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S. aureus Genotyping Kit 2.0

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1. MLST CC Sre SE MEST types associated spa types associated with with this strain ST15 ST582 ST869 t084 t085 t094 t254 t360 cc Cee ST1025 t774 t1509 t1885 CC15 MSSA PVL ST15 t360 CC15 MRSA I CC15 ST199 ST199 MSSA 51199 t774 cc20 CC20 MSSA lukF P83 M CC20 MSSA ST20 ST389 t148 t195 t1023 CC20 MRSA V cc22 CC22 MSSA ST22 t005 t395 t3242 CC22 MSSA PVL t005 t310 t417 t891 CC22 MSSA SCCfus PVL t417 t6101 CC22 MRSA IV ACME UK EMRSA 15 Dublin variant am t022 1032 13185 Irish ARO6 Canadian MRSA 8 t020 t022 t025 t032 t432 t5 CC22 MRSA IV fnbB l 3 A 4 a x K UK EMRSA 1E PR p Spanish PFGE type E13 ST22 A ST22 ST1117 15 t578 t717 t790 t1214 t1 clade of UK ERMSA 15 559 t1802 t2951 Irish ARO6 Canadian MRSA 8 t020 t022 t032 t432 t531 t9 e p Spanish PFGE type E13 ST22 A ST22 81 t1214 t1370 t1802 t1865 clade of UK ERMSA 15 t2235 t3213 t3501 t3505 Irish ARO6 Canadian MRSA 8 Geen ceca emrsa Spanish PFGE type E13 ST22 non ST22 t005 t032 t451 t2951 A clade of UK ERMSA 15 CC22 MRSA IV fusC UK EMRSA 15 Maltese variant CC22 MRSA IV tst1 UK EMRSA 15 Middle ST22 t032 OA t5711 Eastern variant t005 t016 t310 t852 CC22 MRSA IV PVL ST22 2518 12647 CC22 MRSA IV ACME PVL ST22 t2480 CC22 MRSA V CC22 MRSA V PVL t078 t140 t287 t349 t660 CC25 CC25 MSSA ST25 ST28 ST1017 t1521 t6145 CC25 MSSA PVL ST25 ST28 t078 t105
2. MLST CC Srei Se a an spa types associated with WA MRSA 110 CC361 a be ST361 t315 CC395 CC395 MSSA ST395 1426 ST1012 t271 t412 t536 t900 CC395 MRSA IV CC398 CC398 MSSA ST398 t034 CC398 MSSA PVL t034 E osa 008 CC398 MRSA IV ST398 t011 t899 CC398 MRSA V a MRSA sT398 ee ee t1606 t2346 t2510 CC398 MRSA V PVL t034 CC398 MRSA X CC398 ST291 813 ST291 813 MSSA ST291 ST813 t1149 ST291 813 MSSA PVL ST291 t1149 ST425 ST425 MSSA t6386 ST425 MRSA XI t6292 CC479 CC479 MSSA Be inet lukF ST479 ST1275 t2873 t7013 CC509 CC509 MSSA ST509 t375 CC509 ST207 MRSA V ST207 CC522 a lukF ST522 CC599 CC599 MRSA XI ST599 t5930 CC599 MSSA CC692 CC692 MSSA ST692 CC705 a ma lukF CC151 MSSA RF122 ra 3793 t528 t529 CC707 CC707 MSSA ST707 t1458 t3630 CC707 MSSA ccrA B 2 kdp ST707 CC779 CC779 MSSA CC779 MSSA SCCfus t878 CC779 MRSA IV WA MRSA 100 CC779 MRSA V SCCfus ST779 t878 a ss en ST816 ST816 MSSA t1294 ST890 ST890 MSSA t1773 ST913 CC913 MRSA IV t991 CC942 CC942 MSSA ST942 t1445 CC942 MSSA PVL CC1021 CC1021 MSSA ST918 ST1021 CC1021 MSSA PVL S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST CC Sre rE e err cigs Spa types associated with ST1093 ST1093 MSSA ST1290 2481 CC1290 MSSA t131 ST1643 ST1643 MSSA t6385
3. MLST CC an ONES a spa types associated with CC97 CC97 MSSA SCCmer Wood46 ST97 t359 CC97 MRSA I V RANE ST953 ST1174 t267 t1359 CC97 MRSA V ST97 t1234 CC97 MRSA V ACME CC97 ST71 ST71 MSSA ST71 t524 cc1i01 CC101 MSSA ST101 t056 t150 t1312 cci21 CC121 MSSA ST121 t159 t272 t850 t2155 CC121 MSSA PVL ST121 ae ad t433 1018 CC121 MRSA IV hos ST577 t3025 CC121 MRSA V PVL ies yT ST121 t159 CC126 CC126 MSSA bap CC126 MSSA bap CC130 a ae lukF ST700 ST2011 ST2024 t524 t3568 t8403 CC130 MRSA XI ST130 ST1245 ST1764 t843 t6220 CC133 CC133 MSSA ST133 ST2111 t1166 t1181 t4648 t6384 CC133 MSSA capsule type 5 ST2111 t2379 CC133 MSSA lukP83 M ST132 ST133 ST1452 t1403 t2678 CC136 ST136 MSSA ST136 T140 ST140 MSSA ST140 MRSA IV t957 Cc152 CC152 MSSA CC152 MSSA PVL ST152 t355 t4690 CC152 MRSA V CC152 MRSA V PVL WA MRSA 89 ST152 ST377 ST1633 t355 t1096 CC182 CC182 MSSA ST182 t364 CC182 MSSA ccrA B 2 kdp ST182 ST944 364 t493 t1406 t1772 CC188 CC188 MSSA ST188 t189 t3380 CC188 MSSA PVL reed se E CC188 MRSA V CC188 ST1774 ST1774 MRSA IV ACME ST1774 t1081 ST350 ST350 MSSA ST350 t1106 CC361 CC361 MSSA ST361 ee I ST361 ST672 t315 t1309 PE ST672 t1309 CC361 MRSA V S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com
4. NOTE consensus indicates true consensus probes while total indicates a summary for all probes for a given gene to show on one glance whether this gene is present in any known allele or not S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com APPENDIX 4 TYPING INFORMATION Definitions and Explanations The displayed result will yield following typing information e Strain assignment as determined by overall profile and by preset definitions for strains Strains are always defined by clonal complex affiliation see below absence or presence of mecA and by SCCmec type as well as absence or presence of PVL Widely or historically recognised strains might also be defined based on the absence or presence of additional characteristic genes e Strain synonyms These are listed if they can be unambiguously attached to strains as defined above If you use local designations for strains that you want to be included please contact stefan monecke clondiag com e MLST clonal complex affiliation as determined by overall profile and by preset definitions including capsule types and agr groups Isolates can be assigned to clonal complexes as defined by multilocus sequence typing Analysis of hybridisation patterns cannot discriminate sequence types which differ only in single point mutations affecting MLST genes e g ST5 and ST225 or ST59 and ST952 How
5. WA MRSA 14 PVL negative Variant of WA MRSA 109 ST5 t442 CC5 MRSA V sec d j 1 r WA MRSA 87 ST835 t002 CC5 MRSA V sed j r WA MRSA 11 34 35 90 108 ST5 t002 t045 t458 t688 t1265 CC5 MRSA V tst CC5 MRSA V WA MRSA 81 85 86 123 ST5 ST1435 t002 t045 t242 t2666 CC5 MRSA V PVL PVL positive Variant of WA MRSA 109 t002 CC5 MRSA VI New Paediatric Clone ST5 t105 t777 CC5 MRSA VI New Paediatric Clone PVL ST5 MRSA VII SCC JCSC6082 CC5 ST835 MRSA NovelSCCmec WA MRSA 40 46 ST835 t002 CC5 MRSA with atypical SCCmec element CC5 MRSA with atypical SCCmec element CC6 CC6 MSSA ST6 t207 t701 CC6 MSSA PVL CC6 MRSA IV WA MRSA 51 ST6 CC6 MRSA IV V WA MRSA 66 ST6 t701 CC6 MRSA V t5413 CC7 CC7 MSSA ST7 t091 CC7 MRSA IV CC7 MRSA unknown SCCmec WA MRSA 116 ST7 CC7 MRSA V t091 CC7 MRSA VI CC7 ST1048 ST1048 MRSA IV ST1048 t1081 ccs CC8 MSSA ST8 ST254 ST870 t008 t024 t190 t197 t211 t304 t334 t1029 t1854 t2169 t2953 t6021 CC8 MSSA SCCfus t008 t024 CC8 MSSA ACME t008 CC8 MSSA ccrA B2 t024 t5694 CC8 MSSA PVL t008 t400 ST254 MRSA atypical SCCmec Hannover EMRSA ST254 t009 t036 subclone S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureu
6. capsular polysaccharide synthesis enzyme capH5 O antigen polymerase capJ5 capsular polysaccharide biosynthesis protein capK5 capsule type 8 cap 8 total capsular polysaccharide synthesis enzyme capH8 capsular polysaccharide biosynthesis protein capl8 O antigen polymerase capJ8 capsular polysaccharide biosynthesis protein capK8 intercellular adhesion protein A icaA intercellular adhesion protein C icaC biofilm PIA synthesis protein D icaD surface protein involved in biofilm formation bap bone sialoprotein binding protein bbp total bbp consensus bbp COL MW2 bbp MRSA252 bbp Mu50 bbp RF122 bbp ST45 clumping factor A cIfA total cIfA consensus clfA COL RF122 clfA MRSA252 cIfA Mu50 MW2 clumping factor B clfB total clfB consensus clfB COL Mu50 clfB MW2 clfB RF122 collagen binding adhesin cna cell wall associated fibronectin binding protein ebh consensus cell surface elastin binding protein ebpS total ebpS_probe 612 ebpS_probe 614 ebpS 01 1111 ebpS COL enolase eno fibrinogen binding protein 19 kDa fib fib MRSA252 fibronectin binding protein A fnbA total fnbA consensus fnbA COL fnbA MRSA252 fnbA Mu50 MW2 fnbA RF122 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere techno
7. 2 0 version of the assay additional probes for the newly described methicillin resistance gene mecC 3 4 and another marker specific for SCCmec XI blaZ SCCmec XI were introduced S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not Intended for Use in Clinical Diaqnostics This kit allows genotypic characterisation of bacterial cultures from S aureus 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 It cannot be used for other bacteria than S aureus Specifications Upon receipt the assay components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 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 0 For up to date information regarding the kit please visit our website at http www alere technologies com S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com 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 S
8. Ill agrD Ill accessory gene regulator allele IV agrIV total agrB IV agrC IV haemolysin delta hid METHICILLIN alternate penicillin binding protein 2 defining MRSA mecA RESISTANCE AND novel mecA homologue also associated with beta mecC SCCmec TYPING lactam resistance truncated signal transducer protein MecR1 delta_mecR glycerophosphoryl diester phosphodiesterase associated ugpQ with mecA cassette chromosome recombinase genes A 1 ccrA 1 cassette chromosome recombinase genes B 1 ccrB 1 plasmin sensitive surface protein plsSCC COL hypothetical protein from SCCmec elements Q9XB68 dcs cassette chromosome recombinase gene A 2 ccrA 2 cassette chromosome recombinase gene B 2 ccrB 2 potassium translocating ATPase A chain 2 kdpA SCC potassium transporting ATPase B chain 1 kdpB SCC potassium translocating ATPase C chain 2 kdpC SCC sensor kinase protein kdpD SCC KDP operon transcriptional regulatory protein kdpE SCC methicillin resistance gene regulatory protein mecl signal transducer protein MecR1 mecR homolog of xylose repressor associated with SCCmec xylR S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com elements cassette chromosome recombinase gene A 3 ccrA 3 cassette chromosome recombinase gene B 3 ccrB 3 mercury resistance gene operon Hg II reductase merA mercury resistance gene operon alkylmercury lyase merB Putative protein homologue to c
