User guide for the Escherichia coli combined Assay
Contents
1. Position samplelD assaylD comment 1 2013 12345 10313 2 2013 12346 10313 3 2013 12347 10313 4 2013 12348 10313 5 2013 12349 10313 6 2013 12350 10313 7 987654 10313 Isolate referred from Dr J Doe 8 E coli EDL933 10313 Control strain Table 2 Positions in the 96 well format 10 18 26 34 42 50 58 66 74 82 90 11 19 27 35 43 51 59 67 75 83 91 12 20 28 36 44 52 60 68 76 84 92 13 21 29 37 45 53 61 69 77 85 93 14 22 30 38 46 54 62 70 78 86 94 15 23 31 39 47 55 63 71 79 87 95 T O Emo n o gt o0 JXJ O Nn pbj win a ja 16 24 32 40 48 56 64 72 80 88 96 Data Acquisition in the ArrayMate Reader e Insert your memory stick containing the worklist Use any of the USB ports down to the right side of the ArrayMate e Press the button a folder selection dialog will open e Select your worklist path My Computer Removable Disk E coli combined Assay User Guide 13 11 11 001 V1 19 www clondiag com www alere technologies com e Open your selected worklist with Enter or the button Open e Press the button your imported worklist opens in a separate window Proofread If the new window is empty or if it was the wrong worklist repeat the import e Press the button OK the worklist window will close e Leave the memory stick attached to the ArrayMate if you intend to expo2. bfpA bfpA 10 ABO24946 1 cfaC cfa_c_10 outer membrane usher protein M55661 1 cofA cofA 10 major pilin subunit CFA III pilin D37957 1 f17 A f17 A 40 f17 A 50 f17 A 60 major fimbrial subunit protein L77091 1 f17 G f17 G 20 major fimbrial subunit protein pilin G L43372 1 fanA fanA 10 regulatory protein X05797 1 K88ab K88ab 10 major subunit of K88 fimbriae V00292 1 IngA IngA 20 longus pilus structural subunit EF595770 1 IpfA hp_IpfA_611 major fimbrial subunit AYO57066 1 perA perA_10 perA_20 transcriptional activator AF255772 1 major pilu subunit operon regulatory protein prfB prfB_30 X76613 1 sfaS sfaS 10 adhesin minor Shigella fimbriae subunit X16664 4 virulence factor secretion systems cif hp cif 611 cell cycle inhibiting factor type Ill secretion system AY128535 1 espA C rodentium hp espA Crod 611 EspA protein type III secretion system associated with Citrobacter rodentium AF311901 1 hp espA O103H2 611 hp espA O119H6 611 hp espA O127H7 611 hp espA O157H11 611 hp espA O49H12 611 hp espA O55H7 611 espA hp espA O8 611 EspA protein type III secretion system AF054421 1 EspC extracellular serine protease type III secretion espC hp espC 611 system AF297061 1 EspF effector protein type Ill secretion system espF hp espF 611 hp espF 612 AE005174 2 espF C rodentium hp espF Crod 611 EspF effector protein type Ill
3. EspP serine protease autotransporter of espP hp espP 611 Enterobacteriaceae SPATE AF074613 1 Pic serin protease autotransporter of pic hp pic 611 Enterobacteriaceae SPATE U35656 1 RpeA serin protease autotransporter of rpeA hp rpeA 611 Enterobacteriaceae SPATE AY552473 1 SepA serine protease autotransporter of sepA hp sepA 611 Enterobacteriaceae SPATE AY604009 1 SigA serine protease autotransporter of sigA hp sigA 611 Enterobacteriaceae SPATE AF200692 2 Tsh hemoglobin binding protease SPATE tsh hp tsh 611 AJ223631 1 vat hp_vat_611 Vat haemoglobin protease SPATE AF242872 1 EaaA serine protease autotransporter of eaaA hp_eaaA_611 Enterobacteriaceae SPATE AF151674 1 EatA serine protease autotransporter of eatA hp_eatA_611 Enterobacteriaceae SPATE AY163491 2 EpeA serine protease autotransporter of epeA hp_epeA_611 Enterobacteriaceae SPATE AY258503 2 astA astA_consens_10 heat stable enterotoxin consensus sequence virulence factor SPATE serin protease autotransporters cba cba_10 colicin B activity protein M16816 1 ccl ccl_10 colicin activity protein AF540491 1 cdtB cdtB_40 cdtB_50 cdtB_60 cytolethal distending toxin subunit B AJ508930 1 celB celb_10 colicin lysis protein X03632 1 cma cma_20 colicin M activity protein CP000971 1 cnf1 cnf1_20 cytotoxic necrotizing factor
4. Image Quality In case of poor image quality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel In order to determine whether any problems originated from the DNA preparation perform an experiment with the CM Control material This is DNA from the reference strain E coli EDL933 GenBank accession number NC 002655 2 and should be identified by the assay as E coli with O 146 and H 20 If the control experiment yields a valid result and a correct identification there was probably an issue with DNA preparation If the control experiment also fails an error affecting later steps or a degradation of reagents from later steps is likely E coli combined Assay User Guide 13 11 11 001 V1 24 www clondiag com www alere technologies com DNA Quality and RNA contamination control The amount of DNA is crucial because of the linear kinetics of amplification see Introduction DNA should be free of RNA as free RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation A260 readings will cover RNA and other contaminants as well Therefore pure DNA preparations without RNA contaminations are a prerequisite for proper DNA concentration measurement RNase treatment prior to A260 reading therefore is necessary component A2 contains RNAse Additionally the microarray includes probes as an internal control for RNA
5. acetyltransferase streptogramin A acetyltransferase associated with resistance to streptogramin A AF242872 1 aminoglycoside resistance aminoglycoside 3 phosphotransferase locus A and aminoglycoside 6 phosphotransferase associated StrA strB prob strA 611 prob strB 611 with resistance to streptomycin EF090911 1 3 N aminoglycoside acetyltransferase associated with aac aph hp aac aph 611 resistance to gentamycin AE017171 1 hp aac3 611 hp aac3 612 hp aac3 613 hp aac3 614 3 N aminoglycoside acetyltransferase associated with aac3 hp_aac3_615 resistance to gentamycin U90945 1 3 N aminoglycoside acetyltransferase associated with resistance to astromicin gentamicin sisomicin aac3la prob_aac3la_1 U90945 1 3 N aminoglycoside acetyltransferase associated with resistance to apramycin dibekacin gentamicin aac3lVa prob_aac3lVa_1 netilmicin sisomicin tobramycin EU784152 1 hp_aac6_611 hp_aac6_612 hp_aac6_613 hp_aac6_614 aminoglycoside 6 N acetyltransferase associated hp_aac6_616 hp_aac6_617 with resistance to amikacin dibekacin isepamicin aac6 hp_aac6_618 netilmicin sisomicin tobramycin AF162771 1 E coli combined Assay User Guide 13 11 11 001 V1 33 www clondiag com www alere technologies com aminoglycoside 6 N acetyltransferase associated with resistance to streptomycin spectinomycin aac6lb pr
6. e Add 500 ul Buffer AW2 e Centrifuge 14 000 rpm 3 min at room temperature the membrane of the spin column should be dry and all liquid should be in the collection tube e Discard collection tube with liquids e Place the spin column in a clean 1 5 ml tube not provided with the kit e Add 100 ul Buffer AE or PCR grade distilled water directly onto the membrane of the spin column e Incubate at room temperature for 1 min to elute DNA e Centrifuge 8000 rpm 1min at room temperature e Optional add another 100 ul Buffer AE or PCR grade distilled water directly onto the membrane incubate at room temperature for 1 min and centrifuge again e Discard the spin column Please note Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay E coli combined Assay User Guide 13 11 11 001 V1 10 www clondiag com www alere technologies com A contamination with Washing Buffer might occur during elution of prepared DNA by drops adhering to the funnel of the spin columns Thus these funnels should be gently touched and tried with sterile filter paper or wipes prior to the elution step Alternatively prepared DNA can shortly be heated to evaporate ethanol e g 10 min at 70 C e Check for DNA integrity and absence of RNA e g agarose gel If necessary you might perform another digestion step with additional RNase A not provided Measure DNA concentration A so method it shouldn t be less tha
