix three tubes dilute each in 70 ul molecular grade water Store at 20 C Surplus 100 Salm SeroGenoTyping AS 1 Kit 4 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 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 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 n
8. full access to test specific software a default password will be provided together with the ArrayMate device If you log on as User you will obtain raw values only but neither interpretation of positives negatives nor strain assignment The Administrator log on will allow the installation of a new assay specific plug in which can be downloaded at http alere technologies com see p 28 e The user interface will be loaded the ArrayMate performs internal testing It requires slightly less than a minute e Click on the icon New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience e Type in your operator ID optional e You may enter a comment into the memo field optional Worklist A Worklist file allows to link an identifier such as a laboratory or sample number to a position of an array within the ArrayStrip Please respect the rules of confidentiality and data protection Salm SeroGenoTyping AS 1 Kit 19 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com 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 Na etc Create a list with at leas
9. j d 16 hp 3019 FL d j d 17 hp 3020 FL d j d 18 hp 3021 FL d j d j 19 hp 3022 FL d j d 20 hp 3023 FL d j d 21 hp 3024 FL e h e h 22 hp 3025 FL e h e h 23 hp 3026 FL e n x e n x z15 24 hp 3027 FL e n x e n x e n x z15 e n x z15 25 hp 3029 FL g z51 g 251 26 hp 3032 FL i r i 27 hp 3033 FL i r i 28 hp 3034 FL i r i 29 hp 3035 FL i r r 30 hp 3036 FL i r r 31 hp 3038 FL k z k 244 258 32 hp 3039 FL k z 235 z39 765 z10 33 hp 3040 FL k z z35 34 hp 3041 FL k z k 35 hp 3042 FL k z k z41 36 hp 3043 FL k z k 37 hp 3044 FL z z41 38 hp 3045 FL k z z10 39 hp 3046 FL k z z10 40 hp 3047 FL k z 281 41 hp 3048 FL k z 8 z10 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 33 www alere technologies com www alere com 42 hp 3049 FL k z 235 43 hp 3050 FL k z k 258 z44 z41 44 hp 3051 FL k z a z10 45 hp 3052 FL z z41 46 hp 3053 FL k z z81 47 hp 3054 FL k z z35 48 hp 3055 FL k z z35 49 hp 3056 FL k z z35 50 hp 3057 FL k z k 51 hp 3058 FL k z z10 52 hp 3060 FL k z k z41 53 hp 3061 FL k z k 54 hp 3062 FL 1 239 252 z39 55 hp 3063 FL I z39 z52 Lv 1 213 1 228 1 213 228 lw 56 hp 3065 FL 1 239 252 252 57 hp 3066
10. www alere com 87 hp 3118 FL 1 239 252 252 88 hp 3120 FL g z51 g z51 89 hp 3121 FL g z51 g z51 90 hp 3124 FL e n x e n x e n z15 91 hp 3125 FL b z91 b z91 92 hp 3126 FL b z91 b z91 93 hp 3128 FL b z91 b z91 94 hp 3129 FL b z91 b z91 95 hp 3130 FL b z91 b 791 96 hp 3134 FL z 76 97 hp 3135 FL z 76 98 hp 3136 FL z 269 99 hp 3138 FL z z 100 hp 3139 FL z z 101 hp 3140 FL z z 102 hp 3141 FL z z50 103 hp 3142 FL z 2 235 104 hp 3144 FL z 750 105 hp 3145 FL z H 106 hp 3146 FL z z 107 hp 3149 FL l z39 z52 z39 108 hp 3150 FL z 109 hp 3152 FL i r 110 hp 3153 FL l z39 z52 Lv lw 1 213 1 228 1 213 228 1 228 111 hp 3154 FL k z z10 112 hp 3155 FL z4 24 223 24 224 24 232 24 223 113 hp 3157 FL 1 e n x e n x 215 1 2 e n x 26 z 1 11 16 1 12 1 2 1 2 7 1 5 1 7 1 5 7 1 6 114 hp 3158 FL 1 e n x 1 5 7 1 2 e n x 26 e n x 215 z 1 11 16 1 12 1 2 7 1 5 1 7 1 6 115 hp 3161 FL 1 e n x 1 5 1 6 116 hp 3163 FL 1 e n x 1 2 1 12 1 2 7 1 5 7 1 5 1 6 1 7 z 1 11 16 117 hp 3165 manC manC species marker 118 hp 3166 wbyJ 041 119 hp 3167 wbyJ 041 120 hp 3168 manC O16439 O16 O39 121 hp 3169 manC 016 39 O16 O39 122 hp 3170 manC O7 07 123 hp 3171 manC O7 07 124 hp 3172 m
11. 1 0 7 Yes S e enterica Montevideo CDC1904 C1 0 7 6 7 14 8 m p s 1 2 7 C1 0 7 Yes 5 6 enterica Singapore CDC010011 C1 0 7 6 7 k e n x C1 0 7 Yes S e enterica Thompson CDC000342 C1 0 7 6 7 14 k 1 5 C1 0 7 Yes S e diarizonae 6 7 1 v z53 DSM14847 C1 0 7 6 7 1 v 253 C1 0 7 Yes S e enterica Bonn CDC344 C1 0 7 6 7 l v e n x C1 0 7 Yes S e enterica Potsdam CDC876 C1 0 7 6 7 14 l v e n z15 C1 0 7 Yes S e enterica Kenya CDC497 C1 0 7 6 7 1 213 e n x C1 0 7 Yes S e enterica Haelsingborg CDC586 C1 0 7 6 7 m p t u C1 0 7 Yes S e enterica Oranienburg CDC1271 C1 0 7 6 7 14 m t 257 C1 0 7 Yes 5 6 enterica Infantis CDC1428 C1 0 7 6 7 14 r 1 5 C1 0 7 Yes 5 6 enterica Virchow CDC2688 C1 0 7 6 7 14 r 1 2 C1 0 7 Yes 5 6 enterica Bareilly NRL608 C1 0 7 6 7 14 y 1 5 C1 0 7 Yes S e enterica Mbandaka CDC1906 C1 0 7 6 7 14 210 e n 215 C1 0 7 Yes S e enterica Tennessee CDC155 C1 0 7 6 7 14 229 1 2 7 C1 0 7 Yes S e enterica Tienba CDC2425 C1 0 7 6 7 235 1 6 C1 0 7 Yes S e enterica Lille CDC354 C1 0 7 6 7 14 238 C1 0 7 Yes 5 6 enterica Manhattan CDC122 C2 C3 0 8 6 8 d 1 5 C2 C3 0 8 Yes S e enterica Muenchen CDC54 C2 C3 0 8 6 8 d 1 2 C2 C3 0 8 Yes SGSC2243 S e enterica Virginia CDC189 C2 C3 0 8 8 d 1 2 C2 C3 0 8 Yes S e ente
12. 1 Well Position 06 D1 F Assay Name salm pm1 14 13 02 05 5 6 6 Typhimurit B15 13 02 05 S e e Bovismorb Software Version 0 8 Device O4a0006 hintin nncitiva Export of Salm SeroGenotyping Test Reports Two result files in HTML format will be generated The shorter report includes a summary on typing information A longer HTML html result sheet result B res html show information on all probes Possible error messages that might occur in these reports will be explained below see section Troubleshooting Other files that are generated and that can be exported include e Atext file txt with the raw measurements 9 Animage file bmp showing the actual photo of the array A second image file png in which the coordinate grid is superimposed and the recognised spots are circled and e A XML file xml that contains the same information like the HTML result sheets for future export into databases etc e An out file containing output log data which helps our service to trace imgae evaluation errors Please note Only complete runs can be exported The export of individual Salm SeroGenotyping AS 1 Test Reports is not possible e Right click on the selected run a menu appears with the option Export Run Reports Salm SeroGenoTyping AS 1 Kit 24 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com e Right click on
13. 2 241 1 5 D1 0 9 Yes S e enterica Franken CDC2570 D1 0 9 9 12 26 267 D1 0 9 Yes S e enterica Fresno CDC1412 D2 0 9 46 9 46 238 D2 0 9 46 Yes S e enterica Anatum CDC78 1 0 3 10 3 10 15 15 34 e h 1 6 E1 0 3 10 Yes S e enterica Meleagridis NRL737 E1 0 3 10 3 10 15 15 34 e h l w E1 0 3 10 Yes S e enterica Muenster CDC79 E1 0 3 10 3 10 15 15 34 e h 1 5 E1 0 3 10 Yes S e enterica Amsterdam CDC070756 1 3 10 3 10 15 15 34 g m s E1 0 3 10 Yes 5 6 enterica Westhampton CDC326 1 0 3 10 3 10 15 15 34 g s t E1 0 3 10 No Senftenberg S e enterica Bessi CDC1999 E1 0 3 10 3 10 i e n x E1 0 3 10 Yes 5 6 enterica Give CDC495 E1 0 3 10 3 10 15 15 34 1 v 1 7 E1 0 3 10 Yes CDC77 S e enterica London NRL700 E1 0 3 10 3 10 15 I v 1 6 E1 0 3 10 Yes 5 6 enterica Weltevreden CDC147 E1 0 3 10 3 10 15 r z6 E1 0 3 10 Yes 5 6 enterica Orion CDC321 E1 0 3 10 3 10 15 15 34 y 1 5 E1 0 3 10 Yes S e enterica Pietersburg CDC2258 E1 0 3 10 3 10K15 34 269 1 7 E1 0 3 10 Yes Salm SeroGenoTyping AS 1 Kit 45 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com TM S e enterica Senftenberg CDC87 E4 1 3 19
