Manual C.diff-Type AS-1 Kit
Contents
1. 30 UD ret al 30 Quality Controls ETE Ada 31 List of Components for Separate Order 31 Legal banu UOT RE a baba Hal Dabo d r 31 Coco 31 EITERATURE 32 UPDATES AND SOFTWARE c da la 33 APPENDIX 1 pu oan Pepe der ee 34 APPENDIX 2 IMAGES FOR 5 1 35 APPENDIX 3 PROBE TO TARGET TABLE a aa 38 APPENDIX 4 TYPING INFORMATION sn nan aub de eue la dau ha uns 41 Defipitions ahnd Explanations 41 List of Currently recognized Strains ana a 42 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit www alere technologies com www alere com BACKGROUND Clostridium difficile is a component of the human colonic flora If the physiological bacterial flora in the colon is damaged by administration of antibiotics especially of clindamycin fluoroquinolones cephalosporins or amoxicillin clavulanic acid C difficile is able to multiply and to cause antibiotic associated diarrhoea and pseudomembranous colitis 1 Severe cases might progress to toxic megacolon and end fatally C difficile can cause antibiotic associated diarrhoea and a possibility of outb2. positive For Research Use Only Not For Use In Diagnostic Procedures Operator Sample ID Jena 0016 11 E55CD746 D060 4C75 9706 8524558DC191 16045 8 chip Experiment ID Jena 0016 11 E55CD746 D060 4C75 9706 85245580 191 16045 8 chip Date of Result Tue Jul 14 12 50 35 2015 Assay Name C diff Assay ID 16045 Well Position mE Software Version 2015 06 15 Device s StripID T MnesEVena 0016 11 E55CD746 D060 4C75 9706 8524558DC191 16045 8 chip Hybridisation Pattern Hybridisation pattern 35 Score 94 74 Clade IV Corresponding MLST type ST 37 ST 86 Corresponding ribotypes RT 017 Published genome sequences likely to be related CF5 FN665652 002 P50 2011 AGAA 050 P50 2011 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 23 www alere technologies com www alere com or similiar to this strain AGAB M68 FN668375 Experiment Validity passed SPECIES MARKER Marker classification explanation Da rennen oT bise m markers and controls Bacitracin resistance Bacitracin resistance proteinlocus1 locus 1 positive 8 Bacitracin ATP binding cassette transporter dive ABC protein BcrA probe hp1071 Bacitracin ATP binding cassette transporter ABC protein BcrA hp1072 8 Bacitracin ATP binding cassette transporter nase ABC EE en BerA hp1073 8 Lincomycin resist protein ImrBNAP07 hp1097 resist protein ImrBNAP07 hp1097 megti Lincomycin
3. 16 04 0019 VO1 Manual C diff Type AS 1 Kit 25 www alere technologies com www alere com ArrayMate EUR Browse Search results results b raw data segmentatior FI ARCHIVE New Run os Browse For Folder 4 01 01 0 Archive 01 01 01 G ie Desktop a My Documents s 2 Local Disk C S Local Disk D Se Removable Disk E Choose a directory Folder Computer Make New Folder OK Cancel 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 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 Do NOT remove the memory stick as long as the hourglass symbol is visible o Power e Switch off the device by clicking on the Power button left down on the screen Switch off the Screen There is no need to physically switch off the ArrayMate Reader TROUBLESHOOTING In case of trouble always make sure that reagents are within the recommended shelf life and stored under appropriate conditions Should you encounter a problem we will always be happy to support you Please e mail to cct home clondiag com and include a description of the problem as well as the array images bmp files in question Staining Control A staining c
4. 96 B3 Primermix dried Labelling Primermix two tubes dilute each primer mix in 70 ul molecular grade water Store at 20 C Surplus 50 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 4 www alere technologies com www alere com Hybridisation and Detection 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 in 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 StripCaps 24 units e C1 Hybridisation Buffer Store at 18 28 C protect against direct sunlight Surplus 100 96 2 Washing Buffer 1 Store at 18 28 C protect against direct sunlight Surplus 200 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 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 5 www alere technol
5. above Physical Damage to the Array Scratching the array surface with a pipette tip may damage array spots which may lead to the impairment or absence of a valid signal In this case the respective marker will not be assigned as negative but instead the message none appears next to the marker name Report Unavailable If the ArrayMate indicates that no report is available for an array or multiple arrays on one strip please check that the strip was positioned properly into the frame Scratches or drops of condensed water might render the Data Matrix code identifier unreadable please wipe it carefully or try to manually identify the test If no obvious reason for the fault can be discovered please contact the technical service Ambiguous Results Apart from a positive or negative result for the individual markers on C diff Type AS 1 Test Report the result can also be ambiguous In cases affecting resistance genes or virulence factors no definitive answer with regard to the presence of this specific marker than can be given This can be caused by poor sample quality poor signal quality and in case of some resistance associated genes by the presence of plasmids in low copy numbers Allelic variants of some markers differ only in single or few nucleotides This can cause the effect that the actual allele yields a positive signal while other mismatching probes give ambiguous rather than negative results C dif
6. labelled amplification product mix gently Remove the buffer from the well and add the mixture of C1 and labelled amplification product Incubate at 50 C 60 min at 550 rpm Meanwhile login to the ArrayMate device and prepare your worklist see section Data Analysis p 20 Remove the liquid and add 200 ul C2 Washing Buffer Incubate at 45 C 10 min at 550 rpm remove and discard C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 16 www alere technologies com www alere com Add another 200 ul C2 Washing Buffer Incubate at 45 C 10 min at 550 rpm Meanwhile prepare conjugate For each experiment add 1 ul C3 conjugate 100 x HRP to 99 ul C4 Conjugation Buffer This mixture is stable for one working day at room temperature C3 is delivered with a surplus of 100 96 C4 with a surplus of 200 96 Suggested pipetting scheme 1 2 3 4 6 7 10 11 15 16 20 21 30 31 40 well wells wells wells wells wells wells wells C3 1 1 5 ul 3 5ul 7ul 11 ul 16 ul 21 ul 32 ul 42 ul 46 CA 1 148 5 ul i 693 uL 1089 ul 1584 ul 2079 uL 3068 uL 4058 uL Remove the Washing Buffer and add 100 ul diluted conjugate to each well incubate at 30 C 10 min at 550 rpm Remove the conjugate C3 C4 add 200 ul C5 Washing Buffer Incubate at 30 C 5 min at 550 rpm Remove the Washing Buffer add 100 ul of D1 HRP substrate precipitating dye at 25 C see above per