7. incubate twice in 200 ul Buffer C2 45 C 550 rpm 10 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrat D1 25 C i discard Buffer C2 10 min incubate in 100 pl C3 C4 conjugate 30 C 550 rpm 10 min y discard C3 C4 conjugate 5 min incubate once in 200 ul Buffer C5 30 C 550 rpm 5 min i discard Buffer C5 10 min incubate with 100 ul Substrate D1 25 C 10 min discard Substrate D1 analyse ArrayMate 15 min total time requirement overnight MM MasterMix 7 8h PM PrimerMix E coli combined Assay User Guide 13 11 11 001 V1 hands time 5 min 40 min 5 min 2 min 2 min O min 5 min 2 min 2 min 2 min 10 min app 120 min 29 www clondiag com www alere technologies com Protocol Eppendorf Thermoshaker pro hands prepare ArraysStripes prepare DNA cessing on time time AN Grow CLONAL coli isolate over 5 min not part of the kit night isolate genomic DNA 3 4h 40 min not part of the kit label RNA free DNA in thermocycler 2h 5 min 5 ul DNA Cona 0 1 0 4 ug ul rinse ArrayStripes plus MM 3 9 uL B1 0 1 uL B2 200 ul water pipette up and down 4x 1 ul PM Ecoli PM1 y preparing labeled DNA 150 ul Buffer C1 55 C 550 rpm 5 min to 10 uL of labeled DNA add 90 uL 10 min 5 min discard C1 process promptly of Buffer C1 transfer 100 ul labeled DNA to ArrayStripes 2 min 2 min Barcode eo here CEE discard water
8. Wu G Ehricht R Mafura M Stokes M Smith N et al 2012 Escherichia coli isolates from extraintestinal organs of livestock animals harbour diverse virulence genes and belong to multiple genetic lineages Vet Microbiol 160 197 206 UPDATES amp 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 E coli combined Assay User Guide 13 11 11 001 V1 28 www clondiag com www alere technologies com APPENDIX 1 Flow charts The figures summarise the test procedure However please refer to the text section of this user guide at any step of the test protocol for further important details Protocol Quantifoil BioShake iQ pro prepare ArraysStripes prepare DNA cessing time AUN Grow CLONAL E coli isolate over not part of the kit night isolate genomic DNA 3 4 h not part of the kit label RNA free DNA in thermocycler 2h 5 ul DNA cona 0 1 0 4 ug ul rinse Array etripes plus MM 3 9 uL B1 0 1 uL B2 200 ul water 50 C 550 rpm 5 min TIS PM D discard water preparing labeled DNA 150 ul Buffer C1 50 C 550 rpm 5 min to 10 uL of labeled DNA add 90 uL 2 min discard C1 process promptly of Buffer C1 transfer 100 ul labeled DNA to ArrayStripes 2 min Barcode q wot here hybridise 50 C 550 rpm 60 min 60 min discard labeled DNA 20 min
9. class D beta lactamase AY665723 1 blaOXA 60 hp oxa 618 oxacillinase class D beta lactamase AF525303 2 class A beta lactamase PER 1 extended spectrum blaPER 1 hp per1 611 beta lactamase 221957 1 class A beta lactamase PER 2 extended spectrum blaPER 2 hp per2 611 prob per2 1 beta lactamase X93314 1 prob pse1 1pm blaPSE 1 prob pse1 1mm carbenicillinase 218955 1 blaSHF 1 hp sfhi 611 class B metallo beta lactamase AF197943 1 class A beta lactamase consensus sequence for blaSHV genes including extended spectrum beta blaSHV prob shv1 11 lactamases carbapenem hydrolysing beta lactamase Serratia blaSME 1 hp sme1 611 marcescens Z28968 1 metallo beta lactamase carbapenemase blaSPM 1 hp_spm1_611 AY341249 1 class A beta lactamase consensus sequence for blaTEM genes including extended spectrum beta blaTEM prob tem1 1 lactamases E coli combined Assay User Guide 13 11 11 001 V1 35 www clondiag com www alere technologies com blaVEB 1 hp veb1 611 extended spectrum beta lactamase AF010416 1 hp vim 611 hp vim 612 blaVIM hp_vim_613 class B metallo beta lactamase Y18050 2 ble hp_ble_611 bleomycin resistance X01702 1 chloramphenicol resistance chloramphenicol acetyltransferase group A catA1 prob catA1 11 V00622 1 chloramphenicol acetyltransferase group B catB3 prob catB3 11 AJ009818 1 catB8 prob_catB8_12 chloramphenicol acetyltransferase AF227506 1 chloram
10. 11 HBO EE ee 12 General Remarks Handling of Arrays esses eene eene eren entren 12 General Remarks Handling of Liquids eeeeessse sese eene nennen 12 General Remarks the Substrate Precipitating Dye Dl ooocooccconocacionccononcncnonnnononanononanonos 13 General Remarks Thermoshakers viii 14 Protocol for Quantifoil s BioShake iQ iio ete aaa 14 Protocol for Eppendorf s Thermomixer Comfort with microtitre plate adapter 15 Data Analyse ou A 17 Starting the ArrayMate Reader u da 17 edm 18 Data Acquisition in the ArrayMate Reader c ccccsssssecesssstececsssneeecesseneeeesssaaecesseaeeeeseeeneeens 19 A eniin feuis annesacateoetaeudicstaus dad bof csen touto ao ipud SEAN ae eiaa e aaiae elu Aper dieu 21 Export of E coli combined Test Reports nu pere Pop Ra uS de ed ai x ebrei 22 TROUBLESHOOTING nu a a 24 Staining eenig m 24 Image Ould A A A RA ici 24 DNA Quality and RNA contamination control eese esee esee 25 Physical Damage to the Array icon A vu FU dU ze Mb dedu NA Rd UR 25 Report Unavallable coros cnica nd in e 25 ABDITIONALIN FORMS TON aa 26 Watanabe iia 26 Disc ccce 26 Quality Control nai dada 27 tegal Manufacturer is et E E atr ter cs iege eR En adea eee ned 27 Socr R
11. combined Assay User Guide 13 11 11 001 V1 25 www clondiag com www alere technologies com ADDITIONAL INFORMATION Warranty Alere guarantees the performance as described in this user guide Usage of the Assay was successfully tested at ambient temperatures up to 37 C a guarantee is limited to ambient temperatures in the laboratory between 18 C and 28 C Assay 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 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 detection of resistance genes in order to predict phenotypic antibiotic resistances or susceptibilities for the guidance of an antibiotic chemotherapy Since resistances might be caused by genes or mutations not covered by this array or by hitherto unknown genes or mutations any antibiotic chemotherapy MUST be guided by phenotypic susceptibility tests Furthermore we do not accept any liability for damages caused by inappropriate use of the device as a per
12. contamination The automatic software analysis will give a failed if a RNA contamination occurred while DNA isolation DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites DNA should for this reason not be prepared by disrupting E coli 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 Roche High Pure Kit DNA must be free of any traces of ethanol as ethanol strongly influences the amplification It is possible to heat the sample prior to adding it to the labelling mix 5 10 minutes at 70 C Physical Damage to the Array Scratching of the array surface with a pipette tip can lead to the damage of array spots that prohibits the acquisition of a valid signal In this case the respective marker is not assigned as negative but instead the message none appears next to the marker name 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 barcode 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 E coli
13. e 27 E coli combined Assay User Guide 13 04 16 001 V1 www clondiag com www alere technologies com LITERATURE pai iia 28 UPDATES amp SOFTWARE u a AAA AAA AR 28 APPENDOCE FLOWIEHARTS anne 29 APPENDIX 2 PROBE TO TARGET FABLE cosita drid 31 APPENDIX 3 TYPING INFORMATION sisi 41 Definitions amp EXPAN OMG ace esc iet donde he 41 E coli combined Assay User Guide 13 11 11 001 V1 2 www clondiag com www alere technologies com BACKGROUND The CLONDIAG Escherichia coli E coli combined Assay allows DNA based serogenotyping of most known O and H antigens as well as simultaneously the detection of important anti microbial resistance i e blaOXA and virulence genes i e stxA stxB RNA free unfragmented genomic DNA from pure and monoclonal E coli colony material is amplified approximately 50 fold and internally labelled with biotin 11 dUTP using a linear amplification protocol In contrast to standard PCR only one antisense primer per target is used resulting in producing single stranded ss DNA reaction products This allows simultaneous sequence specific labelling and amplification of an essentially unlimited number of targets However sensitivity is lower than in a standard PCR whereas contamination with amplicons is nearly impossible and for that reason the method is restricted to clonal colony material and cannot be performed on samples such as swabs or other patient samples e g faeces Resulting biot