9. MRSA 5 ST1634 MRSA IV WA MRSA ST8 ST576 ST1634 t008 t334 t711 t1677 6 31 83 CC8 MRSA IV ST008 ST507 ST609 USA500 WA MRSA 20 58 ST612 ST1173 t064 t118 451 ST8 MRSA IV PVL ACME WA MRSA 12 Canadian MRSA 10 Es t008 t024 t121 t211 t955 USA300 Spanish PFGE type A t4306 ST8 MRSA IV PVL ACME ACME negative Variant of WA E i MRSA 12 Canadian MRSA 10 ACME negative variant of a 5 A ST8 t008 USA300 Spanish South American Variant of USA300 ST8 MRSA IV putative PVL deletion mutant of USA300 CC8 MRSA IV PVL A ST923 t008 t1635 t9708 sed j k q r WA MRSA 62 CC8 MRSA IV ACME Danish spa t024 ST8 IV Strain 18 wee CC8 MRSA IV SCCfus t008 ST254 MRSA IV V UK EMRSA 10 Hannover ST254 t024 EMRSA CC8 MRSA IV V WA MRSA 92 ST1757 t024 CC8 MRSA V WA MRSA 53 120 ST8 t008 CC8 MRSA V WA MRSA 115 ate toge CC8 MRSA V SCCfus ACME WA MRSA 77 als t008 CC8 MRSA V VI ST983 t008 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST types associated spa types associated with MLST CC Strain Synonymes Kane ccs CC8 MRSA VI SCCfus UK EMRSA 12 13 t064 CC8 MRSA VIII WA MRSA 16 ST8 t024 t190 CC8 ST72 ST72 MSSA ST72 ST72 MSSA SCCfus ST72 MSSA SCCfus kdp ST72 MSSA PVL ST72 MRSA IV USA700 ST72 t126 t324 ST72 MRSA IV PVL WA MRSA 44 ST72 t791 ST72 MRSA V WA MRSA 91 ST72 t3092 S
10. O e Switch off the device by clicking on the Power button left down on the screen F e Switch off the Screen There is no need to physically switch off the ArrayMate Reader S aureus Genotyping Kit 2 0 05 16 04 0001 VO1_Manual S aureus_2 pdf www clondiag com www alere technologies com TROUBLESHOOTING In case of trouble always make sure that reagents are within the recommended shelf life and stored under appropriate conditions In case of trouble we are always happy to support Please contact cct home clondiag com or 49 0 3641 3111 0 and please include a description of the problem as well as the array images bmp files in question Please see Appendix 2 for sample images 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 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 r
11. ORF CM14 WA MRSA 122 CC5 MRSA IV Paediatric clone WA MRSA 03 25 50 71 74 82 105 111 USA800 Spanish PFGE type E7 8 Marseille CF clon STS ST73 ST125 ST575 ST930 ST998 t002 t010 t067 t214 t306 t548 t640 t837 t6183 t7078 CC5 MRSA IV Paediatric clone edinA ST5 ST73 t002 t088 WA MRSA 65 CC5 MRSA IV t002 t045 t067 t088 t509 Paediatric clone sed j r USA800 Spanish PFGE type E7 8 STS t548 t1818 t2173 t6183 CC5 MRSA IV Paediatric clone tst1 CC5 MRSA IV PVL edinA PVL positive Paediatric clone ST5 t311 t3778 WA MRSA 64 121 CC5 MRSA IV fu EES PVL fusC edinA PVL positive Paediatric clone ST5 t1277 rea ete PVL positive Paediatric clone ST5 t311 t1277 CC5 MRSA IV PVL positive Paediatric clone ST5 t002 t311 t450 PVL CC5 MRSA IV SCCfus t447 CC5 MRSA IV SCCfus ST149 t002 t105 Maltese Clone CC5 MRSA IV VI Spanish PFGE type E7 8 ST73 t002 t067 t067 12226 t6475 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST CC Strain Synonymes MLST types associated with this strain Spa types associated with ccs CC5 MRSA Ivar WA MRSA 18 21 48 103 ST5 ST835 t002 t570 CC5 MRSA SCC MRSAZH47 IV V ST641 CC5 MRSA SCC MRSAZH47 IV V PVL ST5 t311 CC5 MRSA V ACME WA MRSA 80 ST5 t071 CC5 MRSA V fusC
12. alere technologies com This is much more DNA than for standard PCR applications see Introduction The DNA specimen needs to be free of RNA and it should not be fragmented This can be determined by agarose gel electrophoresis DNA should not be prepared by disrupting S aureus cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the S aureus Genotyping Kit 2 0 are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend to obey the protocols outlined below We also recommend performing an experiment with Reference DNA from S aureus strain N315 CM reagent when establishing the procedure in your lab This will help to detect the cause of possible problems Extraction of DNA by Spin Columns e Incubate the colony material of the S aureus 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 that is as follows e Add 25 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 Add 200 uL ethanol 96 100 e Vortex the sample and centrifuge briefly e Transfer the complete content of the tube including any precipitate into a spin column that is pl
13. aureus colonies clones requires expertise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed Isolated cell free S aureus DNA may be processed without further biosafety precautions although contamination with S aureus or other bacteria needs to be ruled out Always wear protective clothes as required for laboratory work by your local regulations Material Safety Data Sheets MSDS According to OSHA 29CFR1910 1200 Commonwealth of Australia NOHSC 1005 1008 1999 and the latest anendments to the European Union Directives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet MSDS They do not contain more than 1 of a component classified as hazardous and do not contain more than 0 1 of a component classified as carcinogenic MSDS therefore are not provided Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents In case 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 Precaution
14. isdA other than MRSA252 ImrP other than RF122 probe1 ImrP other than RF122 probe2 ImrP RF122 probe1 ImrP RF122 probe2 www clondiag com www alere technologies com TYPE RESTRICTION type site specific deoxyribonuclease subunit 1st locus hsdS1 RF122 MODIFICATION SYSTEM type site specific deoxyribonuclease subunit 2nd locus SINGLE SEQUENCE SPECIFICITY PROTEIN hsdS2 Mu50 N315 COL USA300 NCTC8325 hsdS2 MW2 MSSA476 hsdS2 RF122 hsdS2 MRSA252 type site specific deoxyribonuclease subunit 3rd locus hsdS3 all other than RF122 MRSA252 hsdS3 COL USA300 NCTC8325 M W2 MSSA476 RF122 hsdS3 Mu50 N315 hsdS3 CC51 MRSA252 hsdS3 MRSA252 type site specific deoxyribonuclease subunit unknown locus MISCELLANEOUS GENES hypothetical protein located next to serine protease operon hsdSx CC25 hsdSx CC15 hsdSx etd Q2FXCO unspecific efflux transporter Q2YUB3 hypothetical protein HYALURONATE LYASE hyaluronate lyase first second locus Q7A4X2 hysA1 MRSA252 hysA1 MRSA252 RF122 and or hysA2 consensus hysA1 MRSA252 RF122 and or hysA2 COL USA300 hyaluronate lyase second locus hysA2 all other than MRSA252 hysA2 COL USA300 NCTC8325 hysA2 all other than COL USA300 NCTC8325 _ probel hysA2 all other than COL USA300 NCTC8325 _ probel hysA2 MRSA252
15. s needed from the pouch e Insert the ArrayStrip s into the white frame Assure the correct orientation Data Matrix code close to row A and proper fit e Pre wash the array in two steps e First PCR grade distilled water 200 uL per well at 50 C 5 min and 550 rpm e Second C1 Hybridisation Buffer 200 uL per well at 50 C 5 min and 550 rpm e Add 90 ul of buffer C1 to each tube with 10 uL labelled amplification product mix gently e Remove the buffer from the well with the array and add the mixture of C1 and labelled amplification product e Incubate at 50 C 60 min and 550 rpm e Remove liquid and wash with 200 uL C2 Washing Buffer pipette up and down four times remove and discard e Add another 200 uL C2 Washing Buffer Incubate at 30 C 10 min and 550 rpm e Meanwhile prepare conjugate For each experiment add 1 uL conjugate 100 x HRP to 99 uL C4 Conjugation Buffer This mixture is stable for one day at room temperature C3 is delivered with a surplus of 100 C4 is delivered with a surplus of 200 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Suggested pipetting scheme 1 well 2 3 wells 4 6 wells 7 10 11 15 16 20 21 30 31 40 wells wells wells wells wells 3 1 5 uL 3 5 uL 7 uL 11 uL 16 uL 21 uL 32 uL 42 uL C4 148 5 uL 346 5 ul 693 uL 1089 uL 1584 uL 2079 uL 3068 uL 4058 uL e Remove a
16. the previous version of the S aureus Genotyping Kit has Assay ID 10248 Assay ID 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 template for pipetting e Safe the worklist as tab separated txt file on the memory stick provided together with the ArrayMate e To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Table 1 Example worklist Position samplelD assaylD comment 1 2013 12345 10620 2 2013 12346 10620 3 2013 12347 10620 4 2013 12348 10620 5 2013 12349 10620 6 2013 12350 10620 7 987654 10620 Isolate referred from Dr J Doe 8 N315 10620 Control strain Table 2 Positions in the 96 well format 1 2 3 4 5 6 7 8 9 10 11 12 A 1 9 17 25 33 41 49 57 65 73 81 89 B 2 10 18 26 34 42 50 58 66 74 82 90 C 3 11 19 27 35 43 51 59 67 75 83 91 D 4 12 20 28 36 44 52 60 68 76 84 92 E 5 13 21 29 37 45 53 61 69 77 85 93 H 6 14 22 30 38 46 54 62 70 78 86 94 G 7 15 23 31 39 47 55 63 71 79 87 95 H 8 16 24 32 40 48 56 64 72 80
17. 20 uL 245104000 2 8 C C1 Hybridisation Buffer 30 mL 245105000 18 28 C C2 Washing Buffer 1 120 mL 245106000 18 28 C C3 HRP Conjugate 100x 200 uL 245107000 2 8 C C4 Conjugate Buffer 30 mL 245108000 18 28 C C5 Washing Buffer 2 120 mL 245109000 18 28 C D1 HRP Substrate 15 mL 245110000 2 8 C ArrayStrips ArrayStrip S aureus 2 0 12 St 240009601 15 28 C StripCaps Covers for unused arrays 24 St 245112000 18 28 C CM Control Material N315 DNA 30 uL 245111000 2 8 C Can be pro en DIR 30 uL vided ion 2 8 C mecC positive request For pricing please contact your local representative or our customer service respectively Legal Manufacturer Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena Germany S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Contact If you require any further information on this product please contact cct home clondiag com LITERATURE Literature quoted in this manual 1 Monecke S Coombs G Shore AC Coleman DC Akpaka P Borg M Chow H Ip M Jatzwauk L Jonas D Kadlec K Kearns A Laurent F O Brien FG Pearson J Ruppelt A Schwarz S Scicluna E Slickers P Tan H L Weber S Ehricht R 2011 A Field Guide to Pandemic Epidemic and Sporadic Clones of Methicillin Resistant Staphylococcus aureus PLoS One 6 4 e17936 2 Monecke S Slickers P Ehricht R 2008 Assignment of Staphylococcus a
