User manual User manual - Check
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1. use reagents after their expiration date e Before use thaw frozen reagents completely at room temperature and vortex briefly to obtain a homogeneous solution After vortexing briefly spin down the solution to avoid contamination when opening the lid Avoid unnecessary freeze thawing of the kit content e Periodically verify the accuracy and precision of pipettes as well as correct functioning of the instruments Prevention of contaminations PCR produces a very high quantity of DNA amplification products amplicons even from minute quantities of starting material Check MDR CT103XL may therefore yields unreliable results if samples become contaminated with amplicons from previous amplification reactions prior to the PCR step B of the protocol Preventive measures to minimize the risk of amplicon contamination must be taken Please read carefully and follow the instructions outlined below Use separate rooms a pre PCR room and a post PCR room e Sample preparation DNA recognition step A and preparation of the amplification step step B is carried out in the pre PCR room e Incubation in the PCR thermocycler of step B is carried out the post PCR room e detection step is also carried out in the post PCR room Alternatively the detection step is carried out in a third room separate from the other two rooms e Never bring the reaction products of step B to the pre PCR room e Never prepare the ligation and or amp2. 10 3 The A mix was not added in step A Please repeat the test 10 4 The BC mix was not prepared properly or was not added to the assay please repeat the test b The amount of bacterial DNA added in step A was probably insufficient the DNA extraction may have failed or the DNA concentration may have been too low Please repeat the test with a new DNA extract 12 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR 11 The software indicates ESBL suspected The software did not find sufficient spots to give a conclusive result Please inspect the picture visually an air bubble or dust particles may interfere with the result Tap the AT gently or pipette the liquid gently up and down and retry to take the image Please repeat the test if the results do not change 12 The software indicates Picture not found or Image capture error Check if the Check Points Tube Reader is properly installed Please contact Check Points Technical Support at support check points com if reinstallation of the reader does not solve this problem 13 The AT image is covered with small spots There may be various causes for this phenomenon Most likely the array dried out during the detection step Please make sure that the AT always contains sufficient amounts of reagents Detection Buffer Blocking Buffer conjugate dilution or Staining Solution This is particularly critical during the incubation
3. e For support concerning results please send the result image along with the desired information to support check points com For this purpose double click on the result s in question in the result summary window A pop up will appear see figure 8 in which your comments may be added to the file Send image to Check Points o REM p tm Image Info Customer CP PB a Place Wageningen NL Date 18 06 2014 ime 12 Tube nr 12345678 Lot re pb ee ED D H Operator pb Sample sample 1 E a Recut ESBL CARBA e e eee ee ee fea ample P ba Resut H H ISWVersion 4823 E Update 126 2014 m email Comments r ptions Your email address youmemail amp iprovider Options IV Send picture directly over internet connection Cancel Send Cancel Send Figure 8 Send image to Check Points pop up Figure 9 Check the box to send pictures directly over the internet Click on the Save Send later button to store the file which will have a cpfe extension on the computer and send it by e mail Alternatively if the computer has internet access check the box Send picture directly over internet connection and enter the reply e mail address followed by clicking on send In both cases feedback should be expected within two working days Support may also be requested at a later stage when the program has been closed For this pur
4. e CT 103XL Check MDR User manual Check MDR CT103XL Molecular Detection and Identification of Carbapenemase AmpC and ESBL genes Version 1 0 Date of issue 01 08 2014 10 0022 V 24 IFU 022 01 EU C I VD U S For Research Use Only Not for use in diagnostic procedures Contents Intended 2 tenn Neen Netware 2 Principle ofthe method ete 3 Kit contents for 24 reactions BEE 4 Materials required but not supplied with the kit EE 4 Shelf life storage and handling 2 nr tia eb Kex 4 Good laboratory ie de 5 Protocol BEN 6 1 DNA recognition Step UR ea dads 6 Z IDNA amipliticstion 6 E 7 Frequently asked questions FAQ amp troubleshooting 12 13 Key tors mbols UsEd TO 14 Appendix 1 Performance 1 lt 15 Appendix 2 CTX M1 group subtyping 17 Appendix 3 Thermocycler d E 17 Appendix 4 Pipetting SCHEMES cccsccscsecescescesssssssssssssscsscsscscssessessessesecarsaesarsorceseass 18 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 e CT 103XL Ch