14. for the cultivation of E coli The test should be performed with colonies harvested from 2xTY or Columbia Blood Agar Other rich media e g Standard 1 or LB may also suffice but have not systematically been tested Liquid media should not been used because contaminations or mixed cultures cannot easily be ruled out e Equipment and consumables needed for the cultivation of E coli incubator inoculation loops Petri dishes e DNA preparation kits The assay has been tested with the DNeasy Blood amp Tissue Kit from Qiagen cat 69504 and High Pure DNA Isolations Kit from Roche Cat No 11796828001 Please note The DNA specimen needs to be free of RNA Recommendation a pre treatment with the Cell Lysis components A1 A2 see below or a standard RNase A treatment while DNA preparation E coli combined Assay User Guide 13 11 11 001 V1 6 www clondiag com www alere technologies com e Equipment needed for DNA isolation e g pipettes centrifuge thermoshaker or automated device see above e Photometer OD 260 nm for measuring the concentration of DNA e Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA e Thermocycler for PCR e Thermoshaker We strongly recommend the BioShake iQ by Quantifoil Instruments http www ginstruments com equipped with a customised heating block designed to fit ArrayStrips Alternatively you may use Eppendorf s Thermomixer Comfort equipped a hea
15. hybridise 55 C 550 rpm 60 min 60min Omin discard labeled DNA 25min 5min incubate twice in 200 ul Buffer C2 40 C 550 rpm 12 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrat D1 25 C y discard Buffer C2 10min 2min incubate in 100 pl C3 C4 conjugate 30 C 550 rpm 10 min y discard C3 C4 conjugate 5 min 2 min incubate once in 200 ul Buffer C5 30 C 550 rpm 5 min y discard Buffer C5 10min 1 min incubate with 100 ul Substrate D1 25 C 10 min discard Substrate D1 analyse ArrayMate 15 min 10 min total time requirement over night app 120 min MM MasterMix 7 8h PM PrimerMix a with heating block for microtitre plates E coli combined Assay User Guide 13 11 11 001 V1 30 www clondiag com www alere technologies com APPENDIX 2 PROBE TO TARGET TABLE Target genes Probes Function Family and Species Marker glyceraldehyde 3 phosphate dehydrogenase A CP000468 1 locus tag APECO1 847 genetic marker gapA prob gapA 611 for family Enterobacteriaceae integration host factor subunit alpha U00096 2 ihfA prob ihfA 611 genetic marker for family Enterobacteriaceae glutamate decarboxylase AE014075 1 locus tag gad gad 10 c4328 genetic marker for genus Escherichia Shigella DNA polymerase III subunit alpha genetic marker for species Escherichia spec and Shigella
16. 1 wzy 0172_11 O antigen 0172 AY545992 1 E coli combined Assay User Guide 13 11 11 001 V1 31 www clondiag com www alere technologies com H serotyping fliC HO1 fliC HO1 11 fliC HO1 12 flagellin C H antigen HO1 AE014075 1 fliC HO2 fliC HO2 11 flagellin C H antigen HO2 AF543692 1 flagellin A H antigen H03 AB128916 1 Note If repressor FIjA positive the gene fliC H16 is flkA H03 flkA HO3 11 repressed H phenotype is HO3 fliC HO4 fliC HO4 11 flagellin C H antigen HO4 AY249989 1 fliC HOS fliC HO5 11 flagellin C H antigen HO5 AY249990 1 flic HO6 fliC HO6 11 flagellin C H antigen HO6 AY249991 1 fliC HO7 fliC HO7 11 fliC HO7 12 flagellin C H antigen HO7 AB028474 1 fliC HO8 fliC HO8 11 flagellin C H antigen HO8 AJ884571 1 fliC HO9 fliC HO9 11 flagellin C H antigen HO9 AY249994 1 fliC H10 fliC H10 11 flagellin C H antigen H10 AY249995 1 fliC H11 fliC H11 11 flagellin C H antigen H11 AY337465 1 fliC H12 fliC H12 11 flagellin C H antigen H12 AY249997 1 fliC H14 fliC H14 11 flagellin C H antigen H14 AY249998 1 fliC H15 fliC H15 11 flagellin C H antigen H15 AY249999 1 flagellin C H antigen H16 AB128919 1 Note If repressor fljA positive fliC H16 might be repressed fliC H16 fliC H16 11 H phenotype is than determined by flkA HO3 fliC H18 fliC H18 11 flagellin C H antigen H18 AY250001 1 fli
17. 17 shiga toxin 2 variant g AJ966782 1 hp stxA2 614 hp stxA2 618 shiga toxin 2 variant a shiga toxin 2 variant c or shiga stx2a c d hp stxB2 614 stx2A 10 toxin 2 variant d X61283 1 subA hp subA 611 subtilase cytotoxin subunit A AF399919 3 hp toxB 611 hp toxB 612 toxB hp toxB 613 cytotoxin B AB011549 2 transcriptional regulator required for transcription of virF virF_20 virB and icsA AF348706 1 virulence factor miscellaneous glutamate 1 semialdehyde aminotransferase hemL hp_hemL_612 U00096 2 intl1 prob intl1 1 class 1 integron integrase AY260546 3 intl2 prob_intl2_11 class 2 integron integrase AY183453 1 siderophore receptor iron regulated outer ireA ireA_20 membrane protein AF320691 1 iroN iroN_10 outer membrane siderophore receptor AF449498 2 iss iss 10 increased serum survival AF042279 1 katP hp katP 611 peroxidase and catalase AB011549 2 hp tir 4051 6 611 hp tir MPEC 611 hp tir O103H2 611 hp tir O111 611 hp tir O157H45 611 hp tir O157H7 611 tir hp tir NTH19 611 translocated intimin receptor consensus E coli combined Assay User Guide 13 11 11 001 V1 40 www clondiag com www alere technologies com APPENDIX 3 TYPING INFORMATION Definitions amp Explanations The displayed result will yield following typing information e Discrimination of the 23 described O serotypes is mainly determined by the genes
18. 2 3 dfrA14 prob dfrA14 21 dihydrofolate reductase type 14 AJ313522 1 dfrA15 prob dfrA15 1 dihydrofolate reductase type 15 Z83311 1 dfrA17 prob dfrA17 11 dihydrofolate reductase type 17 AF169041 1 dfrA19 prob dfrA19 1 dihydrofolate reductase type 19 AJ310778 1 virulence factor adhesins eae consensus 10 eae consensus 20 eae consensus 30 eae consensus eae consensus 40 an outer membrane protein important for the attachment to host cells pathogenesis factor M58154 1 lymphocyte inhibitory factor A adherence factor efal hp_efa1_611 AF159462 2 fasA fasA 10 adhesin fimbrial major subunit M35257 1 fedA fedA 10 adhesin fimbrial major subunit M61713 1 fedF fedF 10 adhesin fimbrial protein Z26520 1 fim41a fim41a_10 adhesin fimbrial protein X14354 1 iha hp_iha_611 adherence conferring protein BA000007 2 chaperone protein required for the expression of nfaE nfaE_10 aggregative adherence fimbria Il S61968 1 EspB protein type Ill secretion system O157 H7 espB O157 espB O157 20 BA000007 2 espB O26 espB O26 40 EspB protein type Ill secretion system 26 H and O15 H AJ287768 1 saa hp saa 611 STEC autoagglutinating adhesin AF399919 3 E coli combined Assay User Guide 13 11 11 001 V1 37 www clondiag com www alere technologies com virulence factor fimbrae BfpA protein essential for apoptosis signalling
19. 28 C protect against direct sunlight Surplus 200 e C5 Washing Buffer 2 Store at 18 C to 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 E coli EDL933 GenBank accession number NC 002655 2 Cona 0 1 0 4 ug uL Store at 2 8 C Sufficient for 5 6 tests E coli combined Assay User Guide 13 11 11 001 V1 5 www clondiag com www alere technologies com Instrumentation amp Software e ArrayMate Reader to be ordered separately for details see below The E coli combined assay may be used on the ArrayMate reader only The alternative devices ATRO1 03 are not suitable for reading ArrayStrip based assays In case of any questions please contact us e conoclust software provided with the reader e Test specific software plug in can be downloaded from Alere Technologies CLONDIAG s website check periodically for updates for details see below Information such as spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is delivered with the reader or can be downloaded from our website These test specific plug ins will occasionally be updated Please check the NEWS section of our website http www alere technologies com Support is available via cct home clondiag com Components required but not provided e Growth media
20. 42 uL C4 150gL 350uL 700uL 1100 uL 1600 uL 2100 uL 3200 uL 4200 uL e Remove and discard Washing Buffer and add 100 ul diluted conjugate to each well incubate at 30 C 10min and 550rpm e Remove liquid and wash with 200 ul C5 Washing Buffer just pipette up and down four times remove and discard E coli combined Assay User Guide 13 11 11 001 V1 16 www clondiag com www alere technologies com e Add another 200 ul C5 Washing Buffer just pipette up and down four times 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 10 min but do not shake e Remove liquid completely e The bottom of the ArrayStrips may be cautiously be cleaned with wipes bubbles may be removed by removing and adding D1 e Scan and process see below Data Analysis Starting the ArrayMate Reader We recommend starting 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 main switch on the rear below the electric cable plug operating switch on the bottom left corner of the front side e Switch on the screen switch right hand below the screen e Log in as R amp D User Research and De