18. 4 t2554 CC30 CC30 MSSA lukF P83 lukM t012 t017 t018 t021 t122 CC30 MSSA ST30 ST39 1338 1363 1797 Phage type 80 81 V8 strain CC30 MSSA PVL Cowan ATCC12598 ATCC25923 ST30 oe edges t300 t318 ATCC49775 Oxford Staph USA200 Irish AR7 0 C di ST36 39 MRSA II re 2 a eo PER t007 t012 t018 t253 t419 UK EMRSA 16 de ve t924 Finland E5 CC30 MRSA IV PVL tst1 CC30 MRSA IV PVL tst1 WA MRSA 68 ST39 t018 t2643 CC30 MRSA IV PVL USA 1100 West Samoan Phage ray aed t019 t021 t122 t300 t318 Southwest Pacific Clone Pattern WSPP Clone t975 t5447 t7561 CC30 MRSA IV SCCfus ST30 t012 t021 CC30 MRSA IV SCCfus PVL CC30 MRSA IV VI WA MRSA 102 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST CC an MES MEST types associated spa types associated with with this strain cc30 CC30 MRSA V CC30 MRSA V PVL WA MRSA 124 CC30 ST34 42 ST34 42 MSSA ST34 ST42 a 6 t166 1582 t015 t026 t050 t065 t116 CCAS agr 1 CARNEA ST45 ST508 ST1008 t133 t339 t362 t383 t390 ST1009 t397 t465 t576 t1331 t1608 t2714 CC45 MSSA egc deletion A ST45 t065 t330 variants CC45 MSSA PVL ST45 CC45 MRSA II USA600 MRSA IV WA MRSA 75 USA600 Belgium E2 ae raoe CC45 MRS
19. 5 from ST22 and ST45 haemolysin gamma component A hIgA Panton Valentine leukocidin F component lukF PV Panton Valentine leukocidin S component lukS PV F component of leukocidin from ruminants lukF PV P83 S component of leukocidin from ruminants lukM leukocidin D component lukD leukocidin E component lukE leukocidin haemolysin toxin family protein lukX leukocidin haemolysin toxin family protein allele from lukY ST30 and ST45 leukocidin haemolysin toxin family protein lukY ST30 ST45 putative membrane protein hl haemolysin alpha hla putative membrane protein hllll consensus Allll other than RF122 VIRULENCE HLB CONV PHAGES VIRULENCE EXFOLIATIVE TOXINS VIRULENCE EPITHEL DIFF INHIB VIRULENCE ACME LOCUS haemolysin beta hlb_probe 1 haemolysin beta hlb_probe 2 haemolysin beta hlb_probe 3 haemolysin beta without phage insertion un disrupted hlb staphylokinase sak chemotaxis inhibiting protein CHIPS chp staphylococcal complement inhibitor scn exfoliative toxin serotype A etA exfoliative toxin serotype B etB exfoliative toxin D etD epidermal cell differentiation inhibitor edinA epidermal cell differentiation inhibitor B edinB epidermal cell differentiation inhibitor C edinC Arginine Catabolic Mobile Element ACME cluster ACME locus arginine deiminase arcA SCC ACME locus ornithincarbamoyltransferase arcB SCC ACME locus carbamatkinase ar
20. 7 Bengal Bay Clone60 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST CC Sie SE MEST types associated spa types associated with with this strain ST772 MRSA V PVL CC1 ST573 772 Bengal Bay Clone ccr mutation deletion t002 t010 t053 t067 t088 ccs CC5 MSSA ST5 ST73 t179 t214 t442 t548 t1062 t1265 CC5 MSSA PVL ST5 t002 t311 ST228 MRSA I Spanish PFGE type E6 9 15 17 18 t001 t023 t041 t062 t110 South German EMRSA Italian UK EMRSA 3 ST5 ST228 143 t149 t811 t892 Clone ST228 MRSA South German EMRSA ST228 truncated SCCmec ST5 MRSA I Geraldine Clone gt t002 CC5 MRSA II ACME WA MRSA 125 ST5 MRSA II tst1 mer Irish AR11 USA100 Canadian MRSA 2 ST5 t045 ST5 MRSA II tst1 USA100 Irish AR7 3 AR7 4 STS t002 t045 New York Japan Clone Canadian MRSA 2 N315 Mu50 CC5 MRSA II trunc kdp ccrA B2 deletion ST5 ST225 MRSA II Rhine Hesse EMRSA New York Japan Clone USA100 Canadian MRSA 2 Irish AR7 3 AR7 4 Finland E1 JH1 JH9 ST5 ST225 ST496 t002 t003 t014 t045 t067 t088 t102 t151 t242 t306 t548 t603 t627 t893 t1062 t1290 t2032 t2302 t2666 t3524 t7053 CCS MRSA III Belgium E3 t002 CC5 MRSA IV ACME CC5 MRSA IV fusC New Zealand AK3 WA MRSA 39 ST5 ST526 t002 t4065 CC5 MRSA IV
21. 88 96 Data Acquisition in the ArrayMate Reader e Insert your memory stick containing the worklist into any of the USB ports down to the right side of the ArrayMate e Press the button ale a folder selection dialog will open e Select your worklist path My Computer Removable Disk e Open your selected worklist with Enter or the button Open e Press the button E 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 the button OK the worklist window will close S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Leave the memory stick attached to the ArrayMate if you intend to export S aureus Genotyping Test Reports afterwards e Press the button Next bottom right on the screen reader opens e Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply any strong force Assure proper fit otherwise the images may be out of focus e Carefully insert the white frame with the array strips into the metallic adapter Assure the correct orientation Position A1 in the frame next to the Data Matrix 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 ArraySt
22. A IV ACME CC45 MRSA IV tst1 ACME CC45 MRSA IV tst1 t2674 CC45 MRSA IV USA600 MRSA IV WA MRSA 75 0195 t004 t007 t015 t040 t302 Berlin EMRSA Belgium E2 t750 t950 t1424 t2135 CC45 MRSA IV SCCfus CC45 MRSA V t015 t1156 CC45 MRSA V ACME WA MRSA 106 CC45 MRSA V tst1 WA MRSA 4 ST45 t123 CC45 MRSA V PVL CC45 MRSA V VI CC45 agr IV CC45 agrIV MSSA CC45 agrIV MSSA capsule STAS 16552 type 5 CC45 agrIV MRSA IV WA MRSA 23 ST45 t727 t1575 CC45 agrlV MRSA IV V CC45 agrIV MRSA VT WA MRSA 84 ST45 t1081 CC45 ST617 ST617 MRSA IV ST617 t305 cc49 CC49 MSSA lukF P83 lukM ST49 t208 CC49 MSSA ST49 t208 CC49 MSSA PVL ST49 CC49 MRSA V ST49 CC50 CC50 MSSA ST50 t246 CC50 MRSA V SCCfus CC59 CC59 MSSA ST59 ST375 t216 t1151 t3736 CC59 MSSA PVL ST59 t437 ST59 MRSA IV PVL negative Variant of WA ST59 t437 WA MRSA 118 MRSA 55 56 ST59 MRSA IV WA MRSA 73 ST59 t172 t528 ST87 MRSA IV ST87 t216 WA MRSA 24 CC59 MRSA IV PVL CC59 USA1000 ST59 t216 t316 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com MLST CC an Ones MEST types associated spa types associated with with this strain ST59 MRSA IV PVL CC59 ST59 1437 WA MRSA 55 56 ST59 MRSA IV V WA MRSA 15 ST59 t976 ST59 MRSA V ST59 t316 t441 CC59 MRSA V PVL ST59 t316 ST59 952 M
23. HIVE E 2013 02 26 13 21 16 New Run in Archive If you click on the plus symbol left on the run name the folder opens and you will see a list of the individual arrays ordered by Sample ID E ArrayMate Browse Q Search m ARCHIVE ee 2013 02 26 13 21 16 New Run D1 A 01 6 FS D1 C Di D Archive D1 E Di F D1 G Di H S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Click on a Sample ID and the S aureus Genotyping Kit 2 0 test report for this array is shown in the window on the right Browse Search results results b raw data segmentation image image print E 3 2013 02 26 13 21 16 For Investigational Use Only Not Intended for Use in Clinical Diagnostics New Run 2 x Operator BS 01 C Sample ID ERIE nE Experiment ID 01 A 01 F Date of Result Tue Feb 26 12 53 02 2013 S Assay Hame Stau_pm7plus Assay ID 10620 Well Position Software Version 2013 02 12 Device staining control oK TYPING SUMMARY Result strain CC130 MRSA XI strain synonyms MLST clonal complex affiliation cc130 sequence types associated with this strain ST130 ST1245 ST1764 spatypes associsted with this strain 1843 16220 assignment score for CC identification 92 70 MRSA meca negative MRSA mecC PYL negative REGULATORY GENES Description O agri total i acc
24. RSA V T PVL na MRSA 9 WA MRSA 52 ST59 ST952 t437 t441 t1950 t2365 Taiwan Clone CC59 MRSA V SCCfus CC80 CC80 MSSA CC80 MSSA PVL ST80 t044 atypical CC80 MSSA ORF CM14 PVL ST80 slv t1849 CC80 MRSA truncated atypical SCCmec CC80 MRSA truncated atypical SCCmec PVL CC80 MRSA IV CC80 MRSA IV PVL t042 t044 t131 t203 t416 WA MRSA 17 30 ST80 ST583 ST728 European caMRSA Clone i t434 CC88 CC88 MSSA ST78 ST88 t186 t729 t730 CC88 MSSA PVL ST88 t693 t729 t1598 t4195 CC88 MRSA IV WA MRSA 2 ST78 ST257 t186 t690 t3205 CC88 MRSA IV etA ST88 t186 t690 t786 CC88 MRSA IV PVL ST88 t690 t692 CC88 MRSA V t186 CC88 MRSA V PVL WA MRSA 117 CC88 MRSA Vtrunc PVL t1764 CC88 MRSA VI CC88 MRSA VII SCC JCSC6082 sites ST93 ST93 MSSA t202 t4178 t4699 t5767 ST93 MSSA PVL 16485 ST93 MRSA IV PVL t1811 t6847 ST93 MRSA IV PVE WA MRSA 7 t202 t4178 Queensland Clone ST93 MRSA V t202 ST93 MRSA V PVL CC96 CC96 MSSA CC96 MSSA PVL ST96 ST154 MRSA IV PVL Central Asian caMRSA WA ST154 ST1930 t667 MRSA 119 CC97 CC97 MSSA lukF P83 lukM ST97 ST352 CC97 MSSA ATCC6538 ST97 ST115 ST464 EN t521 1524 t528 t1234 CC97 MSSA ccrA B2 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com
25. ST1755 ST1755 MSSA ST1852 ST1852 MSSA CC1943 CC1943 MSSA t978 CC1943 MRSA XI ST2361 MRSA XI ST2361 t978 ST2249 T2249 MRSA III t037 T2279 ST2279 MSSA ST2425 ST2425 MSSA ST2479 ST2479 MSSA ST2479 MSSA PVL ST2482 ST2482 MSSA PVL ST2691 ST2691 MSSA En lineage CC75 MSSA ST75 S argenteus lineage ST75 MRSA IV ST75 t 259 31 17 17 17 17 23 CC75 WA MRSA 8 79 17 17 23 17 22 and related S argenteus lineage CC75 ST75 MRSA V S argenteus lineage ST883 ST883 MRSA IV WA MRSA 47 t 259 23 23 17 17 17 23 23 23 17 16 S argenteus lineage CC1223 1594 CC1223 1594 MSSA ST1223 ST1594 ST1719 S argenteus lineage ST1303 MRSA IV t 259 25 17 17 16 16 16 ST1303 WA MRSA 76 16 16 S argenteus lineage ST1304 MRSA IV t 259 31 17 17 17 17 23 CC75 WA MRSA 72 17 17 23 17 22 S argenteus lineage ST2250 2277 ST2250 2277 MSSA ST2250 ST2277 and related S argenteus lineage ST2250 MRSA IV ST2250 2277 WA MRSA 114 ST2250 and related t6675 S argenteus lineage ST2250 2277 MRSA V ST2250 2277 WA MRSA 113 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf
26. Software e ArrayMate Reader to be ordered separately for details see below The S aureus Genotyping Kit 2 0 may be used on the ArrayMate Reader only The older S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com devices ATRO1 03 are not suitable for reading ArrayStrip based assays In case of any questions please contact your local distributor and or Alere Technologies Jena e Iconoclust software provided with the reader e Test specific software plug in that contains information such as spot names marker names positions of the spots on the array This plug in is delivered with the reader or can be downloaded from our website Test specific plug ins will occasionally be updated Please check the NEWS section of our website http www clondiag com Support is available via cct home clondiag com or 49 0 3641 3111 0 Components Required but not Provided e Growth media for the cultivation of S aureus The test should be performed with colonies harvested from Columbia Blood Agar Other media that contain blood may also suffice but have not systematically been tested Media that do not contain blood Mueller Hinton or MRSA selective media usually yield lower DNA concentration they are not recommended As well liquid media should not be used because contamination or culture mixing cannot easily be ruled out e Equipment and consumables needed for the cultivati