7. resist protein ImrBNAP07 hp1096 positive Channel forming haemolysin b Efflux pump ydiC positive 8 Putative lantibiotic ABC transporter permease dtre protein hp1251 Putative lantibiotic ABC transporter permease nase protein hp1252 8 TOXINS Marker classification explanation Toxin detection positive Toxin A allele tcdA CF5 Toxin B detection positive Toxin B allele tcdB CF5 ANTIBIOTIC RESISTANCE Marker classification explanation cat negative chloramphenicol acetyltransferase ermB negative rRNA methyltransferase tetM negative tetracycline resistance protein sat hp1082 positive streptogramin A acetyltransferase sat hp1083 negative OTHER VIRULENCE GENES Marker classification explanation Binary Toxin cdtA B detection negative Binary Toxin Total 080 mann Toxin cdt A B allele tcdE tedE d p1113 positive 8 holin like pore holin like pore forming protein protein putative negative regulator of tedC hp1117 ambiguous pathogenicity locus C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 24 www alere technologies com www alere com Export of C diff Type AS 1 Kit Test Reports Two result files in htmL format will be generated The shorter report will gives a summary on clinically relevant genes virulence markers and genes associated to antibiotic resistance and typing information This includes the affiliation to strains with unique hybridisatio
8. troubleshooting p 37 39 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 RNA reduces the efficiency of amplification and labelling by effectively removing primer from the reaction mix due to competitive hybridisation Azeo readings will cover RNA and other contaminants as well Therefore pure DNA preparations without RNA contamination are prerequisite for proper DNA concentration measurement RNAse treatment prior to A260 reading therefore is necessary component A2 contains RNase DNA must be unfragmented as fragmentation reduces the efficiency of amplification and labelling due to the distance between primer and probe binding sites For this reason DNA should not be prepared by disrupting C difficile cells using bead beaters ultrasonication or C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 27 www alere technologies com www alere com aggressive chemicals such as in alkaline lysis protocols We gained positive 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 Primermix 5 10 min at 70 C Some problems with samples from the Qiagen EZ1 device for example were resolved after heating the samples see
9. 0 Strain 6503 ADEI v QCD 23m63 ABKL C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit ST 11 or related ST127 div s DAzsmes s DAssos tCdArz0291 tcdBeso CdtArz0291 cdtBm120
10. 1030 cdtB hp1031 cdtB hp1038 cdtB hp1039 cdtB hp1040 cdtB hp1041 cdtB hp1103 hly3 holin like pore forming protein hp1113 tcdE 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 38 www alere technologies com www alere com CATEGORY TARGET PROBE ID putative negative regulator of pathogenicity locus hp1117 tcdC CELL WALL PROTEINS C diff Type AS 1 Kit cellwall protein 66 hp1012 cwp66 hp1016 cwp66 hp1020 cwp66 hp1021 cwp66 cellwall protein 84 SplA Surface layer protein A 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit hp1018 cwp84 hp1150 slpA hp1151 slpA hp1153 slpA hp1154 slpA hp1155 slpA hp1156 slpA hp1158 slpA hp1164 slpA hp1166 slpA hp1167 slpA hp1168 slpA hp1169 slpA hp1170 slpA hp1171 slpA hp1173 slpA hp1174 slpA hp1175 slpA hp1176 slpA hp1177 slpA hp1178 slpA hp1180 slpA hp1182 slpA hp1183 sIpA hp1184 slpA hp1186 slpA hp1188 slpA hp1190 slpA hp1191 slpA hp1192 slpA hp1193 slpA hp1195 slpA hp1197 slpA hp1198 sIpA hp1199 slpA hp1200 slpA hp1201 slpA hp1202 slpA hp1203 sIpA hp1204 slpA hp1205 sIpA hp1206 sIpA hp1208 sIpA hp1209 slpA hp1210 slpA hp1211 slpA hp1232 slpA hp1233 slpA hp1234 slpA hp1236 slpA hp1237 slpA hp1239 slpA 39 www alere technologies com www alere com CATEGORY TARGET PROBE ID hp1242 slpA hp1243 slpA hp1249 slpA CELL DIVISON PROT septum formatio
11. 40009610 15 28 C StripCap StripCap 245112000 15 28 C For pricing please contact your local representative or our customer service respectively Legal Manufacturer Alere Technologies GmbH Loebstedter Str 103 105 07749 Jena Germany Contact If you require any further information on this product please e mail to cct home clondiag com C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 31 www alere technologies com www alere com LITERATURE Lubbert C John E von Muller L 2014 Clostridium difficile infection Dtsch Arztebl Int 111 43 723 731 Dingle KE Griffiths D Didelot X Evans J Vaughan A Kachrimanidou M Stoesser N Jolley KA Golubchik T Harding RM Peto TE Fawley W Walker AS Wilcox M Crook DW 2011 Clinical Clostridium difficile clonality and pathogenicity locus diversity PLoS ONE 6 5 e19993 Gawlik D Slickers P Engelmann I M ller E L ck C Friedrichs A Ehricht R Monecke S 2015 DNA Microarray based Genotyping of Clostridium difficile BMC Microbiol doi 10 1186 s12866 015 0489 2 PMID 26242247 http www biomedcentral com 1471 2180 15 158 For further literature please refer to http alere technologies com en science technologies publications downloads html C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 32 www alere technologies com www alere com UPDATES AND SOFTWARE Notifications on database software updates and freeware tools c
12. Aszo HP 06 l N A SA negative pattern tcaBeso cdtBeso B d 7 tcdApz0291 CdtAszo HP 07 1 slpArizgsa trunc RT 054 tcdBso cdtBesp s 7 RT 057 HP 08 l ST 55 SlpAnis RT 070 77 7 RT 094 630 630 RT 002 tcdAg202931 CdtAgso HP 09 108 SIpAr ssa RT 159 tcdBeao cdtBeso var tcdApz0291 CdtAs30 H P 10 m 1 5 tcdBs3o 1 7 7 7 HP 11 CD37 AHJJ I ST 03 s DAzames RT 009 var tcdApzo291 cdtA ayk HP 12 I N A SIP npo8162 RT 103 tcdBaso cdtBeso E 7 RT 014 tcdAg202931 CdtAgso HP 13 70 100 2010 AGAC I ST42 SIPAr12885 RTO 25 Bern z tcdApz0291 CdtAszo 1 DA RT 015 HP 14 SIPAkohn tcdBso edibus C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 42 www alere technologies com www alere com Hybridi Associated 27 Fully sequenced strains Associated DA allel 112 accession no Me 0 ribotypes SIpAkohn tcdArz0291 CdtAcso sipA Pf racas tcdBeso cdtBeso tcdArz0291 CdtAcso slpA IPA snoos041 tcdBe3o cdtBe30 tcdArz0291 CdtAcso IpA SIP MRYos0211 tcdBs3o cdtBe30 tcdAg20201 SIpA trunc P 9952 tcdBeso tcdArz0291 CdtAsao slpA tcdBeso cdtBeso SIPAri3711 tcdArz0291 CdtAcso slpAri3711 tcdBeao cdtBeso tcdArzo291t CdtAcso s pA PARIS tcdBeso cdtBeso S
13. Since resistances might be caused by genes or mutations not covered by this array or by hitherto unknown genes or mutations any antibiotic chemotherapy MUST be guided by phenotypic susceptibility tests Furthermore we do not accept any liability for damages caused by inappropriate use of the device as a personal computer for instance related to the use of additional software to network connections or to a breach of privacy related to the storage of confidential information such as names of patients from whom C difficile was isolated on its hard disk and or to the use of external storage devices that might be contaminated with spyware C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 30 www alere technologies com www alere com Quality Control Each batch is stringently tested for good performance and correctness of results using standard C difficile DNA preparations List of Components for Separate Order If required these reagents for the C diff Type AS 1 Kit may be ordered separately component name Tamoune n storage Br labeling Buffer 0 st 24820000 24 52 ftabelingenme fow 24510400 are ci Hybridisation buffer fomi 245105000 18 28 C 2 Washing ufert_ rom 245106000 1828 cs p onhs eiox aw 24510700 ave a Er G 25 IE 2 8 C ev oaoa soni 2461100 are 1 2 B1 B2 B3 C1 C2 C3 4 C5 D M ArrayStrip C diff Type AS 1 2