14. 3 0 8 6 8 229 e n z15 C2 C3 0 8 Yes 5 6 enterica Corvallis CDC1770 C2 C3 0 8 8 20 24 223 26 C2 C3 0 8 Yes S e enterica Duesseldorf CDC130 C2 C3 0 8 6 8 24 224 C2 C3 0 8 Yes S e enterica Tallahassee CDC196 C2 C3 0 8 6 8 24 232 C2 C3 0 8 Yes S e enterica Gallinarum CDC74 D1 0 9 1 9 12 D1 0 9 Yes DSM13674 S e enterica Berta CDC69 D1 0 9 1 9 12 f g t D1 0 9 Yes S e enterica Miami CDC198 D1 0 9 1 9 12 a 1 5 D1 0 9 Yes SGSC2485 S e enterica Goeteborg CDC696 D1 0 9 9 12 c 1 5 D1 0 9 Yes S e enterica Typhi No 1 D1 0 9 9 12 Vi d D1 0 9 Yes S e enterica Enteritidis CDC64 D1 0 9 1 9 12 g m D1 0 9 No Nitra DSM14221 Blegdam DSM17420 S e enterica Blegdam CDC090361 D1 0 9 9 12 g m q D1 0 9 No Nitra CDC68 Enteritidis S e enterica Dublin CDC10 D1 0 9 1 9 12 Vi g p D1 0 9 No Kiel 0635 Naestved CDC65 Moscow S e enterica Naestved CDC559 D1 0 9 1 9 12 8 p S D1 0 9 No Kiel Dublin SGSC3612 Moscow S e enterica Moscow CDC67 D1 0 9 1 9 12 g q D1 0 9 No Kiel Dublin Naestved S e enterica Panama CDC73 D1 0 9 1 9 12 1 v 1 5 D1 0 9 No Koessen SGSC2496 5 6 salamae 9 l w e n x DSM9220 D1 0 9 9 l w e n x D1 0 9 Yes 5 6 enterica Javiana CDC146 D1 0 9 1 9 12 1 228 1 5 D1 0 9 Yes 5 6 enterica Ottawa CDC1934 D1 0 9 1 9 1
15. 35 35 g m s O 0 35 Yes S e enterica Alachua CDC325 O 0 35 35 24 223 O 0 35 Yes S e enterica Kasenyi NRL878 P 0 38 38 e h 1 5 P 0 38 Yes S e enterica Lansing CDC634 P 0 38 38 i 1 5 P 0 38 Yes S e enterica Inverness CDC171 P 0 38 38 k 1 6 P 0 38 Yes 5 6 enterica Gera CDC1316 T 0 42 1 42 24 223 1 6 T 0 42 Yes S e enterica Niederoderwitz CDC2579 U 0 43 43 b U 0 43 Yes S bongori 66 241 DSM13774 0 66 66 241 0 66 Yes Salm SeroGenoTyping AS 1 Kit 46 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1
16. 54 prob sul1 11 dihydropteroate synthetase type 1 AJ698325 1 55 prob sul2 11 dihydropteroate synthetase type 2 DQ464881 1 56 prob sul3 11 dihydropteroate synthetase type 3 AJ459418 2 57 prob dfrA1 21 dihydrofolate reductase type 1 AJ884723 1 58 prob dfrA1 22 dihydrofolate reductase type 1 AJ884723 1 59 prob dfrV 21 dihydrofolate reductase type 5 AB188269 1 60 prob dfrA7 11 dihydrofolate reductase type 7 AB161450 1 AM237806 1 61 prob dfrA7 12 dihydrofolate reductase type 7 AB161450 1 AM237806 1 62 prob dfr12 11 dihydrofolate reductase type 12 AB154407 1 63 prob dfr13 11 dihydrofolate reductase type 13 synonym A21 50802 3 64 prob dfrA14 21 dihydrofolate reductase type 14 AJ313522 1 65 prob dfrA15 1 dihydrofolate reductase type 15 Z83311 1 66 prob dfrA17 11 dihydrofolate reductase type 17 AF169041 1 67 prob dfrA19 1 dihydrofolate reductase type 19 AJ310778 1 Genes encoding virulence factors No Probes Targets 1 astA consens 10 heat stable enterotoxin consensus sequence 2 prob intl1 1 class 1 integron integrase AY260546 3 3 prob intl2 11 class 2 integron integrase AY183453 1 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 40 www alere technologies com www alere com APPENDIX 3 TYPING INFORMATION Definitions and Explanations The displayed result will yield following typing information e Discrimination of the 46 described O serotypes is m
17. 6115 1 36 prob_oxa1_21 class D beta lactamase blaOXA 1 AY458016 1 37 prob oxa2 11 class D beta lactamases blaOXA 2 blaOXA 15 U63835 1 38 prob oxa7 11 class D beta lactamase blaOXA 7 AY866525 1 39 prob per2 1 class A beta lactamase PER 2 extended spectrum beta lactamase X93314 1 40 prob_psel_1pm class A beta lactamase carbenicillinase 218955 1 41 prob shvi 11 class A beta lactamase consensus 42 prob temi 1 class A beta lactamase consensus 43 prob catA1 11 chloramphenicol acetyltransferase group A V00622 1 44 prob catB3 11 chloramphenicol acetyltransferase group B AJ009818 1 45 prob catB8 12 chloramphenicol acetyltransferase AF227506 1 46 prob cmlA1 11 chloramphenicol transporter EF113389 1 47 prob floR 11 chloramphenicol and florfenicol efflux protein 48 mphA 611 macrolide 2 phosphotransferase EF102240 1 49 ereA 611 type I erythromycin resistance AY183453 1 50 prob_qnr_12 quinolone and fluoroquinolone resistance protein AY931018 1 Salm SeroGenoTyping AS 1 Kit 39 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com 51 prob_qnrB_12 quinolone and fluoroquinolone resistance protein AB281054 1 52 hp qnrD 611 quinolone and fluoroquinolone resistance protein FJ228229 1 53 prob qnrS 11 quinolone and fluoroquinolone resistance protein AM234722 1
18. 7 hp 3276 wzy O7 07 228 hp 3277 wzy O8 08 229 hp 3278 wzy O8 08 230 hp 3279 wzy O18 018 231 hp 3280 SSPAI Paratyphi A 232 hp 3281 SSPAI Paratyphi A 233 hp 3282 Q8ZK10 Typhimurum 234 hp 3287 lygA Enteritidis 235 hp 3288 lygD Enteritidis 236 hp 3289 Q8ZK15 Typhimurium 237 hp 3290 tviA plasmid Vi 238 hp 3292 tviA plasmid Vi 239 hp 3293 stgA Typhi 240 hp 3294 stgA Typhi 241 hp 3297 sefB Enteritidis 242 hp 3298 sefA Enteritidis 243 hp 3299 sefC Enteritidis 244 hp 3300 galF species marker for Salmonella 245 hp 3301 B5FQV7 Dublin 246 hp 3302 B5R5L5 unknown 247 hp 3306 B5R7B6 Gallinarum Weltevreden 248 hp 3307 B5R7C1 unknown 249 hp 3308 ISR1 Infantis 250 hp 3310 ISR1 Infantis 251 hp 3311 Q57QY4 Choleraesuis 252 hp 3312 Q57QY4 Choleraesuis 253 hp 3314 invA species marker for Salmonella 254 hp 3315 invA species marker for Salmonella 255 hp 3316 invA species marker for Salmonella Genes associated with antibiotic resistance No Probes Targets 1 hp aac3 611 3 N aminoglycoside acetyltransferase associated with resistance to gentamycin U90945 1 2 hp aac3 614 3 N aminoglycoside acetyltransferase associated with resistance to gentamycin U90945 1 3 prob aac3la 1 3 N aminoglycoside acetyltransferase associated with resistance to astromicin gentamicin 4 hp_aac6_612 aminoglycoside 6 N N acetyltransferase associated with resistance to amikacin dibekacin 5 hp_aac6_615 aminoglycoside 6
19. 90 hp 3238 wzx 07 07 191 hp 3239 wzx 07 07 192 hp 3240 wzx O8 08 193 hp 3241 wzx O8 O8 194 hp 3242 wzy O13 013 195 hp 3243 wzy O13 013 196 hp 3244 wzy O16 016 197 3245 2 O16 016 198 3246 2 O17 017 199 hp 3247 wzy_ O17 017 200 hp 3248 wzy O18 018 201 hp 3250 wzy O28 Dakar O28 serovar Dakar 202 hp 3251 wzy O28 Dakar O28 serovar Dakar 203 hp 3252 wzy O28 Pomona O28 serovar Pomona 204 hp 3253 wzy O28 Pomona O28 serovar Pomona 205 hp 3254 wzy_03 10 9 46 03 10 O9 46 206 hp 3255 wzy_03 10 9 46 03 10 O9 46 207 hp 3256 wzy_03 10 9 46 03 10 O9 46 208 hp 3257 wzy O30 030 209 hp 3258 wzy O30 030 210 hp 3259 wzy 035 035 211 hp 3260 wzy 035 035 212 hp 3261 wzy 038 038 213 hp 3262 wzy_038 038 214 hp 3263 wzy 041362 041 062 215 hp 3264 wzy 041362 041 062 216 hp 3265 wzy O50 050 217 hp 3266 wzy O50 050 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 37 www alere technologies com www alere com 218 hp 3267 wzy O55 055 219 hp 3268 wzy O55 055 220 hp 3269 wzy 056 056 221 hp 3270 wzy 056 056 222 hp 3271 wzy 058 058 223 hp 3272 wzy 058 058 224 hp 3273 wzy O6 14 06 14 225 hp 3274 wzy O6 14 06 14 226 hp 3275 wzy_O7 07 22