27. T72 MRSA V SCCfus ST72 MRSA V ccrA4r CC8 ST239 ST239 MSSA ST239 MRSA II j A a Australian EMRSA 2 AUS 2 New Vienna Hungarian Brazilian Zealand AKh4 es ST239 ST240 ST241 t019 t030 t037 t363 Clone UK EMRSA 4 7 9 11 Australian ST239 MRSA IlI SCCmer EMRSA 3 AUS 3 Irish Phenotype Vienna Hungarian Brazilian 111 Irish ARO9 14 23 44 Canadian ST239 ST240 ST241 t030 t037 t074 Clone MRSA 3 6 New Zealand AKh4 MRSA Finland E24 Greece 1 ST239 MRSA truncated SCCmec ST239 MRSA III ACME ST239 t037 ST239 MRSA Ill ccrA B4 ST239 MRSA Ilivar delta UK EMRSA 1 Australian EMRSA 3 T239 1037 mecR negat SCCmer AUS 3 Irish ARO1 and AR15 CC9 CC9 MSSA ST9 ST903 t100 t209 t411 CC9 MRSA III CC9 MRSA IV ST9 t1430 CC9 MRSA IV PVL WA MRSA 126 211420 CC9 MRSA IX CC9 MRSA V CC9 MRSA V atyp truncated t899 t1234 CC9 MRSA atypical SCCmec IXvar ST9 WA MRSA 112 CC9 ST834 ST834 MSSA ST834 MRSA IV ST834 formerly ST584 WA MRSA 13 WA MRSA 33 WA MRSA 41 1733 t3029 ST834 MRSA IV PVL t724 t1379 ST834 MRSA VI SCCfus cc10 CC10 MSSA ST10 t166 t864 cc12 CC12 MSSA ST12 ST901 oa ai Ai t4490 CC12 MRSA WA MRSA 59 ST12 t160 CC12 MRSA IV WA MRSA 69 ST12 t160 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com
28. aced in a 2 mL collection tube e Centrifuge at room temperature time and speed need to be determined depending on viscosity of sample and type of centrifuge used All liquid should be collected in the collection tube afterwards e Discard collection tube with liquids S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Place the spin column in a new 2 mL collection tube provided with the kit e Add 500 ul Buffer AW1 e Centrifuge at room temperature e Discard collection tube with liquids e Place the spin column in a new 2 mL collection tube provided with the kit e Add 500 ul Buffer AW2 e Centrifuge at room temperature the membrane of the spin column should be dry and all liquid should be in the collection tube e Discard collection tube with liquids e Place the spin column in a clean 1 5 mL tube provided with the kit e Add 50 ul Buffer AE or PCR grade distilled water directly onto the membrane of the spin column e Incubate at room temperature for 5 min to elute DNA e Centrifuge e Optional add another 50 uL Buffer AE or PCR grade distilled water directly onto the membrane incubate at room temperature for 1 min and centrifuge again e Discard the spin column Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay A contamination with Washing Buffer might occur during elution of prepared DNA by droplets adhered t
29. assette chromosome recombinase A genes ccrAA MRSAZH47 probe 1 ccrAA MRSAZH47 probe 2 cassette chromosome recombinase gene C ccrC 85 2082 cassette chromosome recombinase gene A 4 ccrA 4 cassette chromosome recombinase gene B 4 ccrB 4 RESISTANCE beta lactamase gene blaZ PENICILLINASE beta lactamase gene associated with SCCmec XI blaZ SCCmec XI elements beta lactamase repressor inhibitor blal beta lactamase regulatory protein blaR RESISTANCE MLS rRNA methyltransferase associated with erm A ANTIBIOTICS macrolide lincosamide resistance rRNA methyltransferase associated with erm B macrolide lincosamide resistance rRNA methyltransferase associated with erm C macrolide lincosamide resistance lincosaminide nucleotidyltransferase linA Inu A macrolide efflux pump msr A macrolide efflux protein A mef A macrolide phosphotransferase II npbBM mph C virginiamycin A acetyltransferase vat A acetyltransferase inactivating streptogramin A vat B ABC transporter conferring resistance to streptogramin A vga A and related compounds vga A allele from strain BM 3327 vga A BM 3327 virginiamycin B hydrolase vgb vgB A RESISTANCE aminoglycoside adenyl phosphotransferase aacA aphD AMINOGLYSOSIDES gentamicin tobramycin aminoglycoside adenyltransferase aadD neo kanamycin tobramycin aminoglycoside phosphotrans
30. cC SCC ACME locus arginine ornithine antiporter arcD SCC VIRULENCE PROTEASES VIRULENCE STAPHYLOCOCCAL SUPERANTIGEN ENTEROTOXIN LIKE aureolysin aur consensus aur other than MRSA252 aur MRSA252 serinprotease A splA serinprotease B spIB serinprotease E splE glutamylendopeptidase sspA staphopain B protease sspB staphopain A staphylopain A protease sspP consensus sspP other than ST93 staphylococcal exotoxin like protein SAg gene homolog setC selx SAUSA300_0370 staphylococcal superantigen like protein 1 probes S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf ssl01 set6_probe1_11 ssl01 set6_probe2_11 www clondiag com www alere technologies com GENES SET SSL ssl01 set6_probe1_12 ssl01 set6_probe2_12 ssl01 set6_probe4_11 ssl01 set6_probeRF122 staphylococcal superantigen like protein 1 interpretation alleles ssl01 set6 COL ssl01 set6 Mu50 N315 ssl01 set6 MW2 MSSA476 ssl01 set6 MRSA252 ssl01 set6 RF122 ssl01 set6 other alleles staphylococcal superantigen like protein 2 ssl0O2 set7 ssl02 set7 MRSA252 staphylococcal superantigen like protein 3 ssl03 set8_probe 1 ssl03 set8_probe 2 ssl03 set8 MRSA252 SARO424 staphylococcal superantigen like protein 4 ssl04 set9 ssl04 set9 MRSA252 SARO425 staphylococcal superantigen lik
31. d buffer y Add 100 uL D1 substrate 25 C 6 min strictly no shaking 6 min 6 min Discard substrate analyse ArrayMate 5 min 1 min total time requirement over night 50 80 min 7 8 h S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com APPENDIX 1 FLOW CHART Eppendorf protocol The figure on this page summarises an adapted test procedure for the thermoshaker Thermomixer Comfort by Eppendorf Please always refer to the text section of this manual for further important details pro hands cessing on time time Prepare ArrayStrips Prepare DNA Grow bacteria over 5 min not part of the kit night y Isolate DNA 3 4 h 10 40 min not part of the kit y Rinse ArrayStrips Label DNA in thermocycler 2 3 h 5 min 200 uL water 50 C 550 rpm 5min 5 uL DNA 0 5 2 ug DNA Incubate in Buffer C1 4 9 uL B157 0 1 uL B2 200 uL 50 C 550 rpm 5 min 5 min 5 min discard C1 process promptly Prepare labeled DNA To 10 uL of labeled DNA 5 min 5 min y add 90 uL of Buffer C1 y Transfer DNA to arrays 60 min O min Hybridise 55 C 550 rpm 60 min y Discard DNA wash 200 uL Buffer C2 pipette up and down x4 7 min 2 min 200 uL Buffer C2 30 C 10 min and 550 rpm discard buffer y Incubate with conjugate 100 uL C3 C4 30 C 550 rpm 15 min 15 min 2 min Discard conjugate wash 200 uL Buffer C5 pipette up and down x4 3 min 3 min 200
32. e 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 reference strain N315 GenBank accession number BA000018 and should be identified by the assay as ST5 MRSA II tst1 New York Japan Clone 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 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com 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 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
33. e lysis protocol Bacteriacard for Qiagen s EZ1 e For Qiagen s EZ1 Front row empty elution tubes 1 5 mL second row tip holder with tips third row empty back row sample tube with conical tip 2 mL containing the 200 uL sample volume Set tissue lysis protocol with a set sample volume of 200 uL and an elution volume of 50 uL e Concentrate DNA and evaporate traces of solvents by heating the sample at 70 C for 5 10 min Linear Amplification and Internal Biotin Labelling Please keep in mind the limited surplus of reagents whilst pipetting The surplus of B1 labelling reagent is 25 e Prepare a Master Mix by combining 4 9 uL of B1 labelling reagent version 2 0 Do not use B1 from earlier kit versions and 0 1 uL of B2 Labelling Enzyme per sample e Add 5 ul DNA 0 5 2 ug prepared as described above to a 5 uL aliquot of the Master Mix Do not forget to label the vial S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Perform amplification in a pre programmed thermocycler such as 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 60 sec at 96 C 50 to 55 cycles with 20 sec at 50 C 40 sec at 72 C Cool down to 4 C hold Please note When using a different device some adaptations
34. e protein 5 ssl05 set3_probe 1 ssl05 set3 RF122 probe 611 ssl05 set3_probe 2 612 ssl05 set3 MRSA252 staphylococcal superantigen like protein 6 ssl06 set21 ssl06 NCTC8325 MW2 staphylococcal superantigen like protein 7 sslO7 set1 ssl07 set1 MRSA252 ssl07 set1 AF188836 staphylococcal superantigen like protein 8 ssl08 set12_probe 1 ssl08 set12_probe 2 staphylococcal superantigen like protein 9 ssl09 set5_probe 1 ssl09 set5_probe 2 ssl09 set5 MRSA252 staphylococcal superantigen like protein 10 ssl10 set4 ssl10 RF122 ssl10 set4 MRSA252 staphylococcal superantigene like protein 11 ssl11 set2 COL ssl11 set2 Mu50 N315 ssl11 set2 MW2 MSSA476 ssl11 set2 MRSA252 staphylococcal exotoxin like protein second locus setB3 setB3 MRSA252 setB2 setB2 MRSA252 CAPSULE AND BIOFILM capsule type 1 setB1 cap 1 total ASSOCIATED GENES capsular polysaccharide synthesis enzyme capH1 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com ADHAESION FACTORS GENES ENCODING MICROBIAL SURFACE COMPONENTS RECOGNIZING ADHESIVE MATRIX MOLECULES MSCRAMM GENES O antigen polymerase capJ1 capsular polysaccharide biosynthesis protein capK1 capsule type 5 cap 5 total
35. ec element or an unusual presence of virulence genes such as of PVL in a lineage where it has not been observed before A contamination e g by SCC bearing coagulase negatives needs to be ruled out Re clone and S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com repeat If this message was prompted by a technically faultless experiment and if contamination can be ruled out by repeated cloning please submit the picture and or the strain for further analysis 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 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 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 d