21. 611 class A beta lactamase AY219651 1 blaGIM 1 hp gim1 611 class B metallo beta lactamase AJ620678 1 class A metallo beta lactamase carbapenemase blalMI 3 hp imi3 611 associated with imipenem resistance AY780889 1 hp imp 611 hp imp 612 hp imp 613 hp imp 614 hp imp 615 hp imp 616 class B metallo beta lactamase consensus blalMP hp imp 617 associated with imipenem resistance blaKPC 4 hp kpc4 611 class A beta lactmase carbapenemase EU447304 1 blaLAP 1 hp lapi 611 class A beta lactamase EF026092 1 blaLEN 1 prob leni 11 class A beta lactamase AY743416 1 blaMOX prob mox 1pm class C beta lactamase blaMOX consensus oxacillinase class D beta lactamase blaOXA 1 blaOXA 1 prob oxa1 21 AY458016 1 oxacillinase class D beta lactamases blaOXA blaOXA 2 prob oxa2 11 2 blaOXA 15 U63835 1 oxacillinase class D beta lactamase blaOXA 7 blaOXA 7 prob oxa7 11 AY866525 1 oxacillinase class D beta lactamase blaOXA 9 blaOXA 9 prob oxa9 11 M55547 1 blaOXA 23 hp oxa 611 oxacillinase class D beta lactamase AJ132105 1 blaOXA 40 hp oxa 612 oxacillinase class D beta lactamase AF509241 1 oxacillinase class D carbapenem hydrolyzing beta blaOXA 48 hp oxa 613 lactamase AY236073 2 blaOXA 51 hp oxa 614 oxacillinase class D beta lactamase CP000863 1 blaOXA 54 hp oxa 615 oxacillinase class D beta lactamase AY500137 1 blaOXA 55 hp oxa 616 oxacillinase class D beta lactamase AY343493 1 blaOXA 58 hp oxa 617 oxacillinase
22. Additionally the microarray includes probes as an internal control for RNA contamination The automatic software analysis will give a failed if a RNA contamination occurred while DNA isolation DNA should not be prepared by disrupting E coli cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the E coli combined assay are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend following the protocols outlined below Please note To yield more genomic DNA we recommend the optional cell lysis step with A1 A2 reagent E coli combined Assay User Guide 13 11 11 001 V1 8 www clondiag com www alere technologies com Extraction of DNA by Spin Columns e g Qiagen e Add an inoculating loop full of monoclonal colony material of the E coli isolate to 0 2 ml 1xPBS and vortex thoroughly Loop empty Loop full It is important to harvest enough bacteria this is a prerequisite for extraction of a sufficient amount of DNA Take an inoculating loop of 1mm diameter filled with bacteria as shown in the right picture Optional cell lysis with A1 A2 reagent instead of 1xPBS e Centrifuge A2 tube shortly open it add 0 2 ml of Lysis Buffer A1 to Lysis Enhancer A2 and dissolve e Add an inoculating loop full of monoclonal colony material of the E coli isolate to this A1 A2 reagent and vortex thoro
23. C H19 fliC H19 11 flagellin C H antigen H19 AY250002 1 fliC H20 fliC H20 11 flagellin C H antigen H20 AY250003 1 flagellin C H antigen H21 DQ862122 1 Note If repressor fljA positive fliC H21 might be repressed fliC H21 fliC H21 11 H phenotype is than determined by flmA H54 fliC H23 fliC H23 11 flagellin C H antigen H23 AY250005 1 fliC H24 fliC H24 11 flagellin C H antigen H24 AY250006 1 fliC H25 fliC H25 11 flagellin C H antigen H25 ADUPO1000024 1 fliC H26 fliC H26 11 flagellin C H antigen H26 AY250008 1 fliC H27 fliC H27 11 flagellin C H antigen H27 CU928162 2 fliC H28 fliC H28 11 flagellin C H antigen H28 AY250010 1 fliC H29 fliC H29 11 flagellin C H antigen H29 AY250012 1 fliC H30 fliC H30 11 flagellin C H antigen H30 AY250011 1 fliC H31 fliC H31 11 flagellin C H antigen H31 AY250013 1 fliC H32 fliC H32 11 flagellin C H antigen H32 AY250014 1 fliC H33 fliC H33 11 flagellin C H antigen H33 AY250015 1 fliC H34 fliC H34 11 flagellin C H antigen H34 AY250016 1 fliC H37 fliC H37 11 flagellin C H antigen H37 AY250017 1 fliC H38 fliC H38 11 flagellin C H antigen H38 AY250018 1 fliC H39 fliC H39 11 flagellin C H antigen H39 AY250019 1 flagellin C H antigen H40 AJ865464 1 Note If repressor fljA positive fliC H40 might be repressed fliC H40 fl H40 11 H phenotype is than determined by flkA H53 E coli combined Assay User Guide 13 11 11 001 V1 32 www clondiag com www alere technologies c
24. Germany Contact If you require any further information on this product please contact cct home clondiag com E coli combined Assay User Guide 13 11 11 001 V1 27 www clondiag com www alere technologies com LITERATURE Anjum MF Mafura M Slickers P Ballmer K Kuhnert P et al 2007 Pathotyping Escherichia coli by using miniaturized DNA microarrays Appl Environ Microbiol 73 5692 5697 Ballmer K Korczak BM Kuhnert P Slickers P Ehricht R et al 2007 Fast DNA serotyping of Escherichia coli by use of an oligonucleotide microarray J Clin Microbiol 45 370 379 Geue L Schares S Mintel B Conraths FJ Muller E et al 2010 Rapid microarray based genotyping of enterohemorrhagic Escherichia coli serotype O156 H25 H Hnt isolates from cattle and clonal relationship analysis Appl Environ Microbiol 76 5510 5519 Korczak B Frey J Schrenzel J Pluschke G Pfister R et al 2005 Use of diagnostic microarrays for determination of virulence gene patterns of Escherichia coli K1 a major cause of neonatal meningitis J Clin Microbiol 43 1024 1031 Monecke S Mariani Kurkdjian P Bingen E Weill FX Baliere C et al 2011 Presence of enterohemorrhagic Escherichia coli ST678 0104 H4 in France prior to 2011 Appl Environ Microbiol 77 8784 8786 Schilling AK Hotzel H Methner U Sprague LD Schmoock G et al 2012 Zoonotic agents in small ruminants kept on city farms in southern Germany Appl Environ Microbiol 78 3785 3793
25. aboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents In case any liquids are spilled clean with disinfectant and or laboratory detergent and water Alere assumes no liability for damage resulting from handling or contact with these products If you have any questions please contact our Technical Support see above Shipping Precautions RID ADR Kein Gefahrgut No dangerous goods IMDG No dangerous goods IATA No dangerous goods E coli combined Assay User Guide 13 11 11 001 V1 3 www clondiag com www alere technologies com REAGENTS AND DEVICES Assay 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 user guide 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 have been tested for stability for short term shipment 1 week at ambient temperature 37 C The assay components with a rather limited stability are D1 and C3 The other components have proven to be stable even six months after the assay expiry date has passed Cell Lysis optional e A1 Lysis Buffer cat 245101000 Store at 18 28 C ambient temperature Surplus 5096 e A2 Lysis Enhanc
26. diag com www alere technologies com e Remove liquid and add 200 ul C2 Washing Buffer Incubate at 45 C 10 min and 550 rpm remove and discard e Add another 200 ul C2 Washing Buffer Incubate at 45 C 10 min and 550 rpm remove and discard e Meanwhile prepare conjugate for each experiment add 1 ul conjugate 100xHRP to 100 ul C4 Conjugation Buffer This mixture is stable for around one working day at room temperature C3 is delivered with a surplus of 100 C4 is delivered with a surplus of 200 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells C3 1 5 uL 3 5 uL 7 uL 11 uL 16 uL 21 uL 32 uL 42 uL CA 150uL 350uL 700uL 1100uL 1600 uL 2100 uL 3200 uL 4200 uL e Remove and discard Washing Buffer and add 100 ul diluted conjugate to each well incubate at 30 C 10min and 550rpm e Add 200 ul C5 Washing Buffer Incubate at 30 C 5 min and 550rpm e Remove and discard Washing Buffer add 100 ul of D1 substrate precipitating dye at 25 C see above per well e Incubate at 25 C 10 min but do not shake e Remove liquid completely e The bottom of the ArrayStrips may be cautiously be cleaned with wipes bubbles may be removed by removing and adding D1 e Scan and process see below Protocol for Eppendorf s Thermomixer Comfort with microtitre plate adapter e Switch on the thermoshaker and let it p