20. DC112 B 0 4 1 4 12 27 1 v 1 7 B 0 4 Yes S e enterica Heidelberg CDC16 B 0 4 1 4 5 12 r 1 2 B 0 4 Yes DSM9379 S e enterica Indiana CDC377 B 0 4 1 4 12 z 1 7 B 0 4 No Kiambu SGSC2482 S e enterica Kiambu CDC399 B 0 4 1 4 12 2 1 5 B 0 4 No Indiana S e enterica Haifa SGSC2479 B 0 4 1 4 5 12 210 1 2 B 0 4 Yes 5 6 enterica Stanleyville CDC223 B 0 4 1 4 5 12 27 24 223 1 2 B 0 4 Yes SGSC2518 S e enterica Maska CDC2349 B 0 4 1 4 12 27 241 e n z15 B 0 4 Yes 5 6 enterica Ohio CDC710 C1 0 7 6 7 14 b l w C1 0 7 Yes S e enterica Choleraesuis CDC34 C1 0 7 6 7 c 1 5 C1 0 7 Yes DSM14846 S e enterica Paratyphi C CDC33 C1 0 7 6 7 Vi c 1 5 C1 0 7 Yes SGSC3592 Salm SeroGenoTyping AS 1 Kit 43 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com S e enterica Typhisuis SGSC2527 C1 0 7 6 7 c 1 5 C1 0 7 Yes S e enterica Kambole CDC1863 C1 0 7 6 7 d 1 2 7 C1 0 7 Yes S e enterica Livingstone NRL720 C1 0 7 6 7 14 d l w C1 0 7 Yes S e enterica Braenderup CDC49 C1 0 7 6 7 14 e h e n z15 C1 0 7 Yes S e enterica Nola CDC2206 C1 0 7 6 7 e h 1 7 C1 0 7 Yes S e enterica Rissen CDC955 C1 0 7 6 7 14 f g C
21. Export Run Reports a file browser opens aua Browse Search ED ARCHIVE m 2009 02 05 09 00 42 expe sample ID order37x order37x23 order37x order37x78 order37x order37x82 das order42x01 order42x02 order42x03 Order42x06 order42x07 vee Order78x01 Order78x01 4 Order78x08 New Run Browse For Folder a O Power Choose a directory Desktop i E Se Local Disk D e Removable Disk E 4 Folder My Computer e Click My Computer subsequently on 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 on the OK button data are exported into the new folder on your memory stick e Do NOT remove the memory stick as long as the hourglass symbol is visible O e Switch off the device by pressing Power at the bottom left on the screen res Switch off the screen There is no need to physically switch off the ArrayMate Reader TROUBLESHOOTING In case of trouble always make sure that the 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 contact cct homeQclondiag com and include a description of the problem as well as the array images bmp
22. FL I z39 z52 Lv 1 213 1 228 I w 1 213 228 58 hp 3067 FL y y 59 hp 3068 FL y y 60 hp 3069 FL z29 z29 61 hp 3070 FL z29 z29 62 hp 3071 FL z36 z38 z38 63 hp 3072 FL z36 z38 236 236 238 64 hp 3073 FL z36 z38 236 236 238 65 hp 3074 FL z36 z38 236 236 238 66 hp 3075 FL z36 z38 236 238 238 67 hp 3076 FL z4 24 223 24 223 232 24 224 24 232 68 hp 3077 FL z4 24 224 69 hp 3078 FL z4 24 223 232 70 hp 3080 FL z65 265 71 hp 3085 FL g tg gm p q fg f 8 5 t f 8 t 8 262 g m t 72 hp 3086 FL g f g s t g m s g m s t fg f g t g m t g m p q g t g 262 73 hp 3087 FL g gm p q f g f 8 5 t f g t g t 8 262 g m t 74 hp 3089 FL g f g t mt g m t 75 hp 3090 FL g g m t f g t m t 76 hp 3091 FL g g m t f g t fg f g s t g m p a g t 8 262 g m s g m s t 77 hp 3092 FL g fg f g s t g m p q g t 6 762 f g g m t g m s g m s t 78 hp 3103 FL g fg fgstfgt g m s g m p q g t f g t g m t g m s t g z62 79 hp 3104 FL g fg t g m p q fg f g s t g m s g m t g t g 251 80 hp 3105 FL g 251 g m s t g m t g 262 f g f g s t f g t g m s g m p q g t 81 hp 3106 FL g g m s t g m t 6 762 fg f g s t f g t g m s g m p q g t g z51 82 hp 3107 FL g m t 83 hp 3108 FL g m t 84 hp 3109 FL g m t 85 hp 3113 FL 1 239 252 z39 86 hp 3117 FL 1 239 252 z39 Salm SeroGenoTyping AS 1 Kit 34 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com
23. Mi 13 02 05 5 6 6 Cholerae B11 13 02 05 S e e Infantis 3 B12 HybPM1 13 02 05 5 8 8 Agona 21 B13 13 02 05 S e e Dublin 18 14 HybPM1 13 02 05 5 8 8 Typhimurit DIS 13 02 05 S e e Bovismorb Click on a Sample ID and the Salm SeroGenotyping AS 1 Test Report for this array is shown in the window on the right Salm SeroGenoTyping AS 1 Kit 23 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Browse l Search results results b raw data segmentation image image 5 2013 02 06 13 22 37 print A1 HybPMI 13 02 05 See Kottbus CL fum HybPMI 13 02 05 5 6 6 Choleraest For Investigational Use Only Not Intended for Use in Clinical Diagnostics RUD A3 HybPMI 13 02 05 S e e Infantis 3 A4 HybPMI 13 02 05 5 6 6 Agona 21 Operator elke E AS HybPMI 13 02 05 S e e Dublin 186 A6 13 02 05 5 6 6 Typhimuriur Sample ID 13 02 05 S e e Typhimurium DSM19587 Archive A7 _HybPM1_13 02 05_5 e e _Bovismorbif Experiment ID HybPM1 13 02 05 S e e Typhimurium DSM18587 BE52AEB4 E25D 4606 BB63 03F603341B86 Ap HybPMi 13 02 05 S e e Enteritidis_ A9 HybPMI 13 02 05 See Kottbus CI Date of Result Wed Feb 06 13 48 39 2013 B10 HybPM1 13 02 05 5 8 8 Choleraes B11 HybPM1 13 02 05 5 6 6 Infantis 3 B12 HybPMI 13 02 05 5 6 6 Agona 21 Assay ID 10624 13 13 02 05 See Dublin 18
24. PE acetyltransferase associated with resistance to amikacin dibekacin als ene av Salm SeroGenoTyping AS 1 Kit 38 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com 6 hp_aac6_618 aminoglycoside 6 N acetyltransferase associated with resistance to amikacin dibekacin 7 prob_aac6lb_1 aminaglyc ide 6 N acetyliransferase associated with resistance to streptomycin spectinomycin 8 hp_aadB_611 2 aminoglycoside nucleotidyltransferase L06418 4 9 hp aadB 2 611 2 aminoglycoside nucleotidyltransferase L06418 4 10 hp armA 611 16S rRNA methylase associated with aminoglycoside resistance AB117519 1 11 prob aadA1 1 aminoglycoside adenyltransferase associated with resistance to streptomycin spectinomycin 12 prob aadA2 1 aminoglycoside adenyltransferase associated with resistance to streptomycin spectinomycin 13 prob_aadA4_1 aminoglycoside adenyltransferase associated with resistance to streptomycin spectinomycin 14 prob ant2la 1 aminoglycoside 2 adenylyltransferase associated with resistance to dibekacin gentamicin NAA uj nama d a 15 hp aphA 611 aminoglycoside 3 phosphotransferase kanamycin resistance protein AY260546 3 16 ble 611 associated with bleomycin resistance X01702 1 17 hp sph 611 strep
25. Salm SeroGenotyping Kit may be ordered separately Component Name Jee baam len are cona 0 1 0 4 ug ul For pricing please contact your local representative or our customer service respectively Legal Manufacturer Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena Germany Salm SeroGenoTyping AS 1 Kit 29 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Contact If you require any further information on this product please contact cct home clondiag com LITERATURE Literature quoted in this manual Anonymous 2007 Microbiology of food and animal feeding stuffs horizontal method for the detection of Salmonella EN ISO 6579 2002 Amd 1 2007 Geneva International Organization for Standardization 40 p Braun SD Ziegler A Methner U Slickers P Keiling S et al 2012 Fast DNA serotyping and antimicrobial resistance gene determination of Salmonella enterica with an oligonucleotide microarray based assay PLoS One 7 e46489 UPDATES AND SOFTWARE Notifications on database software updates and freeware tools can be found at http alere technologies com en products lab solutions salmonella html and or http alere technologies com en news html Salm SeroGenoTyping AS 1 Kit 30 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com APPENDIX 1 Flow chart The figure on this page summarises th