14. an be found at http alere technologies com en products lab solutions clostridium difficile html and or http alere technologies com en news html Currently available freeware programs are Alere Result Collector for the conversion of multiple result xml files from the ArrayMate into spreadsheet tables This should make it easier to compare isolates or to determine relative abundances of genes or strains etc e Alere Worklist Generator is a tool which helps you to create a well formatted worklist for the Arraymate Alere Report Generator is a software tool to create reports using the assay software normally used and installed on the ArrayMate It uses an image taken by the ArrayMate or a txt file raw signal data file and generates a report from the raw signal data C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 33 www alere technologies com www alere 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 prepare ArrayStripes prepare DNA cessing on time time grow CLONAL C diff isolate over 5 min not part of the kit night isolate DNA 3 4 h 10 40 min not part of the kit label RNA free DNA in thermocycler 5 YT 2 3h 5 min rinse ArrayStrips 5 ul DNA ca 0 1 0 4 ug uL 200 uL w
15. arkers for isolates within one HP i e for outbreak investigations The list of recognized Hybridization Profiles can be expanded and software updates will likely be provided on an annual basis If you find new Hybridization Profiles if you have array images of a strain not yet covered or if you have additional information on strain you wish to be included please contact stefan monecke clondiag com Clade affiliation as determined by overall profile Clades were defined by Dingle 2011 2 in order as a taxonomic unit that includes related sequence types Allelic variants of several markers depend on Clade affiliation so that the Clade of an unknown isolate can be identified without actual sequencing e Sequence types associated with this strain Hybridization Profile 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 Qclondiag com Ribotypes associated with this strain This information is provided based on a database of isolates that have been typed in parallel by array and by ribotyping It is not directly derived from the actual experiment If you have results you want to be included please contact stefan monecke clondiag com C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 41 www alere technologies com
16. ater 50 C 550 rpm 5 min Mastermix 3 9 pL B1 0 1 uL B2 1 uL B3Cdi preparing labeled DNA to 10 uL of labeled DNA add 90 ul 5 min 5 min of Buffer C1 transfer 100 ul labeled DNA to ArrayStrips 5 min 5 min Barcode here hybridise 50 C 550 rpm 60 min 60 min O min meanwhile Login to the ArrayMate device and prepare your worklist discard labeled DNA 20 min 5 min incubate twice in 200 uL Buffer C2 45 C 550 rpm 10 min prepare C3 C4 conjugate C3 C4 1 100 preheat Substrate D1 25 C discard Buffer C2 10 min 2 min incubate in 100 ul C3 C4 conjugate 30 C 550 rpm 10 min discard C3 C4 conjugate 5 min 2 min incubate once in 200 uL Buffer C5 30 C 550 rpm 5 min discard Buffer C5 6 min 2 min incubate with 100 uL Substrate D1 25 C 6 min discard Substrate D1 analyse ArrayMate 15 min 10 min discard water 200 uL Buffer C1 50 C 550 rpm 5 min discard C1 process promptly total time requirement overnight app 60 min 7 8h C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 34 www alere technologies com www alere com APPENDIX 2 IMAGES FOR TROUBLESHOOTING Comment Result sheets A technically faultless valid experiment Valid results no error messages This image is poor This could be due to low DNA concentration fragmented DNA ethanol trace contaminations in DNA sample or expired reag
17. device If you log in as User you will obtain only raw values but neither positives negatives interpretation nor strain assignment The Administrator log in will allow the installation of a new assay specific plug in which can be downloaded at http alere technologies com see p 31 e The user interface will be loaded the ArrayMate performs internal testing It requires slightly less than a minute Click New Run left upper edge of the screen A suggestion for a run name folder name for the new run appears in the top line of the screen You may modify or change the experiment name at your convenience Type in your operator ID optional Worklist A Worklist file allows linking an identifier such as a laboratory or sample number to a position of an array within the ArrayStrip For privacy reasons arrays should not be identified by patient names Worklists can be generated using spreadsheet software such as EXCEL see below but C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 18 www alere technologies com www alere com must be saved in the txt file format that can be imported into the test specific ArrayMate software Do not use special characters such as etc e Create a list with at least three columns that have headers written in the first line The following headers are obligatory in this order position samplelD assayID Table 1 Positions are consecu
18. diff Type AS 1 Kit reports afterwards Check the flash drive regularly for computer viruses and malware using an appropriate program Press Next at the bottom right on the screen reader is opening Carefully insert the appropriate metallic adapter frame into the ArrayMate Do not apply strong force Ensure proper fit otherwise the images may be out of focus e Carefully insert the white frame with the array strips into the metallic adapter Ensure the correct orientation Position A1 in the frame next to the data matrix barcode on the adapter and proper fit otherwise the images may be out of focus ArrayStrip frame with inserted strips Strips are inserted in accordance with the Worklist Please note ArrayStrips must be clean They should not contain any liquids during analysis Data matrix codes must be clean There must be no Array StripCaps on the wells to be analysed however unused wells should remain capped Press Next at the bottom right on the screen reader closes analysis program starts it takes about 2 10 min depending on the number of strips the reader takes images and automatically analyses the data The progress of the reading is indicated by the following symbols photographed in analysis ready e The reader indicates the end of the entire process with an acoustic signal beep Press Next at the bottom right on the screen reader is opening Remove the white frame wit