27. e with the ArrayStrip s e Press the button Next bottom right on the screen reader is closing Results On the left hand site 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 visible e Press the button Archive left hand 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 reading in this archive Browse 4 Search Ea ARCHIVE 2013 02 06 13 22 37 New Run Archive E coli combined Assay User Guide 13 11 11 001 V1 21 www clondiag com www alere technologies com 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 alphabetically ordered by Sample ID Browse Q search a 59 2013 09 25 8 E coli Bio DNA_gesamt_536_250913 9Fi New Run E coli Bio DNA_gesamt_536_EK_250913 E coli Bio DNA_gesamt_BL21 DE3_250913 pa E coli Bio DNA_gesamt_BL21 DE3_EK_250 E coli Bio DNA_gesamt_CFT073_250913 Archive E coli Bio DMA gesamt CFTO73 EK 25091 E coli Bio DNA gesamt EPEC E2348 69 2 E coli Bio DNA gesamt EPEC E2348 69 El E 2013 09 30 11 04 05 2013 10 07 11 01 33 fl 20 3 1n0 na 13 11 16 BZ Click on a Sample ID and the E coli c
28. er lyophilised cat 245102000 Store at 18 28 C ambient temperature Centrifuge A2 tubes shortly prior to opening Add 200 uL Buffer Al to Lysis Enhancer before use Mix well and store for less than 1 week at 2 8 C Sufficient for 96 isolations DNA Labelling and Amplification e B1 Labelling Buffer Store at 2 8 C Surplus 25 e B2 Labelling Enzyme Store at 2 8 C Surplus 50 e Ecoli PM1 lyophilized Labelling Primer Mix dilute in 50 ul molecular grade water Store at 20 C E coli combined Assay User Guide 13 11 11 001 V1 4 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 C to 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 surface of the microarray at the bottom of the well Do not store or handle unused wells at an air humidity of more than 6096 since this may irreversibly corrode the spots e CapStrips 24 strips e C1 Hybridisation Buffer Store at 18 28 C protect against sunlight Surplus 10096 e C2 Washing Buffer 1 Store at 18 C 28 C protect against direct sunlight Surplus 100 e C3 HRP Conjugate 100x Store at 2 8 C protect against direct sunlight Surplus 100 e C4 Conjugate Buffer Store at 18 C to
29. es GmbH L bstedter Stra e 103 105 D 07749 Jena Germany phone 49 0 36 41 3111 0 fax 49 0 36 41 3111 120 cct home clondiag com E coli combined Assay User Guide 13 11 11 001 V1 2 www clondiag com www alere technologies com Safety Precautions e The assay is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from pure E coli colonies clones requires expertise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed e Isolated cell free E coli DNA may be processed without further biosafety precautions although contamination with E coli or other bacteria needs to be ruled out e 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 amendments to the European Union Directives 67 548 EC and 1999 45 EC the enclosed reagents do not require a Material Safety Data Sheet MSDS They do not contain more than 196 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 l
30. ick on the Ok button data are exported now into the new folder on your memory stick e Do NOT remove the memory stick as long as the hourglass symbol is visible e Switch off the device by clicking on the Power button left down on the screen D e Switch off the Screen There is no need to physically switch off the ArrayMate E coli combined Assay User Guide 13 11 11 001 V1 23 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 in the appropriate way In case of trouble we are always happy to support Please contact cct home clondiag com and please include a description of the problem as well as the array images bmp files in question Staining Control A staining control is included to check whether possible problems originate from the hybridisation or the staining procedure If the staining control has Failed proceed as follows Horseradish peroxidase conjugate may have degraded during storage Add 1uL 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
31. in labelled ssDNA is transferred and hybridised to DNA oligonucleotide microarrays with 393 probes for different genetic markers and a biotin staining control Probes for serogenotyping are printed once whereas probes for antimicrobial resistance AMR and virulence genes are printed in two spots Targets include a variety of species and serotyping markers including genes that code for 23 different O antigens and 47 H antigens Additionally 102 probes targeting AMR genes as well as 88 probes for virulence genes were included Spot recognition is performed automatically based on a digital image of the arrays and results are given as an html file with a description of each analysed target E coli combined Assay User Guide 13 11 11 001 V1 1 www clondiag com www alere technologies com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not Intended for Use in Clinical Diagnostics This assay allows genotypic characterisation of E coli isolates for research and epidemiological applications It must not be used as a substitute for phenotypic susceptibility tests and for the guidance of antibiotic therapy Specifications Upon receipt the assay components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 C to 28 C Technical Support If you require any further information on this product please contact Alere Technologi
32. londiag com www alere technologies com array and the wall of the reagent well If you touch the array surface probes may be scratched off and this may cause an error Pasteur pipette plastic with a flexible tip A T flexible tip oN Pipette use the cavity between array and the wall of the tube do never touch the array Array General Remarks the Substrate Precipitating Dye D1 An appropriate amount of substrate precipitating dye should be filled into an Eppendorf tube and taken out of the refrigerator when starting the procedure allowing it to pre warm to room temperature 25 C Cold D1 may yield weak signals D1 should shortly be centrifuged prior to use to remove bubbles as well as possible precipitates Triggered by peroxidase in case of positive reactions the dye precipitates but it is not covalently bound The precipitate can be dissolved by vigorous shaking Thus the arrays must not be shaken dropped or moved abruptly during the staining procedure and afterwards E coli combined Assay User Guide 13 11 11 001 V1 13 www clondiag com www alere technologies com After completion of staining remove and discard reagent D1 as completely as possible and scan immediately The dye precipitate fades slowly in presence of liquids General Remarks Thermoshakers The correct temperature within the vessels is essential therefore always use the appropriate equipment for heating Because of a possibly i
33. mase blaACC 1 blaACC blaACC prob acc2 11 prob acci 11 2 EF554600 1 blaACT prob acti 11 class C beta lactamase blaACT 1 U58495 2 consensus sequence for blaCMY 13 blaCMY 2 blaCMY 24 blaCMY 35 blaCMY CFE1 blaCMY CFE2 Citrobacter spp blaCMY Cmur Citrobacter murliniae blaCMY Cwer Citrobacter werkmanii hp blaCMY 611 blaCMY Cyou Citrobacter youngae blaCMY HG3 blaCMY hp_blaCMY_612 prob_cmy_11 blaCMY HG4 blaKHM hp blaKHM 1 611 class B metallo beta lactamase AB364006 1 hp blaMOX CMYO9 611 class C extended spectrum beta lactamase precursor hp blaMOX CMYO9 612 associated with resistance to cephalosporins blaMOX CMY9 hp blaMOX CMY9 613 AF381617 1 blaCTX M1 blaCTX M15 prob ctxM1 11 prob ctxM1 12 class A extended spectrum beta lactamase X92506 1 including blaCTX M15 HQ202266 1 blaCTX M2 blaCTX M8 blaCTX M26 prob_ctxM2_11 prob_ctxM26_11 prob_ctxM8_11 class A extended spectrum beta lactamase AMO40709 1 AF518567 2 AY750914 2 ctxM9 prob_ctxM9_11 prob_ctxM9_12 class A beta lactamase AF174129 3 E coli combined Assay User Guide 13 11 11 001 V1 34 www clondiag com www alere technologies com blaDHA 1 prob dha1 1 class C beta lactamase EF406115 1 blaFOX prob fox 11 prob mox 1mm class C beta lactamase blaFOX consensus blaGES 1 hp ges 1
34. n 0 1 ug ul The concentration might be increased by heating and evaporation of water or by using a speed vac centrifuge 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 96 e Prepare a Master Mix by combining 3 9 uL of B1 labelling reagent 1 ul Ecoli PM1 Labelling Primer Mix and 0 1 uL of B2 DNA polymerase per sample e Add 5 ul of E coli DNA cona 0 1 0 4 ug ul prepared as described above to 5 ul of the Master Mix B1 B2 Do not forget to label the vial e Perform amplification in a pre programmed thermocycler e g Eppendorf Mastercycler gradient with heated lid VWR cat No 460 0108 according to following protocol Pre heat cover lid to 105 C 300 sec at 96 C 20 sec at 50 C 50 cycles with 40 sec at 72 C 60 sec at 96 C Cool down to 4 C hold e thesamples can be stored frozen until usage Please note When using another device some adaptations might be necessary Before starting routine use please test the protocol with a few known reference strains E coli combined Assay User Guide 13 11 11 001 V1 11 www clondiag com www alere technologies com Hybridisation General Remarks Handling of Arrays e Never touch the array surface e Avoid complete drying of the array surface during processing e Do not allow it to stay without liquid for more than two minu