36. es from the ArrayMate into spreadsheet tables This should make it easier to compare isolates or to determine relative abundances of genes or strains etc S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com APPENDIX 1 FLOW CHART Quantifoil protocol The figure on this page summarises the test procedure for the thermoshaker BioShake iQ by Quantifoil Please always refer to the text section of this manual for further important details pro hands cessing on time time Prepare ArrayStrips Prepare DNA Grow bacteria over 5 min not part of the kit night Isolate DNA 3 4h 10 40 min not part of the kit Rinse ArrayStrips Label DNA in thermocycler 2 3 h 5 min 200 uL water 50 C 550 rpm 5min 5 uL DNA 0 5 2 ug DNA Incubate in Buffer C1 4 9 pL B157 0 1 pL B2 i f 200 pL 50 C 550 rpm 5 min 5min 5 min discard C1 process promptly Prepare labeled DNA To 10 uL of labeled DNA 5 min 5 min y add 90 uL of Buffer C1 y Transfer DNA to arrays 60 min O min Hybridise 55 C 550 rpm 60 min y Discard DNA wash 200 uL Buffer C2 pipette up and down x4 12 min 2 min 200 uL Buffer C2 30 C 10 min and 550 rpm discard buffer y Incubate with conjugate 100 uL C3 C4 30 C 550 rpm 10 min 10 min 2 min Discard conjugate wash 200 uL Buffer C5 pipette up and down x4 3 min 3 min 200 uL Buffer C5 30 C 2 min and 550 rpm discar
37. essage the long htmL file will display the most similar strain although that identification might be faulty A value of 100 is unlikely because of the mobility of many genes in S aureus 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 such as local synonyms spa types or MLST types please contact stefan monecke clondiag com MLST CC Stain Sun MEST types associated spa types associated with with this strain t127 t174 t398 t559 cc1 CC1 MSSA ST1 ST761 ST762 t1506 t6980 CC1 MSSA lukF P83 lukM CC1 MSSA PVL ST1 t174 CC1 MSSA SCCfus Sanger MSSA476 ST1 t607 t559 t2246 t8989 CC1 MSSA SCCfus PVL ST1 t127 t177 t386 t1784 CC1 MRSA IV f ST1 ST1005 ST1115 t127 t386 t590 t922 WA MRSA 1 57 New Zealand AK1 MRSA strain ST1336 t2601 CC1 MRSA IV PVL MW2 Canadian MRSA 7 New USA400 Zealand WR AK1 MRSA Sl t127 t128 t175 t1784 CC1 MRSA IV SCCfus WA MRSA 1 45 New Zealand AK1 MRSA strain ST1 t127 t2279 CC1 MRSA IV SCCfus PVL New Zealand WR AK1 MRSA CC1 MRSA V CC1 MRSA V PVL t127 CC1 MRSA V SCCfus CC1 MRSA V SCCfus PVL CC1 ST567 ST567 MSSA PVL ST567 t1242 CC1 ST573 772 ST573 772 MSSA ST573 772 MSSA PVL ST772 t1839 ST772 MRSA V ST772 t657 ST573 MRSA V WA MRSA 10 ST573 t5073 ST772 MRSA V PVL ST772 t345 t657 t338
38. essory gene regulator allele agrll total i accessory gene regulator allele Il Power agrlli total positive accessory gene regulator allele Ill Export of S aureus Genotyping Kit 2 0 Test Reports Two result files in htmL format will be generated The shorter report will gives a summary on gene typing information This includes the clonal complex affiliation as derived from the overall hybridisation pattern and the strain affiliation as defined by clonal complex affiliation presence absence and type of SCCmec elements and presence absence of PVL or other relevant markers MLST sequence types and spa types known to be associated with this strain are also displayed Note that this information is derived from a database search see also Appendix 3 not from an actual experiment Furthermore results for virulence markers and genes associated to antibiotic resistance are listed A longer htmL result sheet result_B res htmL provides information on all probes Possible error messages in these reports will be explained below see Troubleshooting S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Other files that are generated and that can be exported include e A txt file with the raw measurements e Animage file bmp showing the actual photo of the array e A second image file png in which the coordinate grid is superimposed and the reco
39. etection 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 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com information such as names of patients from whom S aureus 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 with the use of standard S aureus DNA preparations for good performance and correctness of results List of Components for Separate Order If required these reagents for the S aureus Genotyping Kit 2 0 may be ordered separately Component Name Amount Cat Storage Al Lysis Buffer 30 mL 245101000 18 28 C A2 Lysis Enhancer 96 units 245102000 18 28 C B1 Labelling Buffer Master Mix 700uL 245203000 2 8 C B2 Labelling Enzyme
40. ever there are also sequence types which originate from chromosomal replacements Examples are CC8 ST239 or CC30 ST34 As these events result in different hybridisation patterns such STs can be easily identified Some other STs are also clearly different from parental CCs although recombination is not yet proven In such cases ST affiliation might also be displayed for instance for CC9 ST834 e Sequence types associated with this strain This information is provided based on a database of isolates that have been typed in parallel by array and MLST It is not directly derived from the actual experiment If you have MLST results you want to be included please contact stefan monecke clondiag com e spa types associated with this strain This information is provided based on a database of isolates that have been typed in parallel by array and spa sequencing It is not directly S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com derived from the actual experiment If you have spa results you want to be included please contact stefan monecke clondiag com e Assignment score This is a score for the similarity to the average hybridisation result for a given strain CC Scores below 88 exclude reliable strain identification and could be attributed either to technical reasons or to the presence of a yet unknown strain The short htmL file will display an error m
41. ferase neo kanamycin aphA3 RESISTANCE streptothricin acetyltransferase sat MISCELLANEOUS GENES dihydrofolate reductase mediating trimethoprim dfrS1 resistance dfrA fusidic acid resistance gene far1 fusB fusidic acid resistance gene Q6GD50 fusC isoleucyl tRNA synthetase associated with mupirocin mupA resistance mupR tetracycline efflux protein tet K ribosomal protection protein associated with tetracycline tet M resistance chloramphenicol acetyltransferase S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf cat total cat pC221 cat pc223 cat pMC524 www clondiag com www alere technologies com RESISTANCE EFFLUX SYSTEMS RESISTANCE GLYCOPEPTIDES VIRULENCE TOXIC SCHOCK TOXIN VIRULENCE ENTEROTOXINS cat pSBK203R 23S rRNA methyltransferase phenicols lincosamides cfr oxazolidinones pleuromutilins streptogramin A chloramphenicol florfenicol exporter fexA metallothiol transferase fosB fosB plasmid quaternary ammonium compound multidrug efflux gacA protein C quaternary ammonium compound multidrug efflux qacC total protein A gacC consensus gacC equine qacC SA5 qacC Ssap gacC ST94 putative transport protein tetEfflux sdrM vancomycin resistance gene vanA vancomycin resistance gene from enterococci and vanB Clostridium teicoplanin resistance gene from enterococci
42. gnised spots are circled and e A xmL files providing contains the same information as the html result sheet for future export into databases etc e An out file containing output log data which helps our service to trace image evaluation errors Please Note only complete runs can be exported The export of individual S aureus Genotyping 2 0 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 EE ArrayMate Browse A Search results results b raw data segmentatior ir ARCHIVE eg 2013 02 26 13 21 16 New Run 01 4 1 01 8 Browse For Folder 5 01 C 01 D 3 Archive O1 E Choose a directory o1 F 01 G Oru 3 Desktop Y se Local Disk C se Local Disk D Removable Disk E My Documents Folder My Computer a C e Click on My Computer subsequently on Removable Disk and choose the folder where to save or click on the button Make New Folder on the bottom a new folder icon appears S aureus Genotyping Kit 2 0 05 16 04 0001 V01_ Manual S aureus_2 pdf www clondiag com www alere technologies com 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
43. he presence of plasmids in low copy numbers Please note that for some markers for which allelic variants were to be discriminated bbp cIfA clfB and fnbB as well as some set ss genes isaB mprF and isdA a different approach for analysis was used than for resistance genes or virulence factors In these genes alleles that differ only in single nucleotides are recognised For the sake of creation of identifiable clonal complex specific patterns only the probe with the strongest signal value is regarded as positive provided that it exceeded a defined breakpoint All other allele specific probes are then regarded as ambiguous or if below the breakpoint as negative Therefore it is perfectly normal if a number of allele specific probes for these genes yield ambiguous signals The presence or absence of these genes is indicated by fields such as e g c fA total which are summaries for all probes related to the respective gene 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 carefully or try to manually identify the test If no obvious reason for the fault can be discovered please contact the technical service Error Messages in Result Sheets Please compare Appendix 2 f
44. he staining procedure and afterwards 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 Thermoshakers The correct temperature within the vessels is essential therefore always use the appropriate equipment for heating Because of a possibly inhomogeneous distribution of the temperature within the heating block and because of possible differences between displayed and actual temperatures the use of different brands of thermoshakers might affect test performance We tested the assay with BioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips as well as with Eppendorf s Thermomixer Comfort equipped with a heating block for microtitre plates Thus we S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com recommend the use of either device Accordingly two slightly different protocols are discussed here When using other devices some modifications to the protocol might be necessary Before establishing routine use please test the protocol with a few known reference strains or the control DNA CM supplied with the kit Protocol for Quantifoil s BioShake iQ e Switch on the thermoshaker and pre heat it to 50 C e Remove the amount of ArrayStrip