19. e AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 8 www alere technologies com www alere com This can be determined by agarose gel electrophoresis DNA should not be prepared by disrupting C difficile cells using bead beaters ultrasonication or aggressive chemicals such as in alkaline lysis protocols Most performance problems with the C diff Type AS 1 Kit are due to insufficient amounts or quality of DNA preparation We therefore strongly recommend obeying the protocols outlined below DNA Extraction by Spin Columns e g Qiagen Centrifuge A2 tube shortly open it add 0 2 ml of Lysis Buffer A1 to Lysis Enhancer A2 and dissolve Add culture material of the C difficile isolate as described above to this A1 A2 reagent and vortex thoroughly Incubate the culture material of the C difficile isolate in A1 A2 for 30 60 min at 37 C and 550 rpm in the thermoshaker Proceed with the DNA preparation protocol of the DNA preparation kit For the Qiagen DNeasy Blood amp Tissue Kit it is as follows Add 25 ul proteinase K from Qiagen Kit or equivalent and add 200 ul buffer AL Qiagen Kit Vortex shortly or shake vigorously Incubate for 30 60 min at 56 C and 550 rpm in the thermoshaker Important If A1 A2 reagent is not used add now 4 ul RNase A 100 mg ml mix by vortexing and incubate for 2 min at room temperature before continuing Add 200 ul ethanol 96 100 96 Vortex the sample and centrifuge q
20. e again Discard the spin column Please note Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay Contamination with Washing Buffer might occur during the elution of prepared DNA by drops adhering to the spin columns funnels Therefore these funnels should be gently touched and dried with sterile filter paper or wipes prior to the elution step Alternatively prepared DNA can be heated shortly to evaporate ethanol e g 10 min at 70 C C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 10 www alere technologies com www alere com 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 Az59 method it should not be lower than 0 1 ug ul The concentration might be increased by heating and evaporating water or by using a speed vac centrifuge not recommended when the same preparation shall be used in PCR experiments DNA Extraction by 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 C difficile 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 migh
21. ents The experiment should be repeated with a new DNA preparation If this also fails try an experiment with control DNA CM The system will try to assign the isolate It might yield a putative identification than the closest matching Hybridisation Profile and the clade affiliation will be displayed If this is not possible and or if species markers are missed an error message will be displayed The signal intensity is low so that the image cannot reliably be analysed This could be due to low DNA concentration to fragmented DNA or presence of inhibiting substances It might also be that the DNA was obtained from an isolate of another species than C difficile Species other than C difficile tested or severe user error no DNA confusion of buffers wrong temperatures Check protocol Check species identification and if it was C difficile repeat with a new DNA preparation C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 35 www alere technologies com www alere com Contaminated mixed culture If mutually exclusive alleles of species markers are detected simultaneoulsly i e if clones from different Clades are present an error message will be displayed The experiment is considered invalid due to over staining contamination and or the presence of more than one clone of C difficile An assignment to a strain is not possible If two related clones fro
22. ermomixer Comfort equipped with a heating block for microtiter plates When using other devices some modifications to the protocol might be necessary Before starting routine use please test the protocol with a few known reference strains or the control DNA CM The difference between the protocols for Qlnstrument s BioShake iQ and Eppendorf s Thermomixer Comfort with microtiter plate adapter is only the washing temperature after the hybridisation step Please note The Quantifoil s BioShake iQ has no active cooling function Please use the second pre temperatured passive cooling block to reduce the incubation temperature quickly C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 15 www alere technologies com www alere com Protocol for Quantifoil s BioShake iQ BioShake iQ by Qlnstruments equipped with a customised heating block designed to fit ArrayStrips http www ginstruments com Switch on the thermoshaker and let it pre heat to 50 C Remove the amount of ArrayStrip s needed from the pouch Insert the ArrayStrip s into the white frame Assure the correct orientation data matrix barcode close to row A and proper fit Pre wash the array s in two steps e First PCR grade distilled water 200 ul per well at 50 C 5 min at 550 rpm Remove the water from the well e Second C1 Hybridisation Buffer 200 ul per well at 50 C 5 min at 550 rpm Add 90 ul of C1 buffer to each tube with 10 ul
23. es and it is compared to a database of strain profiles allowing assignment to clonal complexes and strains C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 1 www alere technologies com www alere com GENERAL INSTRUCTIONS FOR USE Intended Use For Research Use Only Not Intended for Use in Clinical Diagnostics This kit allows genotypic characterisation of bacterial cultures from C difficile 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 C difficile Specifications Upon receipt the kit components need to be stored at different temperatures as specified on the package insert The assay is to be performed at an ambient temperature of 18 C to 28 C Technical Support If you require any further information on this product please contact Email cct home clondiag com Phone 49 0 36 41 3111 155 Fax 49 0 36 41 3111 120 For up to date information regarding the kit please visit our website at http www alere technologies com C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 2 www alere technologies com www alere com Safety Precautions The kit is intended for use by personnel that are trained in microbiological and molecular methods Preparation of DNA from pure C difficile colonies clones requires exper
24. f Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 28 www alere technologies com www alere com Error Messages in Result Sheets Please compare Appendix 2 for the possible error messages in Result Sheets and for corresponding array images C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 29 www alere technologies com www alere com ADDITIONAL INFORMATION Warranty Alere Technologies GmbH guarantees the performance as described in this manual Usage of the kit was successfully tested at ambient temperatures up to 37 C A guarantee is limited to ambient temperatures in the laboratory between 18 C 28 C Kit components comprise the arrays and their caps the Lysis Enhancer the reagents for DNA labelling and for detection of labelled DNA products on the array the ArrayMate reader and its software In case one of these components fails within the expiry date due to other reason than misuse contact Alere Technologies GmbH for replacement or refund Terms and conditions apply If you have any 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
25. gnostic Procedures results results b raw data segmentation image image Operator die Sample ID 16045 C Diff 01 D Experiment ID 16045 C Diff O1 E Date of Result Tue Sep 08 11 33 42 2015 01 8 Archive Assay Name C Diff 01 Assay ID 16045 Well Position I Software Version 2015 06 15 Device StripID ID Assays Bilder 16045_C Diff Hybridisation Pattern Hybridisation pattern 13 Score 96 99 Clade I Corresponding MLST type ST42 Corresponding ribotypes RT 014 RT 049 Published genome sequences likely to be related or similiar to this strain SEND AGA Experiment Validity passed SPECIES MARKER Marker classification explanation Biotin positive Species markers and controls Bacitracin resistance protein locus 1 positive Bacitracin ATP binding cassette transporter ABC protein BcrA probe negative hp1071 Bacitracin ATP binding cassette transporter ABC protein BcrA hp1072 Bacitracin ATP binding cassette transporter ABC protein BcrA hp1073 o Lincomycin resist protein ImrBNAP07 hp1097 Lincomycin resist protein ImrBNAP07 lhn1048 positive negative negative Power