35. nhomogeneous 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 and Eppendorf s Thermomixer Comfort equipped with a heating block for microtitre plates When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference strains or the control DNA E coli EDL933 Protocol for Quantifoil s BioShake iQ e Switch on the thermoshaker and let it pre heat to 50 C e Remove the ArrayStrip s from the pouch e Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode close to row A and proper fit e 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 150 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 coli combined Assay User Guide 13 11 11 001 V1 14 www clon
36. ob_aac6lb 1 M21682 1 aminoglycoside adenyltransferase associated with resistance to streptomycin spectinomycin aadA1 prob aadA1 1 EU704128 1 aminoglycoside adenyltransferase associated with resistance to streptomycin spectinomycin aadA2 prob aadA2 1 EU704128 1 aminoglycoside adenyltransferase associated with aadA4 prob_aadA4 1 resistance to streptomycin spectinomycin Z50802 3 aadB hp aadB 611 hp aadB 2 611 2 aminoglycoside nucleotidyltransferase L06418 4 aminoglycoside 2 adenylyltransferase associated with resistance to dibekacin gentamicin kanamycin ant2 prob ant2la 1 sisomicin tobramycin L06418 4 hp aphA 611 hp aphA aminoglycoside 3 phosphotransferase kanamycin aphA var4 611 hp aphA var5 611 resistance protein AY260546 3 16S rRNA methylase associated with gentamicin grm hp grm 611 resistance M55521 1 16S rRNA methylase associated with aminoglycoside armA hp armA 611 resistance AB117519 1 16S rRNA methylase associated with aminoglycoside rmtA hp rmtA 611 resistance AB083212 2 16S rRNA methylase associated with aminoglycoside rmtB hp rmtB 611 resistance DQ345788 1 16S rRNA methylase associated with aminoglycoside rmtC hp rmtC 611 resistance AB194779 2 16S rRNA methylase associated with aminoglycoside rmtD hp rmtD 611 resistance DQ914960 2 16S rRNA methylase associated with aminoglycoside npmA hp npmA 611 resistance AB261016 1 beta lactam resistance class C beta lacta
37. om fliC H41 fliC HA1 11 flagellin C H antigen H41 AY250020 1 fliC H42 fliC HA2 11 flagellin C H antigen H42 AY250021 1 fliC H43 fliC HA3 11 flagellin C H antigen H43 AY250022 1 fliC H45 fliC HA5 11 flagellin C H antigen H45 AY250023 1 fliC H46 fliC H46_11 fliC H46_12 flagellin C H antigen H46 AY250024 1 fliC H48 fliC H48_11 flagellin C H antigen H48 AY250025 1 fliC H49 fliC H49_11 flagellin C H antigen H49 AY250026 1 fliC H51 fliC H51 11 flagellin C H antigen H51 AY250027 1 fliC H52 fliC H52 11 flagellin C H antigen H52 AY250028 1 flagellin A H antigen H53 AB128917 1 Note If repressor FIjA positive the gene fliC H40 is flkA H53 flkA H53 11 repressed H phenotype is H53 flagellin A H antigen H54 AB128918 1 Note If repressor FIjA positive the gene fliC H21 is flmA H54 flmA H54 11 repressed H phenotype is H54 fliC H56 fliC H56 11 flagellin C H antigen H56 AY250029 1 H serotyping additional marker fliC Marker fliC 5p 11 fliC 5p 12 fliC 5p 13 fliC 5p 14 fliC 5p 15 fliC marker consensus sequence for all fliC genes fljA Repressor fIjA_11 FIJA represses the expression of fliC genes if positive the genes flkA HO3 or H53 or flmA H54 represent the H serotype AB128916 1 fliC non motile fl H NM 11 flagellin C from non motile isolates AY337480 1 streptogramin A resistance vatE hp vatE 611 hp vatE 612
38. ombined Assay User Guide 13 11 11 001 V1 22 www clondiag com www alere technologies com e a second image file png in which the coordinate grid is superimposed and the recognised spots are circled and e a xml files that contains the same information as the html result sheets for future export into databases etc Please note only complete runs can be exported The export of individual E coli combined Test Reports is not possible e Right Click on the reading a menu appears with the option Export Run Reports e Right Click on Export Run Reports a file browser opens m Browse Search a ARCHIVE 2009 02 05 09 00 42 expe sample ID order37x order37x23 order37x order37x78 order37x order37x82 ouderdax order42x01 Browse For Folder PR order42x02 4 order42x03 ve Order42x06 Order42x07 s order78x01 ve Order78x01 4 Order78x08 New Run dE O Power Choose a directory 2 Ba E o 3 E D B My Documents a E My Computer Se Local Disk C gt Se Local Disk D E e Removable Disk E o t ES Folder My Computer e Click on My Computer then on Removable Disk and choose the folder where to store or click on the button Make New Folder on the bottom a new folder icon appears e Rename the new folder e g with the experiment name or date e Cl
39. ombined Test Report for this array is shown in the window on the right Browse A Search results results b raw data segmentation image image l a print is ges E For Investigational Use Only Not Intended for Use in Clinical Diagnostics New Run dini Operator ines E coli Bio DNA gesamt BL21 DE3 250913 pl E coli Bio DNA gesamt BL21 DE3 EK 250 Sample ID E coli Bio DNA gesamt 536 250913 E coli Bio DNA gesamt CFTO73 250913 Experiment ID E coli Bio DNA gesamt 536 250913 Archive E coli Bio DNA gesamt CFTO73 EK 25091 9F8F15A6 CBCCAAE9 BA4BC 63D33DE159D 1 E coli Bio DNA gesamt EPEC E2348 69 250913 6AE4231C BAS9 407D AE43 0332B26B009F E coli Bio DN gesamt EPEC E2348 69 FI 9 25 13 39 36 2013 2013 09 30 11 04 05 Assay Name E coli amp 2013 10 07 11 01 33 Assay ID 10313 amp 2013 10 09 13 11 16 des 8 2013 10 16 09 33 57 Well Position 06 01 F E 2013 10 17 12 54 00 Software Version 2013 07 01 E 2013 10 18 09 58 14 Device 0420023 2013 10 18 10 40 52 _ Export of E coli combined Test Reports The generated result files in an html format will show information of all target genes Possible invalid controls that might display in this report will be explained below see Troubleshooting Other files that are generated and that can be exported include e a txt file with the raw measurements e animage file bmp with the actual photo of the array E coli c
40. phenicol acetyltransferase type III catlll prob_catlll_1 AJ249249 1 cmlA1 prob_cmlA1_11 chloramphenicol transporter EF113389 1 floR prob_floR_11 florfenicol export protein AF252855 1 erythromycin resistance ereA hp ereA 611 hp ereA 612 type erythromycin resistance AY183453 1 ereB hp_ereB_611 hp_ereB_612 type Il erythromycin resistance AB207867 1 macrolide resistance rRNA adenine N 6 methyltransferase lincosamide and ermB hp_ermB_611 hp_ermB_612 streptogramin B resistance protein AB089505 1 hp mph2 611 hp mphA 611 hp mphB 611 hp mphBM 611 mphA hp mphD 611 macrolide 2 phosphotransferase EF102240 1 member of macrolide inactivation gene cluster mphA mrx hp mrx 611 mrx mphR AB038042 1 quinolione resistance QepA fluoroquinolone quinolone efflux pump qepA hp qepA 611 AM886293 1 quinolone or fluoroquinolone resistance protein qnrA1 prob qnr 11 prob qnr 12 AY931018 1 quinolone or fluoroquinolone resistance protein qnrB prob qnrB 11 prob qnrB 12 AB281054 1 quinolone or fluoroquinolone resistance protein qnrD hp qnrD 611 FJ228229 1 quinolone or fluoroquinolone resistance protein qnrS prob qnrS 11 AM234722 1 rifampin resistance hp_arr 1_611 hp_arr 4_611 ADP ribosyltransferase associated with resistance to arr hp_arr 5_611 hp_arr 6_611 rifampin AF078527 1 streptomycin resistance sph hp_sph_611 stre
41. ptomycin 3 phosphotransferase U00004 1 streptomycin resistance sul1 prob sul1 11 dihydropteroate synthetase type 1 AJ698325 1 sul2 prob sul2 11 dihydropteroate synthetase type 2 DQ464881 1 sul3 prob sul3 11 dihydropteroate synthetase type 3 AJ459418 2 E coli combined Assay User Guide 13 11 11 001 V1 36 www clondiag com www alere technologies com tetracycline resistance tet37 hp tet37 611 tetracycline resistance protein AF540889 1 tetracycline resistance protein A tetracycline efflux tetA prob tetA 11 protein CP000971 1 tetB prob tetB 11 tetracycline resistance protein A class B V00611 1 tetC prob tetC 11 tetracycline resistance protein A class C EU751612 1 tetD prob tetD 1 tetracycline resistance protein A class D X65876 1 tetE prob tetE 11 tetracycline resistance protein A class E L06940 1 tetG prob tetG 11 prob tetG 12 tetracycline resistance protein A class G AF261825 2 tetX hp tetX 611 tetracycline resistance protein M37699 1 trimethoprim resistance dfrA1 prob dfrA1 21 prob dfrA1 22 dihydrofolate reductase type 1 AJ884723 1 dfrA5 prob dfrV 21 dihydrofolate reductase type 5 AB188269 1 dihydrofolate reductase type 7 AB161450 1 dfrA7 prob dfrA7 11 prob dfrA7 12 AM237806 1 dfrA12 prob dfr12 11 dihydrofolate reductase type 12 AB154407 1 dihydrofolate reductase type 13 synonym A21 dfrA13 prob dfr13 11 25080