26. ainly determined by the genes wzy polymerase and wzx flippase The 114 known H antigens are encoded by two genes fliC phase 1 flagellin and fljB phase 2 flagellin e The probes immobilized on the current array version can discriminate 28 O antigens A 0 2 B 0 4 C1 7 C2 C3 0 8 D1 0 9 D2 0 9 46 E1 E4 0 3 10 0 1 3 19 F 0 11 G 0 13 H 0 6 14 I 0 16 J 0 17 K 0 18 M 0 28 N 0 30 O 0 35 P 0 38 Q 0 39 R 0 40 S 0 41 U 0 43 W 0 45 Z 0 50 O55 O56 058 062 and 066 e The following flagellar antigens can be identified on the array a b c d e h e n x e nxz15 f g f g m t f g s f g t fg 8 s t gm g m p s 8 m t g m q g m s g m s t g m t BD 8 p s 8 p u 5 9 8 5 t t 8 251 8 262 i j k k Iv lw 1 213 1 213 228 1 228 m p t u m t r r i y z 210 229 235 236 236 238 738 739 24 223 24 223 232 24 224 24 232 241 244 247 252 258 76 265 269 7581 291 1 11 AY353292 1 16 AY353263 1 2 1 2 7 1 2 7 1 5 1 5 7 1 5 7 1 6 1 7 e n x e n x z15 e n z15 k l w 1 213 228z z10 235 739 z41 750 and z6 e Probes specifying invA galF and manC that were introduced to confirm the identity of Salmonella and to serve as genus controls e Different probes were used to detect the following antimicrobial resistance genes aac3la aac3le aac lb aac6ll aadA1 aadA2 aadA23b aadA3 aadA5 aadB ant2la aphA armA sp
27. anC 011 O11 125 hp 3173 manC O11 O11 126 hp 3174 manC 018 O18 127 hp 3175 manC 018 O18 128 hp 3176 manC 041 041 129 hp 3177 manC O41 041 130 hp 3178 manC 041 041 131 hp 3179 manC 013 030 043 045 050 013 030 043 045 050 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 35 www alere technologies com www alere com 132 hp 3180 manC 013 030 043 045 050 013 030 043 045 050 133 hp 3181 manC 013 030 043 045 O50 013 030 043 045 050 134 hp 3182 manC 013 030 043 045 O50 013 030 043 045 050 135 hp 3183 manC 013 030 043 045 O50 013 030 043 045 050 136 hp 3184 manC 013 030 043 045 O50 013 030 043 045 050 137 hp 3185 manC 013 030 043 045 050 013 030 043 045 050 138 hp 3186 manC 013 030 043 045 O50 013 030 043 045 050 139 hp 3187 manC 013 030 043 045 O50 013 030 043 045 050 140 hp 3188 manC 02 4 9 3 10 02 04 09 03 10 141 hp 3189 manC 02 4 9 3 10 02 04 09 03 10 142 hp 3190 manC 040 040 143 hp 3191 manC 040 040 144 hp 3192 rfbV 02 9 9 46 02 09 09 46 145 hp 3193 rfbV 02 9 9 46 02 09 09 46 146 hp 3194 rfbV O4 O4 147 hp 3195 rfbV 04 O4 148 hp 3196 wbuH 041 62 041 062 149 hp 3197 wbuH 041 62 041 062 150 hp 3198 weiB_066 066 151 hp 3199 weiB_066 066 152 hp 3200 w
28. c therapy It cannot be used for bacteria other than Salmonella Specifications Upon receipt the kit components need to be stored at different temperatures as specified on the package insert The kit is to be performed at an ambient temperature of 18 28 C Technical Support If you require any further information on this product please contact email cct home clondiag com phone 49 0 3641 3111 155 Fax 49 0 3641 3111 120 For up to date information regarding the kit please visit our website at http www alere technologies com Safety Precautions The kit is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from pure Salmonella colonies clones requires expertise in Salm SeroGenoTyping AS 1 Kit 2 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed Isolated cell free Salmonella DNA may be processed without further biosafety precautions although contamination with Salmonella or other bacteria needs to be ruled out Always wear protective clothing as required for laboratory work according to your local regulations Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest amendments to the European Union Direct
29. d complete drying of the array surface during processing Do not allow it to stay without liquid for more than two minutes Never rinse the wells with distilled water after the hybridisation step use only 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 and leave it 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 Avoid contact of data matrix code 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 Salm SeroGenoTyping AS 1 Kit 14 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent reservoirs We strongly recommend that the liquid is removed by pipetting rather than by inverting the strips and flicking the liquids out Fine tipped so
30. de reliable strain identification and could be attributed either to technical reasons or to the presence of a yet unknown strain List of Currently Recognised Strains If you have array images of a strain not yet covered or if you have additional information on strain you wish to be included please contact cct home clondiag com Results of classical Serotyping Results of microarray based Serotyping Species Serovar Strain Unique Pattern Serogroup Antigenic Formula Serogroup invA galF manC Pate similar to Serovars S e enterica Paratyphi A CDCL A 0 2 1 2 12 a 1 5 A 0 2 Yes 5 6 enterica Nitra CDC1280 A 0 2 2 12 g m A 0 2 No Enteritidis Blegdam S e enterica Kiel CDCO9 A 0 2 1 2 12 g p A 0 2 No Dublin 1879 Naestved CDC674 Moscow S e enterica Koessen CDC2417 A 0 2 2 12 1 v 1 5 A 0 2 No Panama 5 6 enterica Abony CDC103 B 0 4 1 4 5 12 27 b e n x B 0 4 Yes DSM4224 S e enterica Paratyphi B CDC3 B 0 4 1 4 5 12 b 1 2 B 0 4 Yes Salm SeroGenoTyping AS 1 Kit 42 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com S e enterica Wien SGSC2528 B 0 4 1 4 12 27 b l w B 0 4 Yes S e enterica Jericho CDC621 B 0 4 1 4 12 27 c e n z15
31. e linear amplification and internal biotin labelling process Salm SeroGenoTyping AS 1 Kit 12 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Linear Amplification and Internal Biotin Labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 labelling reagent is 40 Salm 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 Salmonella 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 VWR cat 460 0108 according to the following protocol Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 50 C 50 cycles with 40 sec at 72 C 60 sec at 96 C Cool down to 4 C hold e The samples 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 Salm SeroGenoTyping AS 1 Kit 13 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Hybridisation General Remarks Handling of Arrays Never touch the array surface Avoi