5. or the conjugate dilution was not prepared properly Please repeat detection step with new AT 9 The software indicates Hybridisation not OK The software did not find the hybridisation control spots on the AT The hybridisation control is used to check if the hybridisation at 50 C of the PCR product with the AT has been performed properly Causes may be 9 1 The picture is completely dark conjugate dilution was not removed properly Please refer to question 5 9 2 The reaction mixture after completion of step B was not added to the AT Please repeat detection step with new AT 9 3 Hybridisation temperature too high Please verify that the thermomixer temperature was 50 C when the ATs were hybridized 9 4 The BC mix was not prepared properly or was not added to the assay please repeat the test 10 The software indicates Reaction not OK please contact Check Points This message will be displayed in the following two scenarios a The software did not find the reaction control spots on the AT These amplification controls are used to check the performance of the assay in steps A and B Possible explanations are 10 1 The sample DNA was not added to the assay in step A 10 2 The sample DNA contains contaminants inhibiting the reactions These may originate from the growth medium or the DNA isolation method This may be remedied by diluting the DNA extract 10 fold with distilled water Repeat the test with the diluted DNA
6. 16 Filling out the experimental data a Startthe computer b Start software on the computer by double clicking the Check Points desktop icon Ze Check Points c Double Click on Check MDR CT103XL arr in the first screen Array selection as shown in figure 2 Check Points 50 E 1 Array selection 2 Sample information 3 Detection step 4 Results i Pe CT 103XL 1 Array selection eo CT 10 3 Check MDR Please choose array type from the list below Check MDR CT101 arr Check MDR CTI01 12 6 2014 Check MDR CT102 arr Check MDR CT102 12 6 2014 Check MDR CT103_new arr Check MDR CT103 12 6 2014 Please enter lot number of the kit Operator Figure 2 Array Selection 8 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 e CT 103XL Check MDR d Enter the lot number of the kit in the appropriate field and the name of the operator followed by pressing the Next Step button e Insert the sample codes in the screen Sample information see figure 3 Check Points c E 1 Array selection Sample information 3 Detection step 4 Results 2 Sample information ee CT 103XL o Check MDR Please enter your sample codes in the table below 1 2 3 4 5 6 7 8 9 10 11 12 sample 1 sample 3 13 14 15 16 7 18 19 20 2 22 23 24 Figure 3 Sample information Note e Additional remarks may be added per sample optiona
7. CT103XL cannot and does not make any representation or warranty that it is capable of correctly detecting the ESBL AmpC and carbapenemase genes in all gram negative species subspecies or type or in any clinical sample source Results may need to be confirmed by additional methodologies in specific cases e g for regulatory samples Due to the high variability of bacterial genomes it is possible that certain sub types might not be detected The test reflects the state of knowledge of Check Points Health B V The quality of the input DNA is an important factor in obtaining reliable results from Check MDR CT103XL DNA must be extracted from cultured bacteria using a suitable method such as commercially available extraction methods based on columns or beads Methods should be used that extract and purify total DNA i e genomic as well as plasmid DNA The presence of multiple bacterial species in a sample may hamper the interpretation of the test As with other diagnostic assays the results of this test may only be interpreted in 13 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR combination with additional laboratory and clinical data available to the responsible person Use of this assay is limited to appropriately qualified personnel well trained in the test procedure and familiar with molecular biological methods The Check Points software is continuously improving thanks to the expanding global d
8. DNA markers to identify the presence or absence of ESBL AmpC and carbapenemase genes The test detects the presence of the ESBL genes TEM SHV and CTX M PER VEB BEL and GES and it also detects Single Nucleotide Polymorphisms SNPs corresponding to amino acid positions 104 164 and 238 in TEM 238 and 240 in SHV and 170 in GES The nature of the above SNPs in TEM and SHV determines the ESBL phenotype ESBL yes or no for the majority of TEM and SHV types For a detailed explanation see www lahey org studies and M Gniadkowski Clin Microbiol Infect 2008 14 Suppl 1 11 32 Check MDR CT103XL also detects the AmpC genes CMY DHA FOX MOX ACC ACT and MIR which presently represent the clinically most prevalent AmpC genes It should be noted that other AmpC genes are not detected Finally Check MDR CT103XL detects the carbapenemase genes KPC NDM OXA 48 VIM IMP GES GIM SPM OXA 23 OXA 24 and OXA 58 which presently represent the clinically most prevalent carbapenemase genes Check MDR CT103XL requires DNA purified from a colony or bacterial culture Clinical specimens cannot be tested directly The assay has been tested extensively with purified DNA from gram negative bacteria such as Escherichia Salmonella Klebsiella Enterobacter Citrobacter Pseudomonas and Acinetobacter with excellent results However it may never be excluded that other gram negative bacteria or certain strains of the above species will yield poor results Check MDR
9. steps at 50 C In most cases the software will be able to handle these small spots If not repeat the detection step with a new AT 14 The AT was incubated for more than 15 minutes with Staining Solution before taking the image Results may be unreliable due to overstaining Inspect image if spots are very dark please repeat detection step with a new AT 15 Duplicate DNA samples tested with Check MDR CT103XL do not yield identical results 15 1 Inspect images for dust particles Tap or gently shake the tubes to try to remove the particles from the array and rescan the images 15 2 In case of extra spots repeat the test to confirm result 16 May the assay be interrupted and continued at a later stage Reaction mixtures from Step A can be kept at hold at 4 C for a maximum period of two hours Reaction mixtures from Step B can be kept at 20 C for a maximum period of two weeks For further inquiries please contact Check Points Technical Support support check points com 17 The spot intensities on various ATs are weak Please repeat the test Contact Check Points Technical Support at support check points com if results do not improve 18 The AT image contains dust particles The software will correct this in most cases To prevent any interference with the results please take the AT out of the reader and shake it gently until the dust particles have moved to the side of the AT Limitations Check MDR CT103XL uses a range of specific