42. re heat to 55 C e Remove the ArrayStrip s from the pouch E coli combined Assay User Guide 13 11 11 001 V1 15 www clondiag com www alere technologies com e Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode 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 just pipette up and down four times remove and discard e Second C1 Hybridisation Buffer 150 ul per well at 55 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 55 C 60 min and 550 rpm e Remove liquid and add 200 ul C2 Washing Buffer Incubate at 40 C 12 min and 550 rpm remove and discard e Add another 200 ul C2 Washing Buffer Incubate at 40 C 12 min and 550 rpm remove and discard e Meanwhile prepare conjugate for each experiment add 1 ul conjugate 100xHRP to 100 ul C4 Conjugation Buffer This mixture is stable for around one working day at room temperature C3 is delivered with a surplus of 10096 C4 is delivered with a surplus of 20096 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells C3 1 5 uL 3 5 uL 7 uL 11 uL 16 uL 21 uL 32 uL
43. rt E coli combined Test Reports afterwards e Press the button next bottom right on the screen reader is opening 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 barcode on the adapter and proper fit otherwise the images may be out of focus ArrayStrip frame with inserted strips Strips are inserted in accordance to the worklist Please note ArrayStrips must be clean They should not contain any liquids by now Barcodes must be clean There must be no lids on the wells that are to be analysed however unused wells should remain capped e Press the button Next bottom right on the screen reader is closing 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 E coli combined Assay User Guide 13 11 11 001 V1 20 www clondiag com www alere technologies com 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 e Remove the white fram
44. secretion system AF311901 1 hp espF O103H2 611 espF O103H2 hp espF O103H2 612 EspF effector protein type Ill secretion system AJ277443 1 Espl non LEE encoded effector protein LEE EPEC espl hp_espl_611 Locus of Enterocyte Effacement AJ278144 1 EspJ non LEE encoded effector protein LEE EPEC espJ hp espJ 611 hp espJ 612 Locus of Enterocyte Effacement AE005174 2 EtpD type II secretion pathway related protein etpD hp etpD 611 AF074613 1 hp nleA 611 hp nleA 612 NleA non LEE encoded effector protein A type III nleA hp nleA 613 hp nleA 614 secretion system AM421997 1 NleB non LEE encoded effector protein B type III nleB hp nleB 611 secretion system BA000007 2 NleB non LEE encoded effector protein B type III secretion system associated with serotype O157 H7 nleB O157 H7 hp nleB O157H7 611 BA000007 2 E coli combined Assay User Guide 13 11 11 001 V1 38 www clondiag com www alere technologies com nleB Salmonella hp nleB Styp 611 NleB non LEE encoded effector protein B type III secretion system associated with Salmonella enterica AE008894 1 NleC non LEE encoded effector protein C type III nleC hp nleC 611 secretion system AY485823 1 Tir cytoskeleton coupling protein type Ill secretion tccP hp tccP 611 hp tccP 612 system AB275113 1 virulence factor SPATE serin protease autotransporters
45. sed to detect the following antimicrobial resistance and virulence genes see Appendix 2 Probe to Target Table E coli combined Assay User Guide 13 11 11 001 V1 41
46. sonal computer for instance related to the use of additional software to network connections or to a breach of privacy related to the storage of confidential information such as names of patients from whom E coli was isolated on its hard disk and or to the use of external storage devices that might be contaminated with spyware E coli combined Assay User Guide 13 11 11 001 V1 26 www clondiag com www alere technologies com Quality Control Each batch is stringently tested with the use of standard E coli DNA preparations for good performance and correctness of results List of Components for Separate Order If required these reagents for the E coli combined Assay may be ordered separately component name Category au storage Lysis Enhancer lyophylised enzymes 245102000 18 28 Ecoli PM1 PrimerMix Labelling Primer 50 ul tube on request E coli_ combined E coli gesamt o o o o o DE o o o Conjugate Buffer buffered reagents 245108000 18 28 Washing Buffer 2 120 ml 245109000 18 28 HRP Substrate buffered reagents 245110000 2 Control Material E coli EDL933 DNA Cona 30 ul on request 2 0 1 0 4 ug ul ArrayStrips E coli combined AS 2 plugged microarrays 12 St 240008929 15 28 E coli gesamt AS 2 o E o O o p 2 8 2 8 2 8 2 8 8 8 i o o O o E For pricing please contact us Legal Manufacturer Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena
47. spec dnaE hp dnaE 612 hp dnaE 613 U00096 2 16S rRNA genetic marker for species E coli rrs hp rrs 611 hp rrs 612 U00096 2 O serotyping 04 wzy O4 11 O antigen O4 U39042 1 O6 wzx O6 11 wzy O6 11 O antigen O6 AJ426045 2 07 wzx O7 11 wzy O7 11 O antigen O7 AB490074 1 O8 wzx O8 11 wzy O8 11 O antigen O8 AF013583 1 O antigen O9 wbdA O9 D43637 1 wzx O9 09 wzx 09 11 wzy 09 11 AF104912 2 wzy_09 AB031867 1 015 wzx O15 11 wzy O15 11 O antigen O15 CP002291 1 026 wzx O26 11 wzy O26 11 O antigen O26 AF529080 1 052 wzm O52 11 O antigen O52 AY528413 1 053 wzy O53 11 O antigen O53 AF402312 1 055 wzx 055_11 wzy 055_11 O antigen 055 AF461121 1 079 wbdU O79 11 wzx O79 11 O antigen O79 EU294162 1 O86 wzx O86 11 wzy O86 11 O antigen O86 AY670704 1 091 wzx O91 11 wzy O91 11 O antigen O91 AYO35396 1 0101 wz O101 11 wbdA O9 11 O antigen O101 X59852 1 0103 wzx O103 11 wzy O103 11 O antigen O103 AY532664 1 0104 wzx O104 11 wzy O104 11 O antigen 0104 AF361371 1 wbdH O111 11 wbdM O111 11 0111 wzx 0111 11 wzy 0111 11 O antigen 0111 AF078736 1 0113 wzx 0113_11 wzy 0113_11 O antigen 0113 AF172324 1 0114 wzx 0114 11 wzy 0114 11 O antigen 0114 AY573377 1 0121 wzx 0121 11 wzy 0121 11 O antigen 0121 AY208937 1 0128 wzx O128 11 wzy O128 11 O antigen O128 AY217096 1 0157 rfbE O157 11 wzx 0157_11 O antigen 0157 AB008676 1 0172 wzx 0172_1
48. st three columns that have headers written into the first line The following headers are obligatory in this order position sample ID assay ID Table 1 e Positions are continuously numbered from 1 to maximal 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 are strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patient s names should not be used as Sample IDs e Assay IDs allow the system to identify the actual test and to correctly use information on layout spot number and identity etc E coli combined Assay has the Assay ID 10313 Assay ID numbers must not be confused as this could lead to errors or loss of data e You may add further columns and headers with notes and comments at your convenience Information from these columns will not appear on the result screens or the Test Report e Werecommend using a printout of the worklist as template for pipetting E coli combined Assay User Guide 13 11 11 001 V1 18 www clondiag com www alere technologies com 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 Table 1 Example worklist
49. tes e Never rinse the wells with distilled water after the hybridisation step use only C2 Washing Buffer Unused wells should be capped during the whole procedure The strips may be processed up to three times without a loss of quality of properly capped unused arrays Close all wells that will not be used with a cap und leave it there until you use these wells for storage conditions after use see section Assay 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 barcode This may cause an error Barcode label HERE keep it clean Y e Avoid contact of data matrix barcode with organic solvents The ArrayMate needs the information encoded in the data matrix to perform the assay and the analysis afterwards Avoid touching the bottom of the microarray strip and keep it clean General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent reservoirs Please keep in mind the limited surplus of C1 100 We strongly recommend that the liquid is removed by pipetting rather than by inverting the strips and flicking the liquids out Fine tipped soft disposable Pasteur pipettes are suited best such as BRANDT Cat No 612 2856 Always place the pipette tip at the cavity between the E coli combined Assay User Guide 13 11 11 001 V1 12 www c