26. h the ArrayStrip s C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 21 www alere technologies com www alere com Press Next at the bottom right on the screen reader is closing Results On the left hand side of the screen there you will see a list showing all runs stored on the ArrayMate s hard disk A run contains the results from all arrays analysed together within one frame If this list is not displayed Press Archive left hand side and activate the flag Browse at the top left e The runs are organised like folders in Windows Explorer and named by default according to the date of acquisition Example There is one experiment run in this archive Browse Search l FI ARCHIVE 28 2013 02 26 13 21 16 New Run 254 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 Browse 4 Search li FI 3 ARCHIVE 2E 2013 02 26 13 21 16 Mew Run 01 A 01 B 4 01 C 01 D Archive D1 E D1 F 01 G O1 H C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 22 www alere technologies com www alere com Click on a Sample ID and the C diff Type AS 1 Kit test report for this array is shown in the window on the right f x L ArrayMate Browse Search print aj 13 21 16 For Research Use Only Not For Use In Dia
27. ing protocol Pre heat cover lid to 105 C 300 sec at 96 C 60 sec at 96 C 55 cycles with 20 sec at 50 C 40 sec at 72 C Cool down to 4 C hold e The amplification products can be stored frozen until usage Please note When using a different device some adaptations such as an increase of the number of cycles might be necessary Before establishing routine use please test the protocol with a few known reference strains or the control DNA CM supplied upon request We recommend as reference strain the following Clostridium difficile DSM No 27543 Strain designation 630 https www dsmz de catalogues details culture DSM 27543 html C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 12 www alere technologies com www alere com Hybridisation General Remarks Handling of Arrays Never touch the array surface Avoid complete drying of the array surface during processing e Do not allow it to stay without liquid for more than two minutes Never rinse the wells with distilled water after the hybridisation step only use C2 Washing Buffer Unused wells should be capped during the whole procedure The strips may be processed up to three times without a loss of quality of properly capped unused arrays Close all wells that will not be used with a cap und leave them there until you use these wells for storage conditions after use see section Kit compone
28. m one Clade are present this will most likely not be the case The system might then provide a putative identification displaying the closest matching Hybridisation Profile and the Clade affiliation Comment Handling The bottom of the well is contaminated with dust particles Please clean the bottom of the well scan and process again The microarray surface is contaminated with dust particles If the microarray surface is contaminated with particles wash the microarray with double distilled water pipetting water carefully up and down remove scan and process again The bottom of the well is contaminated with a liquid e g buffer Please clean the bottom surface with a cleanroom wipe scan and process again C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 36 www alere technologies com www alere com Substrate D1 residue in the array edges Remove substrate D1 completely with a Pasteur pipette Chip was not in focus during image acquisition Repeat image acquisition after fitting the ArrayStrip in the frame C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 37 www alere technologies com www alere com APPENDIX 3 PROBE TO TARGET TABLE CATEGORY TARGET PROBE ID TOXINS Toxin A hp1132 tcdAp1 hp1134 tcdAp2 hp1135 tcdAp3 hp1247 tcdAp2 Toxin B hp1118 tcdBp1 hp1119
29. mated Device ssssssesesesseeseeeeee nennen nennen nnn 11 Linear Amplification and Internal Biotin Labelling 5 2 11 Hybridisation een ab 13 General Remarks Handling of Arrays rien hae reda orn ae pna roa 13 General Remarks Handling of Liquids 22 14 General Remarks The Substrate Precipitating Dye D1 14 General Remarks Thermoshakers uui iaces ars dA nk a 15 Protocol for Quantifoll s BIGShake IQ ee 16 Data Analysis aa Eton da dI PRI M HM EE DAN MR Eu UMS 18 Starting the ArrayMate Reader erre nennen 18 18 Data Acquisition in the 20 Reus 22 Export of C diff Type AS 1 Kit Test 5 25 TROUBLESHOOTING san araram aaa olayda 26 Sal n CONTIG 26 Mage lib E Da EE a aa 27 C diff Type AS 1 Kit 05 16 04 0019 V01 Manual C diff Type AS 1 Kit www alere technologies com www alere com DNA Quality and RNA Contamination Control esses eene nennen 27 Physical Damage to the Array Lagar ee 28 Report Unavalable ND 28 Ambiguous Result nn ee 28 Error Messages In Result Sheets nn ee 29 ADDITIONAL INFORIVIATION ab a au adada i d 30 rcp
30. mated device DNA Tissue Kit cat 953034 Please note DNA isolation from C difficile 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 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 6 www alere technologies com www alere com e Equipment for DNA gel electrophoresis for quality control of DNA Thermocycler e Thermoshaker We strongly recommend the BioShake iQ by Quantifoil Instruments http www qginstruments com equipped with a customised heating block designed to fit ArrayStrips 4 312 010 BioShake iQ Alere ArrayStrip 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 pL 90 uL 100 uL 200 uL 1000 uL e Multichannel Pipettes for 100 200 uL e Reagent tubes suitable for PCR VWR Cat 732 0098 Ultrapure PCR grade water Pasteur pipettes VWR Cat 612 2856 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 7 www alere technologies com www alere com PROTOCOLS Culturing and Harvesting Bacterial Cells C difficile is a potential pathogen All procedures for cultivation of the bacterium and DNA preparation need to be performed by properly trained staff in a biosafet
31. n initiation protein hp1044 divIC hp1045 divIC hp1046 divIC FLAGELLINS flagellin subunit C hp1141 fliC hp1142 flic hp1144 fliC hp1145 fliC hp1147 lic MISCELLANEOUS GENES C diff Type AS 1 Kit putative endonuclease relaxase Tn1549 like hp1214 CD0425 hp1220 CD0425 hp1228 CD0425 hp1229 CD0425 hp1230 CD0425 hp1231 CD0425 putative calcium binding adhesion protein hp1149 CD2797 hp1212 CD2797 ethanolamine iron dependent alcohol dehydrogenase hp1061 eutG hp1062 eutG leucine Rich repeat containing domain protein 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit hp1227 IPR007253 40 www alere technologies com www alere com APPENDIX 4 TYPING INFORMATION Definitions and Explanations The displayed result will yield following typing information Strains are named HP for Hybridization Profile followed by a chronologically assigned number see Table below Isolates cluster into HPs or strains based on overall hybridization patterns with emphasis to tcdA B and slpA alleles Isolates or strains were regarded as one HP in case of at least 88 identity of positive ambiguous negative classifications for all probe positions covered plus presence of identical tcdA B and 5 alleles Possibly mobile resistance markers are not considered for the definition of hybridization profiles or strains It still needs to be clarified whether these genes could be used as subtyping m