32. e screen reader opens e Remove the white frame with the ArrayStrip s e Press Next at the bottom right on the screen reader closes Salm SeroGenoTyping AS 1 Kit 22 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Results On the lefthand 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 the button Archive lefthand and activate the flag Browse top left The runs are organised like folders in Windows Explorer and named by default according to the date of acquisition Example There is one experiment run in this archive Browse Search EV ARCHIVE 2013 02 06 13 22 37 New Run ES Archive If you click on the plus symbol left of the run name the folder opens and you will see a list of the individual arrays ordered by the sample ID m Browse 0 Search Al Tr 2013 02 06 13 22 37 A1_HybPM1_13 02 05_5 e e _Kottbus_C amp A2 HybPMi 13 02 05 S e e Choleraest New Run A3 HybPM1 13 02 05 S e e Infantis 3 j A4 HybPMi 13 02 05 S e e Agona 21 A5 Hat 13 02 05 5 8 8 Dublin 186 A6 HybPMI 13 02 05 5 6 6 Typhimuriui Archive A7 HybPMi 13 02 05 S e e Bovismorbif A8 HybPMI 13 02 05 S e e Enteritidis A9 HybPMi 13 02 05 S e e Kottbus CL 1 HybP
33. e test procedure However please refer to the text section of this user guide at any step of the test protocol for further important details Salm SeroGenoTyping AS 1 Kit 31 05 16 04 0010 MO Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Pro Prepare ArrayStrips Prepare DNA cessing time Grow CLONAL Salmonella isolate over not part of the kit night lt a Isolate genomic DNA 3 4h E not part of the kit a l Label RNA free DNA thermocycler 5 ul DNA cpya 0 1 0 4 ug ul Rinse ArrayStrips BR PS Seine RARE 200 ul water 55 C 550 rpm 2 min H f Discard water Prepare labeled DNA 150 ul Buffer C1 55 C 550 rpm 4 min to 10 uL of labeled DNA add 90uL 2 min discard C1 process promptly of Buffer C1 Transfer 100 pl labeled DNA to ArrayStrips 2 min Barcollesz PO here Hybridise 55 C 550 rpm 60 min 60 min Discard labeled DNA 5 min incubate twice in 200 ul Buffer C2 45 C 550 rpm 5 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrate D1 25 C Discard Buffer C2 10 min incubate in 100 pl C3 C4 conjugate 30 C 550 rpm 10 min l Discard C3 C4 conjugate 2 min incubate twice in 200 ul Buffer C5 30 C 550 rpm 1 min i Quantifoil BioShake iQ or Eppendorf Thermomixer Discard Buffer C5 6 min incubate with 100 ul Substrate D1 25 C 10 min i Discard Substrate D1 analyse ArrayMate 10 min total time requirement overnight
34. es 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 tubes 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 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 8 000 rpm 1 min at room temperature 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 Salm SeroGenoTyping AS 1 Kit 10 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Contamination with Washing Buffer might occur during elution of prepared DNA by droplets adhering to the funnel of the spin columns Thus these funnels should be gently touched and dried with sterile filter paper or wipes prior to the elution step Alternatively
35. files in your question Salm SeroGenoTyping AS 1 Kit 25 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com 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 wells with Buffer C2 to remove all of Buffer C1 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 CM This contains DNA from the reference strain 5 6 6 Typhimurium LT2 GenBank accession number NC 003197 1 and should be identified by the assay as Salmonella enterica spp enterica serovar Typhimurium If the control experiment yields a valid result and a correct identification there was probably an issue with DNA preparation If the control expe
36. ft disposable Pasteur pipettes are best suited such as VWR cat 612 2856 Always place the pipette tip at the cavity between the array and the wall of the reagent well If you touch the array surface probes may be scratched off and this may cause an error Pasteur pipette plastic with a flexible tip Ce 1 flexible tip MAS Pipette Use the cavity between array and the wall of the tube Do never touch the array Array Salm SeroGenoTyping AS 1 Kit 15 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com General Remarks The Substrate Precipitating Dye D1 It is recommended to fill an appropriate amount of substrate precipitating dye D1 into a reaction tube and taken out of the refrigerator when starting the procedure allowing it to acclimatise it to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged quick spin prior to use to remove bubbles as well as possible precipitates Triggered by peroxidase the dye precipitates in the 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 remove and discard reagent D1 as completely as possible and scan immediately ArrayMate The dye precipitate fades slowly in presence of liquids General Remarks
37. g s t E4 No Westhampton DSM10062 0 1 3 19 0 1 3 19 S e enterica Westerstede CDC607 E4 1 3 19 1 213 1 2 E4 Yes 0 1 3 19 0 1 3 19 S e enterica Missouri CDC2309 F 0 11 11 g s t F 0 11 Yes S e enterica Connecticut CDC2392 F 0 11 11 1 213 228 1 5 F 0 11 Yes S e enterica Rubislaw CDC102 F 0 11 11 r e n x F 0 11 Yes SGSC2511 S e enterica Mississippi CDC154 G 0 13 1 13 23 b 1 5 G 0 13 Yes S e enterica Havana NRL607 G 0 13 1 13 23 f g s G 0 13 Yes S e enterica Idikan CDC1690 G 0 13 1 13 23 i 1 5 G 0 13 Yes S e enterica Kedougou CDC1523 G 0 13 1 13 23 i 1 w G 0 13 Yes S e enterica Poona CDC1243 G 0 13 1 13 22 2 1 6 G 0 13 Yes S e enterica Cubana CDC207 G 0 13 1 13 23 229 G 0 13 Yes S e enterica Ajiobo CDC527 G 0 13 13 23 24 223 G 0 13 Yes S e indica 6 14 a e n x DSM14848 G 0 13 6 14 a e n x G 0 13 Yes S e enterica Blijdorp CDC765 H 0 6 14 1 6 14 25 c 1 5 H 0 6 14 Yes S e enterica Carrau CDC93 H 0 6 14 6 14 24 y 1 7 H 0 6 14 Yes 5 6 enterica Grancanaria CDC2506 0 16 16 239 1 6 0 16 Yes 5 6 enterica Cerro CDC990087 K 0 18 6 14 18 24 223 1 5 K 0 18 Yes 5 6 enterica Pomona CDC2473A M 0 28 28 y 1 7 M 0 28 Yes S e enterica Morocco CDC694 N 0 30 30 1 213 228 e n 215 N 0 30 Yes S e enterica Ealing CDC745 O 0
38. g esses 13 HybridisatiOM MM T daE 14 General Remarks Handling of Arrays sess nennen nnne enn 14 General Remarks Handling of Liquids pe tutae EE nn 15 General Remarks The Substrate Precipitating Dye Di 16 General Remarks Thermoshakers esses aaa aaa aaa aaa nnn nennen 16 Protocol for Quantifoil s BioShake iQ and Eppendorf s Thermomixer Comfort with micr titre plate oks ai AE 17 Data A See 19 Starting the ArrayMate Reader 19 eege G E S 19 Data Acquisition in the ArrayMate Reader AE 21 Dici 23 Export of Salm SeroGenotyping AS 1 Test Reports eesssseseseeeeeneeeee enne 24 AA o HU dna O 25 Staining Controlled o 26 mage QUALITY c 26 e E e RR TT e 26 Physical Damage to the Array 27 Report Unavailable 27 dd 28 Warranty 28 In 28 Salm SeroGenoTyping AS 1 Kit 05 16 04 0010 VO1 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com HENNEN erp tai te dee 29 List of Components for Separate Order ui iia 29 Legal Manufacturer