10. 6 86 90 91 102 105 106 115 120 123 124 126 128 129 134 141 152 153 160 163 165 3 12 15 17 18 21 22 24 26 29 42 43 46 50 52 53 56 60 61 63 6668 71 72 75 85 89 91 94 101 102 106 TEM ESBL 107 109 111 113 115 118 120 121 123 125 129 134 136 138 139 142 144 147 149 151 155 158 165 167 177 187 188 193 195 197 199 205 211 VEB 1 8 ACC 1 2 4 1 3 6 7 9 10 1 2 12 14 38 43 45 49 57 59 63 71 73 80 94 95 99 110 1 3 5 7 FOX 1 10 MIR 1 6 MOX 1 2 4 15 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 SE e De CT 103XL Check MDR Clinical sensitivity and specificity A retrospective study was performed with 163 well characterized bacterial strains covering all B lactamase genes detected with CT103XL All strains were inoculated on TSA plates and grown overnight at 37 The next day bacterial cell suspension of O D 600 1 0 were prepared in MQ water Two hundred microliter of each solution was used to isolate the DNA with the QiaCube robot using the DNA mini bacterial pellet bacterial DNA protocol DNA s were eluted in 200 ul and stored at 20 C until further analysis The samples were processed according to the standard protocol described in this manual Results are depicted in the table below Carbapenemase gene positive Negative Sensitivit Specificit 8 strains tested ains tested Y 100 100 GES Ca
11. AT by pipetting up and down 8 Close the lids of the ATs properly to prevent them from drying out and incubate the tubes for 30 minutes at 50 C 400 rpm 9 After 30 minutes replace the Detection Buffer with 300 ul Blocking Buffer do this with one AT at a time Remove the AT from the thermomixer discard the Detection Buffer using a pipette and immediately replace with 300 ul Blocking Buffer using a new pipette tip Place the AT back in the thermomixer at 50 C and proceed with the next AT until all the AT solutions have been replaced with Blocking Buffer Incubate for 5 minutes at 50 C 400 rpm Optional e Evaporated water condensed in the lid of the AT may be removed with a pipette e g before removing the Detection Buffer containing the sample Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR Note Collect the liquids removed from the ATs in a disposable tube and dispose of it the same day with the other laboratory waste 10 Replace the Blocking Buffer with 300 ul fresh Blocking Buffer Set the temperature of the thermomixer to 30 C and incubate for 10 minutes 400 rpm During this time the thermomixer containing the ATs will cool down from 50 C to 30 C 11 During step 10 prepare a dilution of the Conjugate Solution black 2 ml tube with black cap with Detection Buffer using Appendix 3 at the back of this protocol For this purpose a 1 5 ml tube or a 10 ml tube may be used d
12. CT 103XL Check MDR Appendix 1 Performance Characteristics In silico Specificity The specificity of the Check MDR CT103XL diagnostic test is ensured by the design of optimal target specific probes as well as the selection of stringent reaction conditions Probe sequences were designed to specifically identify the gene variants listed in the Table below A 100 sequence match with the target DNA segment of the probes by in silico analysis was assumed to warrant reliable detection of each of the depicted variants Single mismatches exist in some variants of which we expected that detection would not be compromised This was confirmed by testing such variants in comparison with variants which were 100 homologous GES Carba 2 4 6 13 15 17 18 20 21 GIM 1 IMP 1 3 8 10 13 19 20 24 26 30 KPC 1 17 NDM 1 10 OXA 23 like 23 27 49 73 146 165 171 225 239 OXA 24 like 24 26 33 40 72 139 207 OXA 48 like 48 162 163 181 204 232 244 245 247 370 OXA 58 like 58 96 164 SPM 1 VIM 1 6 amp 8 38 vanes NENNEN BEL 1 3 CTX M1 group See appendix 3 CTX M2 group 2 4 7 20 31 35 43 44 56 59 76 77 95 124 131 9 13 14 16 19 21 24 27 38 45 51 55 57 64 65 67 81 93 102 104 105 106 110 111 112 113 121 122 123 CTX M9 group 126 134 CTX M8 25 group 8 25 26 39 41 63 78 89 91 94 GES ESBL 1 3 7 12 16 19 22 PER 1 7 SHV ESBL 2 5 7 9 10 12 13 15 16 18 23 25 29 30 31 34 3945 46 55 64 6
13. TX M 88 CTX M 132 CTX M 133 CTX M 142 Appendix 3 Thermocycler settings Step A Step B Step Temperature Time mg Step Temperature Time Cycles 1 95 C 3min 1x 1 95 C 10min 1x 2 95 C 30sec 2 95 C 5sec 24x 65 5 min 55 C 30sec 35x 3 98 2min 1x 72 C 30sec Hold 4 3 98 C 2min 1x Hold 4 C Step Melt Step Temperature Time Cycles 1 98 C 2min 1x Hold 4 C 17 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 Appendix 4 Pipetting schemes Pipetting scheme for BC mix Samples ul B 1 60 2 2 90 3 3 120 4 4 210 7 5 210 7 6 210 7 7 300 10 8 300 10 9 300 10 10 390 13 11 390 13 12 390 13 13 510 17 14 510 17 15 510 17 16 600 20 17 600 20 18 600 20 19 690 23 20 690 23 21 690 23 22 780 26 23 780 26 24 780 26 Pipetting scheme for conjugate dilution Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 pl pl en Det Buf 1 3 295 2 4 395 3 5 495 4 10 990 5 10 990 6 10 990 7 15 1485 8 15 1485 9 15 1485 10 20 1980 11 20 1980 12 20 1980 13 25 2475 14 25 2475 15 25 2475 16 30 2970 17 30 2970 18 30 2970 19 35 3465 20 35 3465 21 35 3465 22 40 3960 23 40 3960 24 40 3960 18 ose CT 103
14. XL Check MDR