50. ting block for microtitre plates e Pipettes suitable for 1uL 5uL volumes 90uL 100uL 200uL 1000uL e Multichannel Pipettes for 100 200 uL e Reaction vials suitable for PCR e Ultrapure PCR grade water e RNase A we recommend Qiagen s RNase A solution 100 mg mL Qiagen order 19101 e Pasteur pipettes BRANDT Cat No 612 2856 E coli combined Assay User Guide 13 11 11 001 V1 7 www clondiag com www alere technologies com PROTOCOL Culturing and Harvesting Bacterial Cells Members of the genus Escherichia are potential pathogens All procedures for cultivation of the bacterium and DNA preparation need to be performed by properly trained staff in a biosafety level 2 facility Grow E coli on 2xTY or Columbia Blood agar overnight at 37 C or 48 hrs at room temperature Obtain confirmation of the identification as E coli API VITEK MALDI and make sure that you have a pure monoclonal culture of E coli Contamination with other bacteria especially with other Enterobacteriaceae needs to be strictly avoided as the might carry the same resistance genes as certain E coli strains and thus can introduce false positive signals and patterns Extraction of DNA The required sample type for the E coli combined assay is 0 5 2 ug Cona 0 1 0 4 ug ul of intact genomic DNA from a single clone The DNA specimen needs to be free of RNA and it should not be fragmented This can be determined by agarose gel electrophoresis
51. type 1 CP000243 1 hlyA hlyA 20 hemolysin A AB011549 2 hlyE hlyE_10 hemolysin E AF052225 1 ipaD ipaD_10 IpaD invasin CP000035 1 ipaH ipaH9 8 20 IpaH invasion plasmid antigen CP000039 1 ItcA ItcA 20 heat labile enterotoxin subunit A AB011677 1 mchB mchB 10 microcin H47 activity protein AJ515252 1 mchC mchC 20 member of the microcin operon AJ515252 1 mchF mchF 10 putative microcin L transport protein AJ515252 1 mcmA mcmA 10 microcin M truncated protein AJ515252 1 pet pet 20 enterotoxin SPATE AF056581 1 sat hp sat 611 Sat serine protease SPATE AJ586888 1 E coli combined Assay User Guide 13 11 11 001 V1 39 www clondiag com www alere technologies com senB senB 20 enterotoxin 254195 1 stal sta1_110 heat stable enterotoxin AJ555214 1 sta2 sta2_210 heat stable enterotoxin Il CP000795 1 heat stable enterotoxin Stb enterotoxin B stb stb_10 M35729 1 stx1 consensus stx1A_10 shiga toxin 1 hp_stxA2_611 hp_stxA2_613 hp_stxA2_614 hp_stxA2_615 hp_stxA2_616 hp_stxA2_617 hp_stxA2_618 stx2A_10 hp_stxB2_612 hp_stxB2_613 hp_stxB2_614 hp_stxB2_615 stx2 consensus shiga toxin 2 NOTE variant classification by Scheutz et al 2012 stx2b hp_stxB2_612 shiga toxin 2 variant b AB012101 1 stx2e hp_stxA2_616 shiga toxin 2 variant e X81415 1 stx2f hp stxA2 611 hp_stxA2_613 shiga toxin 2 variant f AJ270998 1 stx2g hp stxA2 6
52. ughly e Incubate the colony material of the E coli isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker e Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit that is as follows e Add 20 ul proteinase K Qiagen Kit or equivalent and add 200 uL buffer AL Qiagen Kit e Vortex shortly or shake vigorously e Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker e important if A1 A2 reagent not used add now 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing e Add 200 ul ethanol 96 100 e Vortex the sample and centrifuge shortly E coli combined Assay User Guide 13 11 11 001 V1 9 www clondiag com www alere technologies com e Transfer the complete content of the tube including any precipitate into a spin column that is placed in a 2 ml collection tube e Centrifuge 8000 rpm 1 min at room temperature time and speed need to be determined depending on viscosity of the sample and type of centrifuge used All liquid should be collected in the collection tube afterwards e Discard collection tube with liquids e Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 ul Buffer AW1 e Centrifuge 8 000 rpm 1 min at room temperature e Discard collection tube with liquids e Place the spin column in a new 2 ml collection tube provided with the kit
53. velopment User for full access to test specific software a default password will be provided together with the ArrayMate device If you log in as User you will obtain only raw values but no interpretation as positives negatives and no strain assignment Administrator log in will allow manipulation of file folders and software and this should be done only upon direct advice of Clondiag s IT team E coli combined Assay User Guide 13 11 11 001 V1 17 www clondiag com www alere technologies com e The user interface will be loaded ArrayMate performs internal testing This will require slightly less than a minute e Click on the icon New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience e Type in your operator ID optional e You may optionally enter a comment into the memo field Worklist A Worklist file allows linking an identifier such as a laboratory sample number to a position of an array within the ArrayStrip For privacy reasons arrays should not be identified by patient names Worklists can be generated using spreadsheet software such as EXCEL see below but 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 lea
54. www clondiag com www alere technologies com User guide for the Escherichia coli combined Assay Array Hybridisation Assay for DNA based serogenotyping and detection of resistance and virulence genes of Escherichia coli For Research Use Only Not Intended for Use in Clinical Diagnostics www clondiag com www alere technologies com CONTENT BACKGROUND errata tM IPS 1 Intendea Use m 2 Specification S 2 acs EIUS 2 Technical SUPPO east 2 Safety Preca tions ccs eode e on bone veeiee aed daxeyaxdsaeiiailes ve nedeGeusaaddaveueeteasee 3 Material Safety Data Sheets MSDS ice en bn MAR ED RR RR RR 3 Shipping PACA Udo NORTE cm 3 REAGENTSAND DEVICES c I 4 Assay Components Storage and Stability un 4 Cell Lysis optional a d YR dx el god AN Rao rd vio eu aa dU EAE UA S 4 DNA Labelling and Amplifieation eii dot testen eee u AS AAA 4 Hybridisation and Detecta 5 Instrumentation amp Software au ern 6 Components required but not provided esses esee eene nnne nnns 6 PROTOCOL MNT eiai 8 Culturing and Harvesting Bacterial Cells ovina aa 8 Extraction OP DNA aan NR 8 Extraction of DNA by Spin Columns e g Qiagen ccoocccnoocccconcccnonnnononcnononnnononcncnonnncnonnncnnnnnos 9 Linear Amplification and Internal Biotin Labelling esses
55. wzy polymerase and wzx flippase The 47 known H antigens are encoded by the gene fliC e The probes immobilized on the current array version can discriminate 23 O antigens O 4 0 6 O 7 O 8 O 9 0 15 0 26 0 52 0 53 0 55 O 79 0 86 O 91 0 101 0 103 0 104 O 111 0 113 0 114 0 121 0 128 0 157 and 0 172 e The following flagellar antigens can be identified on the array H 01 H 02 H 03 H 04 H 05 H 06 H 07 H 08 H 09 H 10 H 11 H 12 H 14 H 15 H 16 H 18 H 19 H 20 H 21 H 23 H 24 H 25 H 26 H 27 H 28 H 29 H 30 H 31 H 32 H 33 H 34 H 37 H 38 H 39 H 40 H 41 H 42 H 43 H 45 H 46 H 46 H 48 H 49 H 51 H 52 H 53 H 54 H 56 e The mix culture control is based on 5 probes located in consensus sequences on the 5 end of the fliC genes These probes were divided in two groups which are correlated with two groups of fliC genes fliC 5bp 11 fliC 5bp 12 and fliC 5bp 13 fliC 5p 14 fliC bp 15 Only one group should be positive if not an E coli mix culture was probed e The gene fljA encodes for a repressor protein which repressed some of fliC genes If the fljA probe positive may two H serotype probes are positive In this case the genes f kA and flmA encodes for the H serotype Such strains are described as biphasic Wang et al 2003 Tominaga 2004 e Probes specifying gapA gad ihfA and dnaE that were introduced to confirm the identity of E coli and to serve as genus controls e Probes u
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