39. h strA strB resistance to various aminoglycosides catA1 catB3 catB8 cmlA floR chloramphenicol tetA tetB tetC tetD tetG tetracyclines sul1 sul2 sul3 dfrA1 dfrA5 dfrA7 dfrA12 dfrA13 dfrA14 dfrA15 dfrA17 dfrA19 sulfonamide trimethoprim ble glycopeptides bleomycin gnrA qnrB qnrD qnrS quinolones acc1 carB2 cmy2 ctxM1 ctxM2 ctxM26 ctxM9 dha1 oxa1 oxa2 oxa10 oxa53 per2 shv tem1 beta lactam Salm SeroGenoTyping AS 1 Kit 41 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com compounds kpc4 carbapenems and ereA mphA macrolides Additionally two probes were designed to determine the presence of genes int 1 and intl2 possibly mediating an integrase function e Using a PatternMatch module a software package was developed to analyze Salmonella serovars directly at the ArrayMate device after scanning and calculating signals of the stained arrays e The detection software used a database comprising 168 reference Salmonella strains representing 132 Salmonella serovars which were classically serotyped Patterns of unknown Salmonella were compared to the whole database and the two best hits were given in an overview result sheets result A and in a detailed result sheet with all probes listed in a table result B e Assignment score This is a score for the similarity to the average hybridisation result for a given strain Scores above 6 5 exclu
40. hat reason the method is restricted to clonal culture material and cannot be performed on samples such as swabs or other patient samples e g faeces Resulting biotin labelled ssDNA is transferred and hybridised to DNA oligonucleotide microarrays with 329 covalently immobilised probes for different genetic markers and a biotin staining control All of them are printed in two duplicate spots The target set consists of a variety of species and serotyping markers including genes encoding 28 O antigens and 86 H antigens H1 and H2 Additionally 77 targets analysing antimicrobial resistance AMR genes were included Braun et al 2012 Spot recognition is performed automatically based on a digital image of the arrays The overall pattern is analysed automatically for the presence or absence of specific genes and it is compared to a database of strain profiles This allows the assignment of the serovar the antigenic formula to Kauffmann White and the AMR profile Salm SeroGenoTyping AS 1 Kit 1 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 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 bacterial cultures from Salmonella isolates for research and epidemiological applications It must not be used as a substitute for phenotypic susceptibility tests and for the guidance of antibioti
41. ically for updates for details see below Information such as spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is delivered with the reader or can be downloaded from our website These test specific plug ins will occasionally be updated Please check the NEWS section of our website http www clondiag com Support is available under cct homeQclondiag com Components required but not provided e Growth media for the cultivation of Salmonella The test should be performed with colonies harvested from 2 x TY Agar Other rich media e g Standard 1 or LB may also suffice but have not systematically been tested Liquid media should also not been used because contaminations or mixed cultures cannot easily be ruled out e Equipment and consumables needed for the cultivation of Salmonella incubator inoculation loops Petri dishes e DNA preparation kits The kit was tested with the DNeasy Blood Tissue Kit from Qiagen cat 69504 and High Pure DNA Isolation Kit from Roche cat 11796828001 Please note The DNA specimen needs to be free of RNA Recommendation a pre treatment with the cell lysis components A1 A2 see below or a standard RNase A treatment while DNA preparation Salm SeroGenoTyping AS 1 Kit 6 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com TM e Equipment needed for DNA isolation e g pipette
42. ith A1 A2 reagent instead of 1 x 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 colony material of the Salmonella isolate to A1 A2 reagent and vortex thoroughly e Incubate the colony material of the Salmonella isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker e Proceed with 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 Qiagen Kit or equivalent and add 200 ul buffer AL Qiagen Kit e Vortex briefly or shake vigorously e Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker e Important If A1 A2 reagent not used add 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing 9 Add 200 ul ethanol 96 100 96 e Vortex the sample and centrifuge quick spin e Transfer the complete content of the tube including any precipitate into a spin column Salm SeroGenoTyping AS 1 Kit 9 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com that is placed in a 2 ml collection tube e Centrifuge at room temperature time 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 tub
43. ives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet MSDS They do not contain more than 1 of a component classified as hazardous and do not contain more than 0 1 of a component classified as carcinogenic MSDS therefore are not provided Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents In case 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 Salm SeroGenoTyping AS 1 Kit 3 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 TM www alere technologies com www alere com REAGENTS AND DEVICES Assay Components Storage and Stability All reagents are provided in surplus see below If necessary all components may also be ordered separately please refer to the order numbers at the end of this manual For pricing please contact your local representative or our customer service respectively The expiry date can be found on each
44. l 350 ul 700 ul 1100 ul 1600 ul 2100 ul 3200 ul 4200 ul e Remove and discard the Washing Buffer and add 100 ul diluted conjugate C3 C4 to each well incubate at 30 C 10 min and 550 rpm e Remove liquid and wash with 200 ul C5 Washing Buffer just pipette up and down once remove and discard e Add another 200 ul C5 Washing Buffer Incubate at 30 C 2 min and 550rpm e Remove and discard Washing Buffer add 100 ul of D1 substrate precipitating dye at 25 C see above per well e ncubate at 25 C 10 min but do not shake e Remove liquid completely e The outside of the bottom of the ArrayStrips may be carefully cleaned with wipes e Scan and process ArrayMate see below Salm SeroGenoTyping AS 1 Kit 18 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Data Analysis Starting the ArrayMate Reader We recommend to start the ArrayMate Reader after starting the hybridisation this allows the convenience of starting the device and importing the worklist file Please note This is a short instruction 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 2 operating switch on the bottom left corner of the front side e Switch on the screen switch righthand side below the screen 9 Log on as R amp D User Research and Development User for