32. n patterns and to MLST defined clades MLST sequence types and ribotypes known to be associated with the respective hybridisation pattern are also displayed as well as related identical genome sequence Note that information on sequence types and ribotypes is derived from a database search not from an actual experiment We are grateful if you could share additional information on sequence types and ribotypes as this could help to improve the software in future 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 Other files that are generated and that can be exported include e A txt file with the raw measurements e An image file bmp showing the actual picture of the array e A second image file png in which the coordinate grid is superimposed and the recognised spots are circled and A xmL file providing the same information as the html result sheet for future export into databases and for using the Result Collector tool 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 C diff Type AS 1 Kit Test Reports is not possible 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 C diff Type AS 1 Kit 05
33. nts Storage and Stability Hybridisation and Detection Always label your ArrayStrips with a laboratory marker at the recommended position Never label them on the bottom or across the data matrix barcode This may cause errors Data Matrix code keep it clean label here 4 Avoid contact of data matrix barcode with organic solvents The ArrayMate needs the information encoded in the data matrix to perform the assay and the analysis afterwards Avoid touching the bottom of the microarray strip and keep it clean C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 13 www alere technologies com www alere com General Remarks Handling of Liquids We recommend the use of a multichannel pipette and reagent reservoirs We strongly recommend that the liquid is removed by pipetting rather than by inverting the strips and flicking the liquids out Fine tipped soft disposable Pasteur pipettes are suited best such as VWR Cattt 612 2856 Always place the pipette tip at the cavity between the array and the wall of the reagent well If you touch the array surface probes may be scratched off and this may cause errors Pasteur pipette plastic with a flexible tip Bm 1 flexible tip I Pipette tip Use the cavity between array and the wall of the tube Do never touch the array N Array General Remarks The Substrate Precipitating Dye D1 An appropriate amount of D1 substrate precipi
34. oducts 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 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 3 www alere technologies com www alere com REAGENTS AND DEVICES Kit Components Storage and Stability All reagents are provided in surplus see below If necessary all components may be ordered separately Please refer to the catalogue reference numbers Cat at the end of this manual For pricing please contact your local representative or our customer service respectively The expiry date can be found on each bottle and on the outer packaging All components were tested for short term shipment lt 1 week at ambient temperature lt 37 C The assay components with limited stability are D1 and C3 The other kit components proved to be stable six months after post expiry Cell Lysis e A1 Lysis Buffer Store at 18 to 28 C ambient temperature Surplus 50 96 e A2 Lysis Enhancer dried Store at 18 to 28 C ambient temperature Centrifuge A2 tubes shortly prior to opening Add 200 ul Buffer A1 to Lysis Enhancer before use Mix well and store for less than 1 week at 2 8 C Sufficient for 96 isolations DNA Labelling and Amplification B1 Labelling Buffer Master Mix Store at 2 8 C Surplus 25 96 e B2 Labelling Enzyme Store at 2 8 C Surplus 50
35. ogies com www alere com Instrumentation and Software e ArrayMate Reader to be ordered separately for details see below The ArrayStrip based C diff Type AS 1 Kit can be used on the ArrayMate reader only The alternative devices ATRO1 03 are not suitable for reading ArrayStrip based assays In case of any questions please contact us lconoclust software provided with the reader Test specific software plug in can be downloaded from Alere Technologies GmbH website check periodically for updates for details see below Information such as spot names marker names location of the spots on the array size of the image taken by the reader s specific camera is delivered with the reader or can be downloaded from our website These test specific plug ins will occasionally be updated Please check the NEWS section of our website http alere technologies com Support is available via cct home clondiag com Components Required but not Provided e Growth media for the cultivation of C difficile for discussion of suitable media see below e Equipment and consumables needed for the cultivation of C difficile incubator anaerobic jars and catalysts inoculation loops Petri dishes e Additional assays for confirmation of species identification e DNA preparation kit The assay was tested with the DNeasy Blood amp Tissue Kit from Qiagen cat 69504 QlAamp Minikit cat 51306 and a DNA preparation kit for Qiagen s EZ1 auto
36. om www alere com Table 1 Example worklist Please note Table header must be written exactly as shown position samplelD assaylD 1 2015 12345 16045 2 2015 12346 16045 3 2015 12347 16045 4 2015 12348 16045 5 2015 12349 16045 6 2015 12350 16045 7 987654 16045 8 C diff 16045 Table 2 Positions in the 96 vvell 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 zo Z moOo n oo gt o IsIloalun s w w r le 16 24 32 40 48 56 64 72 80 88 96 Data Acquisition in the ArrayMate Reader Insert your flash drive containing the worklist into any of the USB ports on the lower right hand side ofthe ArrayMate e Press a folder selection dialogue will open e Select your worklist path My Computer Removable Disk e Open your selected worklist by pressing Enter or Open e Press your imported worklist opens in a separate window Proofread If the new window is empty or if it was the wrong worklist repeat the import e Press OK the worklist window will close C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 20 www alere technologies com www alere com e Leave the flash drive in the ArrayMate if you intend to export C