15. and not applicable to all species Therapeutic failures associated with infections caused by bacteria containing various B lactamase genes are often due to serious problems with interpretation of phenotypic tests Check MDR CT103XL is a rapid molecular test that reliably detects the presence of almost all clinically relevant B lactamase genes in Enterobacteriaceae Pseudomonadaceae and Acinetobacter spp Principle of the method The principle of the Check Points diagnostic system is based on specific molecular recognition of DNA target sequences and subsequent amplification with universal primers Each individual DNA target is recognized by a specific probe that contains a unique ZIP code corresponding to a unique position address on the microarray These ZIP codes are used for detection on the microarray after amplification Probes consist of two segments probe arms which are joined by a DNA ligase when they match perfectly with the target DNA Only connected probe arms will result in amplification products Probes that differ from the target DNA will not give amplification products even in the case of a single nucleotide difference Amplification products are hybridized to the microarray and visualized by colorimetric detection The microarrays are contained in so called CP Array Tubes which are inserted in the Check Points Tube Reader upon completion of the detection reaction This generates an array image that is analyzed by dedicated software to
16. atabase of available results In order to further improve the software original result images including specific data are shared with this database through a default setting in the software by means of an encrypted file This data will be used solely for software improvements All information will be kept strictly confidential and will not be shared with any third parties The specific data included are the number identifying the CP Array Tube the kit lot number the software version used the operator and the customer name All patient data and sample information is removed in the encrypted file Despite the utmost care in the development and preparation of the protocol Check Points cannot take any responsibility for errors omissions and or future changes herein Literature Citation When describing a procedure for publication using this product please refer to it as the Check MDR CT103XL Key to symbols used Symbol Definition EE For In Vitro Diagnostic Use Catalog number LOT Batch code IFU number Use before YYYY MM Consult instructions for use Manufacturer Temperature limitation lt q E Contains sufficient for lt n gt tests Check Points Health BV Tel 31 317 453 908 Binnenhaven 5 Fax 31 317 210 147 6709 PD Wageningen info check points com The Netherlands www check points com 14 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger
17. ction tube Next add 10 ul DNA solution of each sample For concentrated DNA solutions a smaller amount may be required in that case adjust volume to 10 ul with MQ water Please write down the sample reference for each tube 4 Close the tubes mix well by tapping against each tube and spin down briefly using the mini centrifuge 5 Place the tube s in the thermocycler and run the CP step A program total sample volume 18 ul The program will run for approximately 2 5 hours The step A program is outlined in Appendix 2 Note e reaction tubes are NOT supplied with the kit e When closing the tube s don t use excessive pressure as the cap may distort and the sample may then evaporate during steps A and B e Refer to Appendix 2 if the settings of the thermocycler are lost 2 DNA amplification step B Important point before starting the preparation of the step B reaction is carried out in the pre PCR room Procedure 1 Briefly spin down the reaction tubes when step A has been completed 2 Foramplification step B prepare BC mix in a 1 5 ml or 10 ml tube Take Solution B white cap O from the freezer Thaw properly at room temperature mix well and spin down briefly before use Then take Solution C red cap 6 from the freezer Prepare the required amount of BC mix using the pipetting scheme in Appendix 3 First add the required amount of Solution B to the tube Then dispense Solution C in Solution B by pipetting up and down 3 t
18. eck MDR Intended use Check Direct CT103XL is a qualitative in vitro diagnostic test for the molecular detection and typing of a wide range of clinically important B lactamase genes in gram negative bacteria The test detects the following extended spectrum B lactamase ESBL AmpC and carbapenemase genes TEM SHV CTX M PER VEB BEL GES CMY DHA FOX MOX ACC MIR ACT KPC NDM OXA 48 VIM IMP GIM SPM OXA 23 OXA 24 OXA 58 Check MDR CT103XL is also able to classify CTX M gene families and subclasses and to discriminate between ESBL and non ESBL variants of TEM and SHV and between ESBL and carbapenemase variants of GES The assay involves a two step amplification process followed by microarray detection of the reaction products using DNA extracted from bacterial cells Check MDR CT103XL is intended to confirm the presence of ESBL AmpC or carbapenemase genes in bacteria suspected to carry one or more of these genes The test is also intended to classify and type bacterial strains carrying ESBL AmpC and or carbapenemase genes for epidemiological and infection control purposes Check MDR CT103XL is not intended to diagnose infections with Enterobacteriaceae producing ESBL AmpC or carbapenemase genes nor to guide or monitor treatment for these infections Introduction The TEM SHV and CTX M genes encode the clinically most prevalent extended spectrum B lactamases ESBLs These three groups of ESBLs are generally capable of hydrolyzing firs