45. mputer viruses and malware using an appropriate program on a regular basis Press the button Next at the bottom right on the screen reader opens Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply strong force Assure proper fit otherwise the images may be out of focus Salm SeroGenoTyping AS 1 Kit 21 05_16_04_0010_V02_Manual_Salm SeroGenoTyping AS 1 www alere technologies com www alere com TM e Carefully insert the white frame with the ArrayStrips into the metallic adapter Ensure the correct orientation position A1 in the frame next to the data matrix code on the adapter and proper fit otherwise the images may be out of focus ArrayStrip frame with strips inserted in accordance with the Worklist Please note ArrayStrips must be clean They should not contain any liquids during analysis Data matrix codes must be clean There must be no StripCaps on the wells to be analysed however unused wells should remain capped e Press the button 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 right on th
46. oclonal culture of Salmonella Contamination with other bacteria especially with other Enterobacteriaceae needs to be strictly avoided as they might carry the same resistance genes as certain Salmonella strains and thus can introduce false positive signals and patterns Extraction of DNA The required sample quantity is 0 5 2 ug Cpna 0 1 0 4 ug ul of intact genomic DNA from a single clone The DNA specimen needs to be free of RNA and it should not be fragmented This can be determined by agarose gel electrophoresis DNA should not be prepared by disrupting Salmonella cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the Salmonella Serogenotyping kit are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend to obey the protocols outlined below Salm SeroGenoTyping AS 1 Kit 8 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com DNA Extraction by spin columns e g Qiagen e Add an inoculating loop full of monoclonal colony material of the Salmonella isolate to 0 2 ml 1 x PBS and vortex thoroughly loop empty loop full It is important to harvest enough bacteria this is prerequisite for extraction of a sufficient amount of DNA Take an inoculating loop 1 mm in diameter filled with bacteria as shown in the righthand picture Optional cell lysis w
47. ot store or handle unused wells at more than 60 relative humidity since this may irreversibly corrode the spots e StripCaps 24 units e 1 Hybridisation Buffer Store at 18 28 C protect against sunlight Surplus 150 e C2 Washing Buffer 1 Store at 18 28 C protect against direct sunlight Surplus 200 96 e C3 HRP Conjugate 100 x Store at 2 8 C protect against direct sunlight Surplus 100 96 e C4 Conjugate Buffer Store at 18 28 C protect against direct sunlight Surplus 200 96 e C5 Washing Buffer 2 Store at 18 28 C protect against direct sunlight Surplus 200 96 e D1 Horseradish Peroxidase Substrate Store at 2 8 C protect against direct sunlight Surplus 50 e optional CM Reference DNA from 5 9 6 Typhimurium LT2 GenBank accession number NC 003197 1 Cpna 0 1 0 4 ug ul Store at 2 8 C Sufficient for 5 6 tests Salm SeroGenoTyping AS 1 Kit 5 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Instrumentation and Software e ArrayMate Reader to be ordered separately for details see below The Salm SeroGenoTyping AS 1 Kit may 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 website check period
48. prepared DNA can 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 concentration A so method it should not 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 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 heat lysis 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 Salmonella 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 Add 0 25 ul RNase A not provided see above with a stock concentration of 1 mg ml e ncubate at 37 C 5 min at 550 rpm in a thermoshaker Salm SeroGenoTyping AS 1 Kit 11 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com TM e Use 5 ul of this DNA suspension for th
49. ray 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 is not 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 positioned properly into the frame Scratches or drops of condensed water might render the data matrix code identifier unreadable please wipe it carefully or try to manually identify the test If no obvious reason for the fault can be discovered please contact the technical service Salm SeroGenoTyping AS 1 Kit 27 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 TM 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 to 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 reasons other than misuse contact Alere Technologies GmbH for replacement or refund Te
50. rica Kottbus CDC52 C2 C3 0 8 6 8 e h 1 5 C2 C3 0 8 Yes S e enterica Newport CDC2434 C2 C3 0 8 6 8 20 e h 1 2 C2 C3 0 8 Yes S e enterica Emek SGSC2477 C2 C3 0 8 8 20 g m s C2 C3 0 8 Yes 5 6 enterica Kentucky CDC2590 C2 C3 0 8 8 20 i z6 C2 C3 0 8 Yes Eng196 5 6 enterica Lindenburg CDC334 C2 C3 O 8 6 8 i 1 2 C2 C3 0 8 Yes 5 6 enterica Blockley CDC448 C2 C3 0 8 6 8 k 1 5 C2 C3 0 8 Yes Eng23 Eng24 S e enterica Litchfield CDC000462 C2 C3 0 8 6 8 1 2 C2 C3 0 8 Yes 5 6 enterica Manchester Eng205 C2 C3 0 8 6 8 1 7 C2 C3 0 8 Yes S e enterica Breukelen CDC1699 C2 C3 0 8 6 8 1 213 228 e n 215 C2 C3 0 8 Yes S e enterica Goldcoast NRL852 C2 C3 0 8 6 8 r l w C2 C3 0 8 Yes S e enterica Bovismorbificans CDC2201 C2 C3 0 8 6 8 20 r i 1 5 C2 C3 0 8 Yes S e enterica Hidalgo CDC2359 C2 C3 0 8 6 8 r i e n z15 C2 C3 0 8 Yes Salm SeroGenoTyping AS 1 Kit 44 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com S e enterica Hadar CDC347 C2 C3 0 8 6 8 210 e n x C2 C3 0 8 No Istanbul S e enterica Istanbul CDC1466 C2 C3 0 8 8 z10 e n x C2 C3 0 8 No Hadar S e enterica Uno CDC1697 C2 C
51. riment fails as well an error affecting later steps or a degradation of reagents applied in later steps is likely DNA Quality The amount of DNA is crucial because of the linear kinetics of amplification see introduction DNA should be free of RNA as RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation A260 Salm SeroGenoTyping AS 1 Kit 26 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com readings will cover RNA and other contaminants as well Therefore pure DNA preparations without RNA contamination are prerequisite for proper DNA concentration measurement RNase treatment prior to A260 reading therefore is necessary component A2 contains RNAse DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites For this reason DNA should not be prepared by disrupting Salmonella cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols We evaluated the manual QIAGEN DNeasy kit and the Roche High Pure Kit DNA must be free of any trace 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 to evaporate the ethanol Physical Damage to the Array Scratching of the ar