37. ontrol is included to check whether possible problems originate from the hybridisation or the staining procedure If the staining control has Failed proceed as follows C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 26 www alere technologies com www alere com Horseradish peroxidase conjugate may have degraded during storage Add 1 ul buffer C3 C4 to 9 ul D1 substrate If the solution turns green within 3 5 seconds the horseradish peroxidase still has sufficient enzymatic activity Enzymatic reaction is inhibited by carryover of buffer C1 Ensure proper washing of the wells with C2 buffer to remove all C1 buffer prior to adding horseradish peroxidase conjugate If the staining control has Passed refer to the following hints Image Quality In case of poor image quality we recommend to re check DNA quantity and quality first by loading leftover DNA on an agarose gel In order to determine whether any problems originated from the DNA preparation perform an experiment with the Control material CM This is DNA from the C diff reference strain details strain ID corresponding GenBank number etc upon request If the control experiment yields a valid result and a correct identification there was probably an issue with DNA preparation If the control experiment also fails an error affecting later steps or a degradation of reagents from later steps is likely See also Appendix 2 Images for
38. reaks in hospital settings warrants molecular typing The microarray based assay facilitates rapid and high throughput genotyping of clinical C difficile isolates including toxin gene detection and strain assignment The C diff Type AS 1 Kit allows DNA based detection of resistance genes and pathogenicity markers of C difficile and assignment of unknown C difficile isolates to known strains RNA free unfragmented genomic DNA from pure and monoclonal C difficile colony material is amplified 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 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 gen
39. t be divided into two aliquots and used for DNA preparation of two samples 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 When the cells are lysed proceed by performing the tissue lysis protocol Bacteriacard for Qiagen s EZ1 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 4096 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 11 www alere technologies com www alere com Prepare a Master Mix by combining 3 9 uL of B1 labelling reagent 0 1 uL of B2 labelling Cdi enzyme and 1 0 ul of B3 primer mix per sample 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 e Perform amplification in a pre programmed thermocycler such as Mastercycler gradient with heated lid VWR cat 460 0108 according to the follow
40. tating dye should be transferred into an Eppendorf tube and taken out of the refrigerator when starting the procedure allowing it to acclimatise to room temperature 25 C Cold D1 may yield weak signals D1 should be centrifuged prior to use to remove bubbles as well as possible precipitates quick spin C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 14 www alere technologies com www alere com Triggered by peroxidase the dye precipitates in case of positive reactions but it is not covalently bound The precipitate can be dissolved by vigorous shaking Thus the arrays must not be shaken dropped or moved abruptly during the staining procedure or thereafter After completion of staining 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 Qlnstruments see picture below http www qinstruments com equipped with a customised heating block designed to fit ArrayStrips and Eppendorfs Th
41. tcdBp1 hp1121 tcdBp4 hp1122 tcdBp4 hp1124 tcdBp2 hp1126 tcdBp2 hp1127 tcdBp2 hp1129 tcdBp3 hp1130 tcdBp3 ANTIBIOTIC RESISTANCE chloramphenicol acetyl transferase cat hp1014 catD rRNA methyl transferase ermB hp1022 ermB tetracycline resistance protein hp1105 tetM hp1108 tetM hp1109 tetM hp1110 tetM hp1112 tetM streptogramin A acetyltransferase hp1082 sat hp1083 sat lincomycin resist protein ImrBNAPO7 hp1096 ImrB lincomycin resist protein ImrB630 hp1097 ImrB LANTIBIOTIC RESISTANCE lantibiotic two component sensor histidine kinase hp1079 spaK hp1080 spaK putative lantibiotic ABC transporter permease protein hp1251 spaE hp1252 spaE EFFLUX SYSTEMS bacitracin resistance protein locus 1 hp1013 bacA1 bacitracin ATP binding cassette transporter hp1071 bcrA hp1072 bcrA hp1073 bcrA ABC type transporter ATP binding protein hp1084 CD0456 putative efflux pump hp1087 cme MATE efflux family protein hp1063 matE efflux pump antibiotic resistance protein hp1098 ydic hp1244 ydiC vexP1 vncS ABC type transport system permease Tn1549 hp1055 lvexP1 hp1059 l vexP1 two component sensor histidine kinase Tn154c hp1051 vncS hp1052 vncS OTHER VIRULENCE GENES C diff Type AS 1 Kit C difficile binary toxin component A hp1023 cdtA hp1026 cdtA hp1027 cdtA hp1029 cdtA C difficile binary toxin component B channel forming hemolysin hp
42. tise in microbiology and the local regulations for handling of pathogenic microorganisms biosafety level 2 are to be obeyed Isolated cell free C difficile DNA may be processed without further biosafety precautions although contamination with C difficile 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 regulations EC 1272 2008 CLP and 1907 2006 REACH the enclosed reagents do not require a Material Safety Data Sheet MSDS except Hybridisation Buffer C1 The MSDS can be downloaded via our website from any lab solutions product page e g http alere technologies com en products lab solutions html All other reagents do not contain more than 1 of a component classified as hazardous and do not contain more than 0 1 of a component classified as carcinogenic Nevertheless the buffers may cause irritation if they come into contact with eyes or skin and may cause harm if swallowed The regular precautions associated with laboratory work should be obeyed e g wear protective goggles gloves and lab coat and avoid contact with the reagents If liquid is spilled clean with a disinfectant and or laboratory detergent and water Alere assumes no liability for damage resulting from handling or contact with these pr
43. tively numbered from 1 to a maximum of 96 Position 1 would correspond to A1 8 to H1 9 to A2 and 96 to H12 Table 2 Do not leave empty lines in the worklist If you use EXCEL position numbers should be entered into column A e Sample IDs are strain sample laboratory numbers such as exported from your LIMS or assigned in any different way Patients names should not be used as sample IDs e Assay ID allows the system to identify the current test and to correctly use information on layout spot number and identity etc The E C diff Type AS 1 Kit has the Assay ID 16045 Please note Assay ID numbers must not be confused as this could lead to errors or loss of data e You may add further columns and headers with notes and comments at your convenience Information from these columns will not appear on the result screen or in the Test Report We recommend using a printout of the worklist as a template for pipetting e Save the worklist as tab separated txt file on the memory stick provided together with the ArrayMate e To avoid confusion make sure that worklists are named unambiguously or that worklists from earlier experiments are deleted You may use the software tool Worklist Generator to create a worklist easily http alere technologies com en products lab solutions software tools worklist generator html C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 19 www alere technologies c