19. epending on the amount required Dispense the Conjugate Solution in the Detection Buffer by pipetting up and down 3 times Mix well by vortexing for 30 seconds Note e Conjugate Solution is stored at 20 C but is not frozen and can be used directly e Conjugate dilutions have to be made fresh and should be used on the day of preparation e At30 C remove solutions from all ATs before proceeding with adding new solutions to the ATs 12 Remove the Blocking Buffer completely from all ATs Then add 150 ul conjugate dilution to each AT Incubate for 15 minutes at 30 C 400 rpm 13 Remove the conjugate dilution from the ATs and add 600 ul Detection Buffer Incubate the tubes for 2 minutes at 30 C 400 rpm 14 Replace the Detection Buffer with 600 ul fresh Detection Buffer and incubate the tubes again for 2 minutes at 30 C 400 rpm 15 Remove the Detection Buffer from the ATs and add 150 ul Staining Solution to each AT Incubate for 15 minutes at room temperature outside the thermomixer to complete the staining procedure Continue with the image analysis immediately after the 15 minutes incubation time Do not incubate with Staining Solution for more than 15 minutes because images may become too dark During the 15 minutes incubation time complete the required sample information and relevant test data in the Check Points software as outlined in point 16 a g Note e Store the bottle with Staining Solution in the dark after use
20. ettes amp disposable preferable filter tips e for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes Disposable laboratory powder free gloves Pipettes amp disposable preferable filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 10 ml tubes 10 ml tubes PCR tubes suitable for Thermocyclers used contact your local representative for specifications Shelf life Storage and Handling Please check the individual components for optimal storage conditions immediately after delivery of the kit and store components accordingly Reagents stored at the appropriate storage conditions can be used until the expiration date indicated on the boxes Please visually inspect the boxes upon initial opening to ensure that their content is intact Do not use when damaged Please contact the Check Points office at support check points com if you have any questions or in case shipping has taken more than 2 days Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR Good laboratory practices Recommendations for best results The quality of the results depends on strict compliance with the following good laboratory practices especially concerning PCR The test must be performed by adequately trained personnel e Spinning down for a few seconds is done in the various steps to ensure that all material is collected at the bottom of the tubes
21. g very few therapeutic options The carbapenemase genes detected by Check MDR CT103XL are briefly described in the paragraph below Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR Klebsiella Pheumoniae Carbapenemases are plasmid encoded Ambler class A carbapenemases that were first identified in Klebsiella pneumoniae isolates in North America Currently these enzymes have spread to many other parts of the world and are found in several other Enterobacteriaceae species including Escherichia coli New Delhi Metallo B lactamase NDM was first reported in 2009 when found in a Klebsiella pneumoniae and Escherichia coli strain from a Swedish patient repatriated from a New Delhi hospital Recent reports indicate that it has already spread to various European countries notably the U K and that NDM may be prevalent in the Indian subcontinent Carbapenem hydrolyzing oxacillinases are class D f lactamases An important group is the OXA 48 type carbapenemase occurring predominantly in Enterobacteriaceae and originating from Shewanella species This gene group consists of 10 members with OXA 48 and OXA 181 being the most prevalent variants It seems to be most prevalent in Turkey and the North African subcontinent The second important group of OXA type carbapenemases are found predominantly in Acinetobacter species and consist of 3 type members OXA 23 OXA 24 and OXA 58 Each type has several subtypes ou
22. imes Mix very well by vortexing and spin down briefly 3 Add 30 ul of the freshly prepared BC mix to each reaction tube Close the tubes mix by tapping each tube and spin down briefly 4 Transfer the tubes to the post PCR room 5 Place the tube s in the PCR thermocycler and run the CP step B program total sample volume 48 ul The program will run for approximately 1 5 hours The step B program is outlined in Appendix 2 6 Briefly spin down the reaction tubes after amplification step B is completed 7 After completion of step B the reaction tubes may be stored at 4 C for maximum two hours e g in the PCR thermocycler Alternatively store the reaction mixtures at 20 C for a maximum period of two weeks Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR 3 Detection step met L A L Ge Figure 1 A the CP Array Tube AT and B the Check Points Tube Reader Important points before starting the detection step is carried out in the post PCR room Procedure 1 Start preparing the required number of CP Array Tubes ATs for detection approximately 10 minutes before the end of step B One AT is required for every reaction tube Remove the ATs from their package s and place them in the thermomixer 2 Add 300 ul Detection Buffer to each AT Switch on the thermomixer and heat to 50 C It is not necessary to close the tubes Note e Be careful when removing or addi