45. ing and Harvesting Bacterial Cells S aureus is a potential pathogen 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 S aureus on Colombia blood agar overnight at 37 C or 48 hrs at room temperature Obtain confirmation of the identification as S aureus by katalase coagulase clumping factor assays or by other means and make sure that you have a pure monoclonal culture of S aureus Contamination with other bacteria especially with other staphylococci needs to be strictly avoided as they might carry the same resistance genes as certain S aureus strains and thus can introduce false positive signals and patterns e Centrifuge A2 tube briefly open it add 0 2 mL of Lysis Buffer Al to Lysis Enhancer A2 and dissolve e Add an inoculating loop full of monoclonal colony material of the S aureus isolate into this A1 A2 reagent vortex 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 Extraction of DNA The required sample type for the S aureus Genotyping assay is 0 5 2 ug of intact genomic DNA from a single clone of S aureus S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www
46. issues such as poor signal quality overstaining or conta mination The isolate could also represent a new Strain within a known clonal complex might appear in the short file The long file will yield an approximate identification but individual probes might yield false negative results Error message in the short html file The long file yields Coagulase negative Staphylococci other bacteria or very poor signal quality Check identification by biochemical means or MALDI TOF or repeat experiment and shows positive results only for resistance genes and or genes associated with SCCmec S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com APPENDIX 3 TARGET GENES SPECIES MARKER domain 1 of 23S rRNA rrnD1 S aureus glyceraldehyde 3 phosphate dehydrogenase locus 1 gapA katalase A katA coagulase coA thermostable extracellular nuclease nucl staphylococcal protein A spa lgG binding protein sbi REGULATORY GENES staphylococcal accessory regulator A sarA histidine protein kinase sae locus saes sensor protein vraS accessory gene regulator allele agrl total agrB I agrC I agrD I accessory gene regulator allele Il agrll total agrB Il agrC Il agrD Il accessory gene regulator allele III agrlll total agrB Ill agrC
47. lity tnea e a n A AA A las la aa 26 DNA Quality aonta sio 27 Physical Damage to the Array cccccccccccccscssssssssecececeesssesssseeceeeescsesesaeseseceecseseseseaeaeeeeeesesesessaeeas 27 Ambiguous Results tt tn Ai ES id 27 Report Unavailable vicios tad ASA AAA A IE 28 Error Messages i Result Sheets iii dai 28 ADDITMTONALIN FORMATO N yuri une 30 Wa NVidia A ALA A A AA E AAA 30 Disclaimer uud hie 30 Quality Controla ln eine iR Re cin 31 List of Components for Separate OFder ccccccccccccsssssssscececesseesssseseeeeeescessessaeeeaeeeeeeseeesessnaees 31 t gal Manufacturera nie la tac 31 Contato is 32 LITERATURE usar ee een 32 UPDATES AND SOFTWARE ocn a 32 APPENDIX 1 FLOW CHART A Ea 33 APPENDIX 2 IMAGES FOR TROUBLESHOOTING una aaa sede attecdtuacartcs 35 APPENDIX 3 TARGET GENES a 2 ctetiitesdatet tacit E e 36 APPENDIX 4 TYPING INFORMATION ne a 44 Definitions and Explanations sc 2c2ccceccsccedescesbesvenddevcokeesnds ceeddiudescdedsdavendddvesdsedadavadedvecadebedveegandiva 44 List of Currently Recognised Strains u itee aa ER 45 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com BACKGROUND The CLONDIAG S aureus Genotyping Kit 2 0 allows DNA based detection of resistance genes and pathogenicity markers of Staphylococcus aureus and assignment of unknown S aureus isolates to known strains RNA free unfragmented genomic DNA from pure a
48. logies com IMMUNODOMINANT ANTIGEN B DEFENSIN RESISTANCE TRANSFERRIN BINDING PROT PUTATIVE TRANSPORTER fibronectin binding protein B fnbB total fnbB COL fnbB COL Mu50 MW2 fnbB Mu50 fnbB MW2 fnbB ST15 fnbB ST45 2 major histocompatibility complex class Il analog protein Extracellular adherence protein eap map total map COL map MRSA252 map Mu50 MW2 Staphylococcus aureus surface protein G sasG total sasG COL Mu50 sasG MW2 sasG other than MRSA252 RF122 Ser Asp rich fibrinogen bone sialoprotein binding protein C sdrC total sdrC consensus sdrC B1 sdrC COL sdrC Mu50 sdrC MW2 MRSA252 RF122 sdrC other than MRSA252 RF122 Ser Asp rich fibrinogen bone sialoprotein binding protein D sdrD total sdrD consensus sdrD COL MW2 sdrD Mu50 sdrD other van Willebrand factor binding protein immunodominant antigen B defensin resistance gene protein transferrin binding protein hypothetical protein similar to integral membrane protein LmrP S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf vwb total vwb consensus vwb COL MW2 vwb MRSA252 vwb Mu50 vwb RF122 isaB isaB MRSA252 mprF COL MW2 mprF Mu50 MRSA252 isdA consensus isdA MRSA252
49. mpted by a technically faultless experiment and if contamination can be ruled out by repeated cloning please submit the picture and or the strain for further analysis It might be an unknown strain that cannot be identified because it was not included into the database If this is the case we will use multilocus sequence typing MLST for further characterisation and might include this strain into future database updates This error message in the short result sheet accompanied by positive signals only for resistance and SCCmec associated genes indicates the presence of different staphylococcal species Staph epidermidis Staph haemolyticus etc The long result_B res htmL htmL result sheet should provide this information occasionally a faulty identification as Staph argenteus lineage CC75 albeit at a low score might occur Another error message An assignment to a strain is not possible although the clonal complex was recognised This might be caused by technical issues such as poor signal quality overstaining or contamination The isolate could also represent a new strain within a known clonal complex i e a strain carrying an unusual SCCmec element or an unusual set of virulence genes If this appears to be the case please submit the array image and or the isolate in question to Alere Technologies might appear instead of the typing information in an otherwise normal result file This could indicate an unusual SCCm
50. nd discard the Washing Buffer and add 100 uL diluted conjugate to each well incubate at 30 C 10 min and 550 rpm e Remove liquid and wash with 200 uL C5 Washing Buffer pipette up and down four times remove and discard e Add another 200 uL C5 Washing Buffer Incubate at 30 C 2 min and 550 rpm e Remove and discard Washing Buffer add 100 uL of D1 substrate precipitating dye at 25 C see above per well e incubate at 25 C 6 min but do not shake e Remove liquid completely e The outside of the bottom of the ArrayStrips may cautiously be cleaned with wipes gently pipetting up and down the D1 e Scan and process ArrayMate see below Adapted Protocol for Eppendorf s Thermomixer Comfort e Switch on the thermoshaker and pre heat to 55 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 code close to row A and proper fit e 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 e Second C1 Hybridisation Buffer 200 uL per well at 55 C 5 min and 550 rpm S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Add 90 ul of Buffer C1 to each tube with 10 uL of labelled amplification product mix gently e Remove the Washing Buffer from the well with the array and add the mixtu
51. nd monoclonal S aureus colony material is amplified approximately 40 fold and internally labelled with biotin 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 that reason the method is restricted to colony material and cannot be performed on samples such as swabs or pus The resulting biotin labelled ssDNA is transferred and hybridised to DNA oligonucleotide microarrays with 336 probes for different genetic markers and a biotin staining control Most of them are printed in two duplicate spots The target set consists of a variety of species markers virulence associated genes including genes encoding exotoxins antibiotic resistance genes genes encoding microbial surface components recognizing adhesive matrix molecules MSCRAMMs various enzymes and other types of markers 1 Spot recognition is performed automatically based on digital images 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 allowing assignment to clonal complexes and strains 1 2 In the recent
52. nnnnonnnnnnnnnnnnnnnnnonnnnnnnnnos 11 Linear Amplification and Internal Biotin Labelling ccccccoonococonnnnnnnnnanoncnononnnnncnnnnarnnnnos 11 Hybridisation AA E A EET E E A E A E EE 12 General Remarks Handling of Arrays ccccsccscccccceesssesssaeeceeecessesseaeeeceeseeseeesaseaeeeeeeseesses 12 General Remarks Handling of LiquidS cccccccccccecessessseeceeecessnsseaeeeceeeeeseeeeasaaeeeeeeesesees 13 General Remarks the Substrate Precipitating Dye Dl oocoocccnoccccnocccoonnncnonnnononcncnnnnnonn 14 General Remarks Thermoshakers ccccssssscecesssseececsesnececeseeeececseaaececssaeeeseseeaeeecesseaaeeeees 14 Protocol for Ouantifoil S BioShake TO cani a ea ee kn 15 Adapted Protocol for Eppendorf s Thermomixer Comfort c cccccccccessssscececeeesesssssseseeeeeees 16 Data Anal iii id dial 18 Starting the ArrayMate Reader cooconcocccncncncnononononnnnnnnnnnonononnnnnnnnonnnnnnnrnnnnnnnnnnnnnannnn iaiia 18 World i 19 Data Acquisition in the ArrayMate Reader ccooooccccnonoccconononnnononancnnnnnnnncnnnononanonnnnn non nncnnnnnnnnos 20 Res a dd a ia 22 Export of S aureus Genotyping Kit 2 0 Test Reports o cccccncconononocnnnnnnnonnoananonnnnnnonnnananononons 23 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com TROUBLESHOOTING oriee Ra u Eee 26 Staining CONTO leccion into at A E a AA IRA ea 26 image Q a