44. train 630 AM180355 slpA tcdArz0291 CdtAcso Strain 6534 ADEJ tcdBeao cdtBeso tcdArz0291 ST 35 SIDA npos037 1 63 N A SIDA1446 N A SIPAr13700 ST 58 or tedAcrs Strain 6407 ADEH ld SIDAs407 tcdBeao N A SIPAs407 SIpA negative probe 1164 tCAArz20291 sometimes 1 ambiguous CD196 FN538970 11 FN668941 CIP107932 ABKK SIPArz0291 QCD 76vv55 ABHE QCD 97b34 ABHF R20291 FN545816 2007855 FN665654 QCD 32g58 AAML SIPArz0291 QCD 37x79 ABHG QCD 66c26 ABFD tcdArz0291 CdtArzo291 tCABra0291 CdtBr20291 tcdArzo291t CdtArzo291 tCABr20291 CdtBr20291 tcdArzo291 CdtAciade m IpA SIPAr12884 tcdBe3o COtBr20201 SIDAr12884 tCdArz0291 CdtAciade m trunc tcdBe3o CdtBr20291 CF5 FN665652 002 P50 2011 AGAA 050 P50 2011 AGAB M68 FN668375 tcdAcrsk H P 35 tcdBcrs C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 43 www alere technologies com www alere com Hybridi sation profile Fully sequenced strains GenBank accession no M120 FN665653 NAPO7 ADVM ADNX Associated sequence types ST11 SIpA allele SIDAzscss SIPAr13540 Associated ribotypes tcdAcrs tcdBers tcdAr20291 tcdBeso CdtArz0291 cdtBunzo Strain 6466 ADDE ST 11 or related SIPAr13540 tcdAr20291 tcdBeso CdtArz0291 cdtBm120 HP 4
45. uick spin Transfer the complete tube content including any precipitate into a spin column that is placed in a 2 ml collection tube Centrifuge 8 000 rpm 1 min at room temperature Time and speed need to be determined depending on the sample viscosity and the type of centrifuge used All liquid should be collected in the collection tube afterwards C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 9 www alere technologies com www alere com Discard collection tube with liquid Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 ul Buffer AVV1 Centrifuge 8 000 rpm 1 min at room temperature e Discard collection tube with liquid Place the spin column in a new 2 ml collection tube provided with the kit e Add 500 ul Buffer AW2 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 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 Centrifuge 8000 rpm 1 min at room temperature Optional Add another 100 ul Buffer AE or PCR grade distilled water directly onto the membrane incubate at room temperature for 1 min and centrifug
46. well Incubate at 25 C for 10 min but do not shake Remove liquid completely The bottom of the ArrayStrips outside surface may be cleaned cautiously with wipes Bubbles may be removed by removing and adding D1 Scan and process ArrayMate see below Please note Check immediately all images for cleanliness i e absence of dust particles residual liquids and for good focus Dust particles and residual fluids inside the vial can be removed by cautiously washing twice with 200 ul PCR grade distilled water If necessary scan and process again For Troubleshooting see p 26 and 35 37 C diff Type AS 1 Kit 05 16 04 0019 VO1 Manual C diff Type AS 1 Kit 17 www alere technologies com www alere com Data Analysis Starting the ArrayMate Reader We recommend starting the ArrayMate Reader after starting the hybridisation this allows the convenience of starting the device and to importing the worklist file Please note that this is a short instruction only For more detailed information please refer to the ArrayMate User Manual e Switch on the ArrayMate 1 main switch on the rear below the electric cable plug 2 9 operating switch on the bottom left corner of the front side e Switch on the screen switch is on the right hand side below the screen Log in as R amp D User Research and Development User for full access to test specific software a default password will be provided together with the ArrayMate
47. www alere com e Assignment score This is a score for the similarity of the actual experimental result to the predefined strain Hybridisation Profiles Scores below 87 2596 exclude reliable strain identification and could be attributed either to technical reasons or to the presence of a yet unknown strain but the the most similar strain and the Clade affiliation will be displayed In case of a score below 83 96 the experiment is considered invalid and both html files will display error messages A value of 100 is unlikely because of the mobility of some genes in C difficile and the possibility of ambiguous results for mismatching allelic probes List of Currently recognized Strains Hybridi Fully sequenced strains MLST Associated Associated tcdA B cdtA B sation sequence s pA allele cat erm B tet M R GenBank accession no Clade ribotypes alleles alleles profile types RT 001 BI 9 FN668944 tcdArzo291t CdtAcso HP 01 Qcp 63442 ABHD l d slpAus RT 015 0 cdtB pap J RT 072 RT 001 tcdA cdtAsao HP 02 ATCC43255 ABKI 5745 ST 46 sIpAs o RT 013 CAArz0291 COLAs30 _ _ tcdBeso cdtBeso RT 087 RT 011 HP 03 l ST 58 SIPAs4o7 RT 049 7 var var RT 056 630 630 RT 137 tcdAkzoz rk CdtAszo 1 5 04 DA HP 04 Pee RT 150 tcdBeso cdtBez0 tCdAr20291 CdtAs30 HP 05 I N A slpApinsos7e RT 163 ied Unident tcdApz0291 Cdt
48. www alere technologies com www alere com Manual C diff Type AS 1 Kit Array Hybridisation Kit for DNA based detection of resistance genes and pathogenicity markers of Clostridium difficile and assignment of unknown C difficile isolates to known strains Kit order number 246100096 96 reactions ArrayStrip format For Research Use Only Not for Use in Diagnostic Procedures www alere technologies com www alere com CONTENT BACKGROUND NN ER nv 1 GENERAL INSTRUGTIONS FOR USE na dada Oams 2 Intended se NN 2 SBEC C L ORSL be ad dayananda 2 Technical Support n la dalbadal las 2 Safety PRG Ca OIA LA ee NE 3 Material Safety Data Sheets MSDS an 3 Shipping Preca tionS EEE adaya asd Aka ad da ab nam mda 3 REAGENTSAND DEVICES a A Ra 4 Kit Components Storage and Stability eee 4 Cell Lysis optional order EN acutus da s 4 DNA Labelling and Ampl fiestion y aaa aa 4 Hybridisation anid Detection aaa 5 Instrumentation and Software mans snakke Ee Ub SO M TUS edad 6 Components Required but not Provided eese eene eee nnns 6 PROTOCOL c O e T P 8 Culturing and Harvesting Bacterial Cells iicet ae 8 D d Y YY d d d 8 DNA Extraction by Spin Columns e g lagen za venu AL A a 9 DNA Extraction by Auto
49. y level 2 facility C difficile can be isolated under anaerobic growth conditions from faecal samples using pre treatment with ethanol in order to kill off other bacteria and or cycloserinethanol cephoxitin fructose agar e g Oxoid Cat Nr PB5054A Pre reduction of the agar under anaerobic conditions or in an incubator with increased CO concentration will improve recovery rates Since a comparatively high amount of DNA is required because of the linear amplification see below single colonies from a primary culture usually do not suffice Therefore a single colony should be picked and used to inoculate several plates of blood or Schaedlar agar or more conveniently a vial of Schaedler broth The subculture should be incubated again 48 hrs at 37 C and under anaerobic conditions and cultures be harvested or respectivel centrifuged e Centrifuge A2 tube briefly open it add 0 2 mL of Lysis Buffer A1 to Lysis Enhancer A2 and dissolve e Add some inoculatings loops full of monoclonal culture material of the C difficile isolate or the sediment of centrifuged liquid culture into this A1 A2 reagent vortex DNA Extraction The required sample type for the C diff Type AS 1 Kit is 0 5 2 ug Cona 0 1 0 4 ug ul of intact genomic DNA from a single clone 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 C diff Typ
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