23. l when sample references have been filled out Double click on one of the sample reference fields A pop up will appear see figure 4 allowing remarks to be added to individual or all samples by marking the box es of the sample s in question Check Points eut 1 Array selection 2 Sample information 3 Detection step l 4 Results e CT 103 Cl 1O3XL 2 Sample information Check MDR Please enter your sample codes in the table below Sample information 1 9 10 11 12 sample 1 Aditional sample information 12 3 4 5 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 13 21 22 23 24 Select all EE Close Deselect all Figure 4 Pop up for additional sample information f Gotothe Detection step screen see figure 5 by clicking the Next Step button g Thesoftware will now indicate the samples to be analyzed first Check Points ie 1 Array selection 2 Sample information 3 Detection step 4 Results l 3 Detection step ee CT 103 XL Cannot initialize AT reader Make sure reader is connected to PC Check MDR Please enter CPTube LOT number place tube in reader and Confirm Sample s sample 1 CPTube LOT number Results Figure 5 Detection step Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 SE e De CT 103XL Check MDR 17 Enter the AT lot number in the appropriate field when the 15 minutes incubation time has been comple
24. lification steps in the post PCR room To keep laboratory free of PCR product contamination e Use pipettes with hydrophobic filter tips e Make sure to always use a new pipette tip when adding solutions or samples to a reaction tube to avoid contamination e Follow proper pipette dispensing techniques to prevent aerosols Use separate equipment pipettes thermocyclers sample holders lab coats gloves disposables and reagents that are assigned to these rooms Never transfer items from the post PCR room to the pre PCR room e Wear a clean lab coat and clean gloves during all steps of the test e Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or during sample preparation e Change gloves whenever you suspect that they are contaminated e Keep the tubes of all kit components and samples closed as much as possible e Clean the lab benches and all equipment regularly with 0 596 sodium hypochlorite solution Users are strongly advised to read the full protocol before starting the test Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 Dee aaa ee e CT 103XL Check MDR Protocol The protocol consists of the following steps 1 DNA recognition step A in which the specific probes hybridize to their target DNA if present When present the probe arms are joint allowing exponential PCR amplification in the next step 2 DNA amplification step B in which
25. ligated probe arms are exponentially amplified and labelled to yield sufficient amplicons for microarray detection on the CP Tube 3 The Detection Step in which the labelled amplicons are hybrized to the microarray and visualized by colorimetric detection The procedure requires nanogram quantities of DNA of high quality We recommend using state of the art commercial DNA extraction methods known to yield high quality DNA Bacterial cells grown in nutrient broth or on nutrient agar plates can best be used as input material for DNA extraction Methods should be used that purify total DNA i e genomic as well as plasmid DNA Generally such methods co purify DNA and RNA from bacterial cells A nucleic acids concentration of 5 50 ng ul should be used Please contact Check Points for suitable DNA extraction methods that have been validated for use with Check MDR CT103XL Store DNA extracts according to the manufacturer s instructions for the DNA extraction method used 1 DNA recognition step A Important points before starting step A is completely carried out in the pre PCR room Procedure 1 Thaw all reagents i e Solution A DNA samples if kept at 20 C mix well and keep on ice 2 For x number of samples add the following to a 1 5 ml tube x 1 2 5 ul Solution P purple cap x 1 5 ul Solution A brown cap 9 Mix well and spin down briefly The solution should have a uniform blue color 3 Add 7 5 ul of this mixture to each rea
26. ng liquids with the pipette to or from the Do not touch the microarray at the bottom of the tube at any time Pipette all material in or out of the AT at the side of the bottom of the tube without touching the array e At 50 C remove and add solutions to one AT at a time to prevent the ATs from drying out When the thermomixer has reached 50 C incubate the tubes for 2 minutes 400 rpm Remove the Detection Buffer from the ATs and repeat step 3 Replace the Detection Buffer with 300 ul fresh Detection Buffer Take the samples from step B Samples stored for longer than 2 hours after step B was completed should be heated in the PCR thermocycler at 98 C for 2 minutes CP Melt program of the PCR thermocycler see Appendix 2 Briefly spin down the reaction mixture 7 Transfer 10 ul reaction mixture from each reaction tube to the corresponding AT Please note that one reaction tube corresponds to one AT The total volume of the AT will be 310 ul The lid of the AT should be labelled for reference gv utm o Note e Samples may contain a white colored precipitate This is due to denaturation of one of the reaction components a protein stabilizer The presence of this precipitate has no effect on the result of the detection step and may be ignored when adding the sample e When adding samples to the AT do not remove the AT from the thermomixer to prevent the buffer from cooling down Add the sample directly into the Detection Buffer of the
27. omonas hydrophilia see table 1 Generally plasmid encoded AmpC genes give rise to significantly increased B lactamase production compared to the chromosomally located AmpC genes Hence it is useful for the clinician to be able to discriminate between plasmid and chromosomally encoded AmpC genes The presence of a plasmidic AmpC gene is indicated when detected in a species that does not have a chromosomal AmpC e g Klebsiella pneumoniae or when the genotype of the AmpC gene differs from the inherent chromosomal gene of the species For example Morganella morganii contains a chromosomal copy of the DHA family Detection of this AmpC gene in a species other than M morganii indicates the presence of plasmid mediated AmpC Table 1 AmpC genes and the species from which they originate AmpC gene Origin of chromosomal gen ee CMY II Citrobacter freundii DHA Morganella morganii FOX Aeromonas caviae ACC Hafnia alvei ACT MIR Enterobacter cloacae amp asburiae CMY I MOX Aeromonas hydrophilia Bacteria carrying ESBL or AmpC genes generally stay susceptible to carbapenem antibiotics During the last decade various carbapenemase genes have been reported associated with elevated or complete resistance against carbapenem antibiotics Strains carrying such genes are generally resistant to all B lactam antibiotics They often have additional B lactamase genes and genes for resistance against quinolones and aminoglycosides leavin