53. notyping Kit 2 0 e B2 Labelling Enzyme Store at 2 8 C Surplus 50 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies 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 protect them against humidity and dust and store them at a dark place Avoid any touching or scratching of the microarray surface at the bottom of the well Do not store or handle unused wells at more than 60 relative humidity since this may irreversibly corrode the spots e StripCaps 24 units e C1 Hybridisation Buffer Store at 18 28 C protect against direct sunlight Surplus 100 e C2 Washing Buffer 1 Store at 18 28 C protect against direct sunlight Surplus 200 e C3 HRP Conjugate 100 x Store at 2 8 C protect against direct sunlight Surplus 100 e C4 Conjugate Buffer Store at 18 28 C protect against direct sunlight Surplus 200 e C5 Washing Buffer 2 Store at 18 28 C protect against direct sunlight Surplus 200 e D1 Horseradish Peroxidase Substrate Store at 2 8 C protect against direct sunlight Surplus 50 e CM Reference DNA from S aureus strain N315 GenBank accession number BA000018 0 1 ug uL Store at 2 8 C Sufficient for 5 6 tests Instrumentation and
54. o the funnel of the spin column Thus these funnels should be touched gently and dried with sterile filter paper or wipes prior to the elution step Alternatively prepared DNA can be heated briefly to evaporate ethanol e g 10 min at 70 e Check for DNA integrity and absence of RNA e g by agarose gel If necessary you might perform another digestion step with additional RNAse not provided Measure DNA concentration Azg9 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 S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Extraction of DNA by Automated Device The assay was tested with Qiagen s EZ1 Other systems also can be used as well However performance should be checked with some known reference strains prior to routine use Incubate the colony material of the S aureus isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker as described above depending on the input sample volume required by the device you actually use the A1 A2 mixture might be divided into two aliquots and used for DNA preparation of two samples e Add 10 ul proteinase K and add 100 uL buffer AL e Vortex briefly or shake vigorously e Incubate sample 45 60 min at 56 C and 550 rpm in the thermomixer e When the cells are lysed proceed by performing the tissu
55. on of S aureus incubator inoculation loops Petri dishes e Clumping factor coagulase assays for confirmation of S aureus e DNA preparation kit The assay was tested with the DNeasy Blood Tissue Kit from Qiagen cat 69504 QlAamp Minikit cat 51306 and a DNA preparation kit for Qiagen s EZ1 automated device DNA Tissue Kit cat 953034 Please note DNA isolation from S aureus requires a pre treatment with the Cell Lysis components A1 A2 see below e Equipment needed for DNA isolation e g pipettes centrifuge thermoshaker or automated device see above e Photometer for measuring the concentration of DNA S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Equipment for DNA gel electrophoresis for quality control of DNA e Thermocycler e Thermoshaker We strongly recommend the BioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips Alternatively you may use Eppendorf s Thermomixer Comfort equipped with a heating block for microtitre plates e Pipettes suitable for volumes of 1 uL 5 uL 90 uL 100 uL 200 uL 1000 uL e Multichannel Pipettes for 100 200 uL e Reagent tubes suitable for PCR e Ultrapure PCR grade water S aureus Genotyping Kit 2 0 05 16 04 0001 VO1_Manual S aureus_2 pdf www clondiag com www alere technologies com PROTOCOL Cultur
56. or images If strains cannot be identified error messages are displayed instead of the short htmL result sheet In order to facilitate searching for the cause of the error the long result_B res htmL htmL result sheet will be generated although it might be faulty However it might give a hint what the cause of the problem was S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com One possible error message is Identification is not possible This could be due to technical issues such as poor signal quality overstaining or to contamination Please re clone the culture confirm its identity as Staphylococcus aureus and its purity and repeat the experiment Identification is also not possible for strains that are not covered by the database If this is likely i e if your isolate is repeatedly un identifiable or if you have additional typing data suggesting an unknown strain please submit the array image and or the isolate in question to Alere Technologies This will appear for instance when the pattern is entirely irregular or if mutually exclusive alleles are detected simultaneously The long result_B res htmL htmL result sheets might show in the latter case that several agr types or capsule types 5 and 8 were detected in one sample This can be caused by massive unspecific staining or by contamination mixed culture Re clone and repeat If this message was pro
57. ping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com e Scan and process ArrayMate see below Data Analysis Starting the ArrayMate Reader We recommend to start the ArrayMate Reader after having started the hybridisation this allows you to conveniently start the device and to import the worklist file see below Please note that this is a short instruction only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate 1 st main switch on the rear below the electric cable plug 2 nd operating switch on the bottom left corner of the front side e Switch on the screen switch righthand below the screen e Log on as R amp D User Research and Development User for full access to test specific software a default password will be provided together with the ArrayMate device If you log on as User you will obtain raw values only but no identification as positives negatives and no strain assignment Administrator log on will allow manipulation of file folders and software this should be done only upon direct advice of Alere s IT team e The user interface will be loaded 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 e
58. probe binding sites For this reason DNA should not be prepared by disrupting S aureus cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols We made good experiences with the manual QIAGEN DNeasy Kit and the automated device EZ1 DNA must be free of any 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 min at 70 C Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples see above Physical Damage to the Array Scratching of the array surface with a pipette tip can lead to damage of array spots which in turn prohibits the acquisition of a valid signal In this case the respective marker is not assigned as negative but instead the message none appears next to the marker name Ambiguous Results Apart from a positive or negative result for the individual markers on the S aureus Genotyping Test Report the result can also be ambiguous S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com In cases affecting resistance genes or virulence factors no definitive answer with regard to this specific marker can be given This can be caused by poor sample quality poor signal quality and especially in some resistance associated genes such aacA aphD by t
59. re of C1 and labelled amplification product e Incubate at 55 C 60 min and 550 rpm e Remove liquid and wash with 200 uL C2 Washing Buffer pipette up and down four times remove and discard e Add another 200 uL C2 Washing Buffer Incubate at 30 C 5 min and 550 rpm e Meanwhile prepare conjugate For each experiment add 1 uL conjugate 100 x HRP to 99 uL C4 Conjugation Buffer This mixture is stable for one day at room temperature C3 is delivered with a surplus of 100 C4 is delivered with a surplus of 200 Suggested pipetting scheme 1 well 2 3 wells 4 6 wells 7 10 11 15 16 20 21 30 31 40 wells wells wells wells wells C3 1 5 uL 3 5 uL 7 uL 11 ul 16 uL 21 ul 32 uL 42 uL C4 148 5 uL 346 5 ul 693 uL 1089 uL 1584 uL 2079 uL 3068 uL 4058 uL e Remove and discard Washing Buffer and add 100 uL diluted conjugate to each well incubate at 30 C 15 min and 550 rpm e Remove liquid and wash with 200 uL C5 Washing Buffer pipette up and down four times remove and discard e Add another 200 uL C5 Washing Buffer Incubate at 30 C 2 min and 550 rpm e Remove and discard Washing Buffer add 100 uL of D1 substrate precipitating dye at 25 C see above per well e incubate at 25 C 6 min but do not shake e Remove liquid completely e The outside bottom of the ArrayStrips may cautiously be cleaned with wipes gently pipetting up and down the D1 S aureus Genoty
60. rips must be clean They should not contain any liquids during analysis Data Matrix codes must be clean There must be no ArrayStripCaps on the wells that are to be analysed however unused wells should remain covered e Press the button Next bottom right on the screen reader closes analysis program starts it takes ca 2 10 min dependent on the number of strips reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analysis ready e The reader indicates the end of the entire process with an acoustic signal beep e Press the button Next bottom right on the screen reader is opening S aureus Genotyping Kit 2 0 05 16 04 0001 V01_ Manual S aureus_2 pdf www clondiag com www alere technologies com e Remove the white frame with the ArrayStrip s e Press the button Next bottom right on the screen reader closes 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 by default named according to the date of acquisition Example There is one experiment run in this archive Browse 4 Search a ARC