52. rms and conditions apply If you have any 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 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 resistances might be caused by genes or mutations not covered by this array or by hitherto unknown genes or mutations any antibiotic chemotherapy MUST be 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 from whom Salmonella was isolated on its hard disk and or to the use of external storage devices that might be contaminated with spyware Salm SeroGenoTyping AS 1 Kit 28 05 16 04 0010 VO2 Manual Salm SeroGenoTyping AS 1 www alere technologies com www alere com Quality Control Each batch is stringently tested with the use of standard Salmonella DNA preparations for good performance and correctness of results List of Components for Separate Order If required these reagents for the
53. s centrifuge thermoshaker or automated device see above e Photometer OD gt 60 nm for measuring the concentration of DNA e Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA e Thermocycler for PCR e Thermoshaker We strongly recommend the BioShake iQ by Quantifoil Instruments http www qinstruments com equipped with a customised heating block designed to fit ArrayStrips Alternatively you may use Eppendorf s Thermomixer Comfort equipped a heating block for microtitre plates e Pipettes suitable for 1 5 ul volumes 90 ul 100 ul 200 1000 e Multichannel pipettes for 100 200 e 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 Salm SeroGenoTyping AS 1 Kit 7 05_16_04_0010_V02_Manual_Salm SeroGenoTyping AS 1 www alere technologies com www alere com PROTOCOL Culturing and Harvesting Bacterial Cells Serovars of the genus Salmonella 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 Salmonella on 2 x TY agar overnight at 37 C or 48 hrs at room temperature Obtain confirmation of the identification as Salmonella EN ISO 6579 2002 Amd 1 2007 Anonymous 2007 and make sure that you have a pure mon
54. t three columns with obligatory headers in the following order position sample ID assay ID table 1 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 Sample ID is strain sample laboratory number such as exported from your LIMS or assigned in any different way Patient name should not be used as sample ID The Assay ID enables the system to identify the current test and to correctly use information on layout spot number and identity etc The Salm SeroGenotyping AS 1 Kit has the Assay ID 10624 Please note Assay ID numbers must not be confused as this could lead to errors or loss of data You may add further columns and headers with notes and comments at your convenience Information from these columns will not appear on the result screen or in the Test Report We recommend using a printout of the worklist as a template for pipetting Save the worklist as tab separated txt file on the memory stick provided together with the ArrayMate To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted Table 1 Example worklist Please note Table header must be written exactly as shown Position Sample ID Assay ID Comment
55. tomycin 3 phosphotransferase associated with resistance to streptomycin U00004 1 18 prob strA 611 aminoglycoside 3 phosphotransferase locus A associated with resistance to streptomycin 19 prob strB 611 aminoglycoside 6 phosphotransferase associated with resistance to streptomycin EF090911 1 20 prob tetA 11 tetracycline resistance protein A tetracycline efflux protein CP000971 1 21 prob tetB 11 tetracycline resistance protein A class B V00611 1 22 prob tetC 11 tetracycline resistance protein A class C EU751612 1 23 prob tetD 1 tetracycline resistance protein A class D X65876 1 24 prob tetG 11 tetracycline resistance protein A class G AF261825 2 25 hp kpc4 611 carbepenem hydrolyzing beta lactamase 26 blaCMY 611 consensus sequence for blaCMY 13 blaCMY 2 blaCMY 24 blaCMY 35 27 prob cmy 11 consensus sequence for blaCMY 13 blaCMY 2 blaCMY 24 blaCMY 35 28 prob acci 11 class C beta lactamase blaACC 1 EF554600 1 29 prob acc2 11 class C beta lactamase blaACC 2 EF554600 1 30 hp per2 611 class A beta lactamase PER 1 extended spectrum beta lactamase Z21957 1 31 prob ctxM1 11 class A extended spectrum beta lactamase X92506 1 including blaCTX M15 HQ202266 1 32 prob ctxM2 11 class A extended spectrum beta lactamase AY517475 33 prob ctxM9 11 class A beta lactamase AF174129 3 34 prob ctxM26 11 class A extended spectrum beta lactamase AF518567 35 prob dha1 1 class C beta lactamase EF40
56. www alere technologies com www alere com Manual Salm SeroGenoTyping AS 1 Kit Array Hybridisation Assay for DNA based serogenotyping and detection of resistance genes of Salmonella and assignment of unknown Salmonella isolates to known strains Kit order number 245700096 96 reactions ArrayStrip format For Research Use Only Not Intended for Use in Clinical Diagnostics www alere technologies com www alere com CONTENT dx Tp 1 GENERAL INSTRUCTIONS FOR USE Eug io Poe uh eoa AE Gd 2 Us naar ida 2 rae A aE E EEE AEA Ea E EEEa A RAE a a TEE a a eai 2 ai c ada 2 Safety udi ao inka GG a 2 Material Safety Data Sheets MSDS Divino KE rid ger aad gU Gnd 3 Shipping Precautions EE 3 RENGER 4 Assay Components Storage and Stability sss eene 4 Cell Lysis optional order EE 4 DNA Labelling and Amplificaci n 4 5 Instrumentation and nins rl e EEN 6 Components required but not provided E 6 PROTOCOL PUTET 8 Culturing and Harvesting Bacterial Cells 1 erret nhan aan eo radar 8 Extraction of DNA ai HH 8 DNA Extraction by Spin Columns e g Qiagen sseu soo aa aaa aaa eene 9 DNA Extraction DY Heat sis indios 11 Linear Amplification and Internal Biotin Labellin
57. zx_013 013 153 hp 3201 wzx_013 013 154 hp 3202 wzx_016 016 155 hp 3203 wzx_016 016 156 hp 3204 wzx_017 017 157 hp 3205 wzx_017 017 158 hp 3206 wzx O18 O18 159 hp 3207 wzx O18 O18 160 hp 3208 wzx O249 O2 O9 161 hp 3209 wzx 0249 02 O9 162 hp 3210 wzx O28 Dakar O28 serovar Dakar 163 hp 3211 wzx O28 Dakar O28 serovar Dakar 164 hp 3212 wzx O28 Pomona O28 serovar Pomona 165 hp 3213 wzx O28 Pomona O28 serovar Pomona 166 hp 3214 wzx 03 10 03 10 167 hp 3215 wzx_03 10 03 10 168 hp 3216 wzx O30 030 169 hp 3217 wzx 030 030 170 hp 3218 wzx 035 035 171 hp 3219 wzx 035 035 172 hp 3220 wzx O4 04 Salm SeroGenoTyping AS 1 Kit 05_16_04_0010_vV02_Manual_Salm SeroGenoTyping AS 1 36 www alere technologies com www alere com 173 hp 3221 wzx O4 04 174 hp 3222 wzx O4 04 175 hp 3223 wzx_041 62 041 062 176 hp 3224 wzx_041 62 041 062 177 hp 3225 wzx_050 050 178 hp 3226 wzx O50 050 179 hp 3227 wzx O55 055 180 hp 3228 wzx_055 055 181 hp 3229 wzx_056 056 182 hp 3230 wzx_056 056 183 hp 3231 wzx O58 O58 184 hp 3232 wzx O58 O58 185 hp 3233 wzx O6 14 06 14 186 hp 3234 wzx_06 14 06 14 187 hp 3235 wzx_066 066 188 hp 3236 wzx_066 066 189 hp 3237 wzx_07 07 1

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