28. pose the Send image to Check Points pop up see figure 9 may also be accessed from the database viewer DBview shortcut that is located on the desktop Open the database by clicking on File followed by Open the database is located in C Images by default and scroll down to the results which require support By clicking on Save the Send image to Check Points pop up will appear just like in the result summary window 11 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 e CT 103XL Check MDR Frequently asked questions FAQ amp Troubleshooting 1 Thethermocycler states an error in step A or B Please contact Check Points Technical Support support check points com 2 During the PCR programs step A or B sample s have partly evaporated Tubes may not have been closed properly Please restart the procedure from step A 3 Ihave left Solutions A B and or C out of the 20 C 4 storage These reagents must be stored at 20 C 4 F for proper performance of the test The performance of the product cannot be fully guaranteed if these solutions were left out of 20 C 4 F for longer than 24 hours 4 Staining Solution turned blue after adding it to the AT Conjugate dilution was not properly removed by washing steps 13 and 14 detection step Continue incubation with Staining Solution for 15 minutes and take the image as described in the protocol If the image is too dark please refer to
29. question 5 5 Thepicture of the array is very dark The conjugate dilution detection step 12 was not properly removed by washing steps 13 and 14 detection step Please replace the Staining Solution with Detection Buffer and take image immediately If the image is still too dark please repeat detection step with new AT 6 Software indicates Background intensity too high See question 5 7 Thesoftware indicates Image too weak The intensity of the spots is too low Causes may be 7 1 The conjugate dilution was not prepared and or added properly Please repeat detection step with new AT 7 2 Incubation time with the Staining Solution was shorter than 15 minutes Continue incubation up to 15 minutes 7 3 Detection Buffer was not removed properly before adding Staining Solution Please repeat detection step with new AT 8 The software indicates Reference spots not found The software did not find the reference spots on the AT Causes may be 8 1 An air bubble is interfering with the result Tap the AT gently or pipette the liquid gently up and down and then retry to take the image 8 2 The picture is very dark conjugate dilution was not removed properly Please refer to question 5 8 3 The picture is completely white Staining Solution was not added Please add Staining Solution again and proceed from detection step 15 If the results do not improve then the staining has failed Most likely no conjugate dilution was added
30. rba 13 150 10096 10096 GIM 2 161 10096 10096 IMP 10 153 9096 10096 KPC 6 157 10096 10096 NDM 8 155 10096 10096 OXA 23 like 7 7 10096 10096 OXA 24 like 5 158 10096 10096 OXA 48 like 7 156 10096 10096 OXA 58 like 4 159 10096 10096 SPM 1 163 10096 10096 152 10096 10096 EES Negative ER CTX M1 group 136 10096 100 CTX M2 group 4 159 100 100 CTX M9 group 10 153 100 100 CTX M8 25 group 2 161 100 100 GES ESBL 15 148 100 100 PER 7 156 100 100 SHV ESBL 22 141 100 100 TEM ESBL 18 145 100 100 VEB 9 154 100 100 Positive Negative Ge e ACC 4 159 10096 10096 ACT MIR 11 152 100 100 CMY 1 MOX 2 161 100 100 CMY 2 7 156 10096 10096 DHA 7 156 10096 10096 FOX 2 161 10096 10096 16 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 EE e CT 103XL Check MDR Appendix 2 CTX M1 group subtyping The Check Points software reports four subgroups belonging to the CTX M1 group CTX M1 like CTX M15 like CTX M3 like and CTX M32 like CTX M1 group CTX M1 subgroup CTX M15 subgroup CTX M3 subgroup CTX M32 subgroup CTX M 1 CTX M 15 CTX M 3 CTX M 32 CTX M 23 CTX M 28 CTX M 10 CTX M 53 CTX M 52 CTX M 29 CTX M 11 CTX M 55 CTX M 58 CTX M 33 CTX M 12 CTX M 57 CTX M 60 CTX M 71 CTX M 22 CTX M 69 CTX M 61 CTX M 72 CTX M 30 CTX M 79 CTX M 62 CTX M 96 CTX M 34 CTX M 114 CTX M 68 CTX M 101 CTX M 36 CTX M 116 CTX M 103 CTX M 37 CTX M 136 CTX M 107 CTX M 42 CTX M 139 CTX M 108 CTX M 54 CTX M 109 CTX M 66 CTX M 117 C
31. t second third and fourth generation cephalosporins penicillins and monobactams thereby limiting treatment options TEM and SHV ESBL subtypes are derived from their parental sequences by point mutations leading to amino acid substitutions www lahey org studies These amino acid substitutions may extend the substrate spectrum to hydrolyze a wide range of B lactam antibiotics i e ESBL The CTX M genes originate from Kluyvera species and are presently the most prevalent ESBLs They can be divided into 5 different groups according to their amino acid sequence CTX M 1 CTX M 2 CTX M 9 CTX M 8 and CTX M 25 The VER PER BEL and GES ESBL gene families are generally less prevalent B lactamase types Some of these are found locally BEL while others are spread internationally VEB PER and GES They are typically found in Pseudomonas aeruginosa and Acinetobacter baumannii however there are numerous reports of VEB GES and PER in Enterobacteriaceae as well The AmpC cephalosporinases are able to hydrolyze almost all B lactam antibiotics including penicillins cephalosporins and monobactams Unlike ESBLs they are not inhibited by clavulanic acid Many gram negative bacteria carry a chromosomal copy of an AmpC gene and plasmid encoded AmpC genes have emerged from the chromosomes of these Enterobacteriaceae Species carrying a chromosomal AmpC gene include Citrobacter freundii Morganella morganii Aeromonas caviae Hafnia alvei Enterobacter spp and Aer