61. s RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com REAGENTS AND DEVICES Kit Components Storage and Stability All reagents are provided in a certain surplus amount see below In case of need all components may also be ordered separately please refer to the order numbers at the end of this 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 package All components were been tested for stability for short term shipment lt 1 week at ambient temperature lt 37 C The kit components with a rather limited stability are D1 and C3 All other components proved to be stable even six months after passing the kit expiry date Cell Lysis e Al Lysis Buffer Store at 18 28 C ambient temperature Surplus 50 e A2 Lysis Enhancer lyophilised Store at 18 28 C ambient temperature Centrifuge A2 tubes briefly 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 B1 Labelling Buffer Master Mix Store at 2 8 C Surplus 25 Do not use B1 Labelling Buffer Master Mix from earlier kit versions for S aureus Ge
62. s_2 pdf www clondiag com www alere technologies com MLST CC A Ones MEST types associated spa types associated with with this strain UK EMRSA 5 8 17 Irish AR22 ccs ST247 MRSA I North rish New 02 Spanish PFGE type ST247 t051 t052 t194 t3503 German Iberian EMRSA E1 Belgium E1 Finland E7 E10 France C ST250 MRSA I Early Ancestral MRSA rish ARO2 Irish Phenotype II ST250 ST985 t008 ST8 MRSA IIA B D A r Irish AR13 14 rish AROS Irish 01 Irish New03 ST8 t190 ST8 MRSA IIA B D SCC M1 A e Irish AR13 14 rish AROS Irish 01 Irish NewO3 ST8 ST609 t064 t190 t2196 ST8 MRSA IIC E A y Irish AR13 14 rish AROS Irish 01 Irish NewO3 ST8 t190 ST8 MRSA IIC E SCC M1 A E A Irish AR13 14 rish AROS Irish 01 Irish New03 ST8 t190 rish AR43 Irish 02 Subclone with ST8 MRSA IVF ccrA B 4 VI SCCmec IV F ST8 MRSA IV F WA ST8 t190 MRSA 16 ST8 MRSA IVG E ccrA B 4 UK EMRSA 12 13 rish 02 ST8 MRSA IV G E ST8 ST94 t190 t4691 Irish AR43 CC8 MRSA IV sea Lyon Clone UK EMRSA 2 France A France B ST8 ST995 ST1337 t008 t024 t068 t121 t967 t2047 t2206 t4268 CC8 MRSA IV Lyon Clone sea neg variant WA MRSA 88 CC8 MRSA IV sea N315 CC8 MRSA IV sea UK EMRSA 6 CC8 MRSA IV tst1 WA MRSA 104 tst1 positive Variants of WA MRSA 5 31 ST576 ST8 MRSA IV ST576 MRSA IV CC8 MRSA IV UK EMRSA 14 WA
63. 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 Hybridisation General Remarks Handling of Arrays Never touch the array surface Avoid 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 hybridisation 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 until you use these wells for storage conditions after use see section Kit Components Storage and Stability Hybridisation and Detection Always label your array strips with a laboratory marker at the recommended position Never label them on the bottom or across the Data Matrix code This may cause errors S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Data Matrix code keep it clean label here Avoid contact of Data Matrix code with organic solvents The ArrayMate Reader needs the information encoded in the data matrix to perform the assay Avoid touching the bottom of the ArrayStrip and keep it clean General Remarks Handling of Liquids We recommend the
64. uL Buffer C5 30 C 2 min and 550 rpm discard buffer y Add 100 uL D1 substrate 25 C 6 min strictly no shaking 6 min 6 min Discard substrate analyse ArrayMate 5 min 1 min total time requirement over night 50 80 min 7 8 h S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com APPENDIX 2 IMAGES FOR TROUBLESHOOTING Image Comment This image is overstained The experiment should be X repeated This image is poor This could be due to low DNA concentration fragmented DNA ethanol trace conta minations in DNA sample or expired reagents The experi ment should be repeated with a new DNA preparation If this also fails try an experiment with N315 control DNA CM Species other than S aureus tested Check identification by other means Result sheets Valid results no error messages There might be no error messages although individual probes might yield false positives The error message An assignment to a strain is not possible although the clonal complex was recognised This might be caused by technical issues such as poor signal quality overstaining or conta mination might appear if false positives hinder strain identification The error message An assignment to a strain is not possible although the clonal complex was recognised This might be caused by technical
65. ureus isolates to clonal complexes based on microarray analysis and pattern recognition FEMS Immunol Med Microbiol 53 237 251 3 Garcia Alvarez L Holden MT Lindsay H Webb CR Brown DF Curran MD Walpole E Brooks K Pickard DJ Teale C Parkhill J Bentley SD Edwards GF Girvan EK Kearns AM Pichon B Hill RL Larsen AR Skov RL Peacock SJ Maskell DJ Holmes MA 2011 Meticillin resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark a descriptive study Lancet Infect Dis 11 8 595 603 4 Shore AC Deasy EC Slickers P Brennan G O Connell B Monecke S Ehricht R Coleman DC 2011 Detection of staphylococcal cassette chromosome mec type XI carrying highly divergent mecA mecl mecR1 blaz and ccr genes in human clinical isolates of clonal complex 130 methicillin resistant Staphylococcus aureus Antimicrob Agents Chemother 55 8 3765 3773 For further literature please refer to http alere technologies com en science technologies publications saureus htmL UPDATES AND SOFTWARE Notifications on database software updates and freeware tools can be found at http alere technologies com en science technologies publications downloads htmL and or http alere technologies com en news htmL Currently available freeware programs are e spa type mapper for the analysis of spa sequences e Alere S aureus Results Collector for the conversion of multiple result xmL fil
66. use of a multichannel pipette and reagent reservoirs Please keep in mind the limited surplus of C1 100 We strongly recommend to remove the liquid 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 cat 612 2856 Pasteur pipette plastic with a flexible tip CC T flexible tip S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Always place the pipette tip in 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 be pipette tip Use the cavity between array and the wall of the tube Do never touch the array array 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 to acclimatise it to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged briefly prior to use to remove bubbles as well as possible precipitates 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 t
67. vanz toxic shock syndrome toxin 1 tst1 consensus tst1 human allele tst1 bovine allele from RF122 enterotoxin A entA sea enterotoxin A allele from strain 320E sea 320E enterotoxin A allele from strain N315 enterotoxin P sea N315 enterotoxin B entB seb enterotoxin C entC sec enterotoxin D entD sed enterotoxin E entE see enterotoxin G entG seg enterotoxin H entH seh enterotoxin entl sei enterotoxin J entJ sej enterotoxin K entK sek enterotoxin L entL sel enterotoxin like gene protein M sem entM selm enterotoxin like gene protein N sen entN seln consensus seln other than RF122 enterotoxin like gene protein O seo entO selo enterotoxin gene cluster seg i selm n o u egc enterotoxin Q entQ seq enterotoxin R entR ser enterotoxin like gene protein U seu entU selu enterotoxin like protein ORF CM14 ORF CM14_ probe1 VIRULENCE HLG AND LEUKOCIDINS enterotoxin like protein ORF CM14 haemolysin gamma leukocidin component B F ORF CM14_ probe2 lukF haemolysin gamma leukocidin component C S lukS S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com VIRULENCE HAEMOLYSINS haemolysin gamma leukocidin component C S allele JukS ST22 ST4
68. www clondiag com www alere technologies com S aureus Genotyping Kit 2 0 Array Hybridisation Kit for DNA based detection of resistance genes and pathogenicity markers of Staphylococcus aureus and assignment of unknown S aureus isolates to known strains For Research Use Only Not for Use in Diagnostic Procedures www clondiag com www alere technologies com CONTENT BACKGROUND ae S 1 GENERACINSTRUETIONS FOR USE aa 2 Intended Useni iia 2 Specific E E E E EE T E EA OO E A TE 2 Technical Support aa a aan eats E A E a a iia e E REN 2 Safety Precautionsi naar a O E a aaa 3 Material Safety Data Sheets MSDS ne ae een 3 SHIPPINE Precision 3 REAGENTS AND DEVICES a iR nba la ais 4 Kit Components Storage and Stability an ta 4 GEN EYSiISt nn a er ak SURG stu a wad DEE R DET ARAS E AAT 4 DNA Labelling and Amplification ccccccccsccccececessesssscseeceeeceesssaeseeeceeecesesaeeuseseeeeeesenssessaaees 4 Hybridisation and Detection ae ee He a a 5 Instrumentation and Software aiii enable 5 Components Required but not Provided cccconcnconocncnnncnonononannnnnnnnnnonononnnnnonnnnnnnnnnnnnnnnnnnnnnnnnnnns 6 PROTOCOL a ii 8 Culturing and Harvesting Bacterial CellS o ooooooocnnoncnonononacannnnnnnnnnnononononcnncnnnnnnrnnonnnnnnnnnnnnnns 8 Extraction O EDNA ida a AAA AA 8 Extraction of DNA by Spin Col MNSning i ieee A EEE AE E E 9 Extraction of DNA by Automated Device cccccccnnononocnnnnnnnnnnonononcnnn
69. xperiment name at your convenience e Type in your operator ID optional e You may enter a comment into the memo field optional S aureus Genotyping Kit 2 0 05 16 04 0001 V01_Manual S aureus_2 pdf www clondiag com www alere technologies com Worklist A Worklist file allows to link an identifier such as a laboratory sample number to a position of an array within the ArrayStrip Please respect the rules of confidentiality and data protection 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 etc e Create a list with at least three columns with obligatory headers in the following order position sample ID assay ID Table 1 e Positions are continuously numbered from 1 to a maximum of 96 Position 1 would correspond to A1 8 to H1 9 to A2 and 96 to H12 Table 2 Do not leave empty lines in the worklist If you use EXCEL position numbers should be typed into column A e Sample ID is strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patient name should not be used as Sample ID e The Assay ID enables the system to identify the current test and to correctly use information on layout spot number and identity etc S aureus Genotyping Kit 2 0 has the Assay ID 10620 whereas

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