32. table 2 for a list of the genes that can be reported Please note that for the CTX M 1 group additional information regarding the subgroup will be displayed see Appendix 1 Click on the Quit button to end this run of analyses Check Points 0 1 Array selection 2 Sample information l 3 Detection step f sults 4 Results vi e CT 103XL Check MDR Presence Sample name CARBA ESBL AMPC CARBA gene AMPC gene CTX M group SHV SNP TEM SNP ESBL other 1 CPTube 12345678 Y 48 1 type 15 like mp CPTube 34567890 WP sample3 In case assistance is needed please double click on sample name to send picture to Check Points Please refer to appendix 1 in user manual Quit Print results Analyse picture Figure 7 Summary of the results 10 Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 ger CT 103XL Check MDR Table 2 6 lactamase genes reported by the Check Points software carbapenemases i ESBLs Minor ESBLs AmpCs GES CTX M 1 group TEM wt SHV wt BEL ACC GIM CTX M 1 subgroup TEM 104K SHV 238A GES ACT MIR IMP CTX M 2 group TEM 164C SHV 238S PER CMY I MOX KPC CTX M 3 subgroup TEM 164H SHV 240K VEB CMY Il NDM CTX M 8 group TEM 1645 DHA OXA 23 CTX M 9 group TEM 238S FOX OXA 24 CTX M 15 subgroup OXA 48 CTX M 25 group OXA 58 CTX M 32 subgroup VIM SPM Note
33. ted optional use the Barcode Reader to scan the AT lot numbers Next insert the AT with open cap into the reader close the lid of the reader and click on the Confirm button in the software Then click on Scan image the results will be displayed immediately see figure 6 Finally click on Save Results to save the results in the database The software will now indicate which sample should be analyzed next Repeat this step until all ATs are analyzed Check Points olm 1 Anay selection 2 Sample information 3 Detection step 4 Results Scan ready 3 Detection step e 1 3 XL Reader found test Check MDR Please enter CPTube LOT number place tube in reader and Confirm Sample s sample 1 CPTube LOT number 12345678 Results ESBL Figure 6 Presentation of the final result Note e It is important to adhere to the 15 minutes incubation time with Staining Solution as much as possible step 15 A shorter incubation time may lead to faint images exceeding the 15 minutes incubation time may lead to overstaining In both cases incorrect results may be obtained 18 When all ATs have been analyzed a new window with the summary of the results will appear see figure 7 which may also be printed click on the Print results button The results summary will display the sample ID and the presence absence of carbapenemases plasmidic AmpC and ESBL as well as combinations thereof see
34. tlined in Appendix 1 of this manual VIM carbapenemase is a metallo B lactamase first reported in a Pseudomonas aeroginosa isolate found in Verona in 1997 Since then it has transferred to E coli and K pneumoniae and many other Enterobacteriaceae Presently a total of 38 variants of the VIM gene have been reported of which the DNA sequences may differ significantly Therefore VIM should be regarded as a family of homologous gene variants The IMP group of metallo B lactamases presently consists of 44 members IMP 1 was one of the first carbapenemases to be reported and was detected in Japan in 1990 in a Pseudomonas aeruginosa isolate As with VIM the gene sequences of these IMP types may vary significantly They are spread worldwide and have been found in various Enterobacteriaceae including E coli Klebsiella and Enterobacter GIM 1 and SPM 1 are less frequently found metallo B lactamases GIM 1 originates from Germany and was reported in P aeruginosa Enterobacteriaceae and Acinetobacter spp SPM 1 producing P aeruginosa is endemic in Brazilian hospitals and is associated with numerous outbreaks The GES gene family consists of both ESBL types and carbapenemase types Both types are detected and discriminated by CT103XL Appendix 1 depicts the various types and there B lactamase properties Conventional methods for detection of B lactamases rely on phenotypic identification which is time consuming frequently inconclusive
35. yield a definitive and objective assay result Check MDR CT103XL User manual Version 1 0 Issued 01 08 2014 e CT 103XL Check MDR Kit contents for 24 samples Components Mat No Storage conditions Box Room Temperature Detection Buffer 9 0007 1 bottle 80 ml Room temperature Blocking Buffer 9 0008 1 bottle 20 ml Room temperature Staining Solution 9 0014 1 bottle 5 ml Room temperature store in the dark CP Array Tubes 10 0003 6 bags of 4 each total 24 Manual 9 0039 Leaflet download from website Not critical Room temperature Solution P 9 0035 1 tube purple cap 100 ul 20 C Solution A 9 0021 1 tube brown cap 600 ul 20 C Solution B3 9 0019 1 tube white cap O 1600 ul 20 C Solution C 9 0024 1 tube red cap 120 ul 20 C Conjugate Solution 9 0027 1 black tube amp cap 120 ul 20 C Positive and negative controls are built into the system It is however strongly recommended to use a positive and negative control for each series of reactions Materials required but not supplied with the kit Thermocycler Thermocycler Vortex Mixer Vortex Mixer Mini centrifuge Mini centrifuge Equipment Thermomixer with active cooling Check Points Tube Reader with E Ads software Computer with USB and internet connection Barcode Reader optional Supplies Disposable laboratory powder free gloves Pip
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