User manual - Check
Contents
1. Sample s 5000001 S000002 CPTube LOT number 5000003 Confirm 25850001 eoa TT e Se lel PETE D ECCO EE 8 3 aioe EE 16 Figure 7 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 8 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 table 2 for a list of the genes that can be reported Click on the Quit button to end this run of analyses Version 2 1 Issued 1 October 2012 os Po Check MDR CT 101 Send image to Check Points Check Points DER usage RE Sse ee LS oe een 4 Results e Check MDR CT 101 Presence Typing Sample name CPTube 25850001 S000001 238S 240K Figur e 9 s000002 238S 240K H i oe 000003 2108 240K Send image to Check Points pop up ___ PTube 25850001 S000004 000005 CMY H DHA WT 000006 CMY II WT l ee 1 1 lt lt lt Comments lt lt lt lt lt2. and 4 Analysis is not possible without sample codes g The software will now indicate the samples to be analyzed first Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 DER lt 1 Check Points 3 Detection 1 Array selection 2 Sample information 3 Detection step Reader found BM000805 e e Check MDR 6 CT 101 Please enter CPTube LOT number place tube in reader and Confirm Sample s S000001 000002 CPTube LOT number 000003 SY 5000001 No result S000002 No result 5000003 No result Figure 6 Detection step 17 Enter the AT lot number in the appropriate field when the 15 minutes incubation time has been completed 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 7 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 MDR CT101 User manual O Check MDR CT 101 DER lt 1 Check Points 3 Detection 1 Array selection 2 Sample information Scan ready 3 Detection step Reader found test Check MDR CT 101 Please enter CPTube LOT number place tube in reader and Confirm
3. Klebsiella Pneumoniae Carbapenemases KPC 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 is a new type of carbapenemase that 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 Conventional methods for detection of B lactamases rely on phenotypic identification which is time consuming frequently inconclusive and not applicable to all species Therapeutic failures associated with infections caused by bacteria containing various B lactamase genes 2 are often due to serious problems with interpretation of phenotypic tests Therefore the capability of accurate and objective molecular detection is becoming more and more important Check MDR CT101 is a microarray based diagnostic test that identifies the presence of TEM SHV CTX M KPC NDM and AmpC genes as well as mutations leading to extended spectrum types of TEM and SHV enzymes Check MDR CT101 detects TEM mutations at positions E104K R164S H and G238S and SHV muta
4. 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 in the post PCR room e The 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 amplification steps in the post PCR room To keep laboratory free of PCR product contamination Use pipettes with hydrophobic filter tips Make sure to always use a new pipette tip when adding solutions or samples to a reaction tube to avoid contamination 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 Wear a clean lab coat and clean gloves during all steps of the test Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or during sample preparation Change gloves whenever you suspect that they are contaminated Keep the tubes of all kit components and samples closed as
5. 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 AT by pipetting up and down e With the completion of step 7 three samples have been added to one AT 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 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 Note 10 11 e Collect the liquids removed from the ATs in a disposable tube and dispose of it the same day with the other laboratory waste Replace the Blocking Buffer with 300 ul fresh Blocking Buffer Set the temperature of the thermomixer to 30 C and incubate
6. Buffer 9 0008 1 bottle 20 mi Staining Solution 9 0014 1 bottle 5 ml OR e store in the dark Box 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 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 Materials required but not supplied with the kit Equipment Supplies Pre PCR Thermocycler Vortex Mixer Mini centrifuge 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 contact your local representative for specifications Post PCR Thermocycler Vortex Mixer Mini centrifuge Thermomixer with active cooling Check Points Tube Reader with E Ads software Computer with USB drive and internet connection Barcode Reader optional Disposable laboratory powder free gloves Pi
7. 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 16 e Store the bottle with Staining Solution in the dark after use Filling out the experimental data a Start the computer b Start software on the computer by double clicking the Check Points desktop icon e e e p Check Points c Double Click on Check MDR CT101 arr in the first screen Array selection as shown in figure 2 one Check MDR CT 101 Check Points 1 Array selection Check Points 2 Sample information 3 Detection step 3 Detection 4 ld 1 Array selection 2 Sample information 3 Detection step 3 Detection 4 gt 1 Array selection 2 Sample information ose Check MDR CT 101 ose Check MDR CT 101 Please choose array type from the list below C amp T Salmonella arr C amp T definition 02112011 Check MDR CT101 arr Check MDB CT101 26 4 2012 Check MDR CT102 arr Check MDR CT102 26 4 2012 Check MDB CT103 arr Check MDB CT103 26 4 2012 Please enter your sample codes in the table below S000001 S000004 S000007 Please enter lot number of the kit 5000002 60
8. for 10 minutes 400 rpm During this time the thermomixer containing the ATs will cool down from 50 C to 30 C During step 10 prepare a dilution of the Conjugate Solution black 2 ml tube with black cap with Detection Buffer using Appendix 2 at the back of this protocol For this purpose a 1 5 ml tube or a 10 ml tube may be used depending on the amount required Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 Dispense the Conjugate Solution in the Detection Buffer by pipetting up and down 3 times Mix well by vortexing for 30 seconds Note 12 13 14 15 e The 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 At 30 C remove solutions from all ATs before proceeding with adding new solutions to the ATs 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 Remove the conjugate dilution from the ATs and add 600 ul Detection Buffer Incubate the tubes for 2 minutes at 30 C 400 rpm Replace the Detection Buffer with 600 ul fresh Detection Buffer and incubate the tubes again for 2 minutes at 30 C 400 rpm 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
9. 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 or the conjugate dilution was not prepared properly Please repeat detection step with new AT of 20 C 4 F for longer than 24 hours 9 The software indicates Hybridisation not OK The software did not find the hybridisation control spots on the AT The hybridisation Staining Solution turned blue after adding it to the AT control is used to check if the hybridisation at 50 C of the PCR product with the AT has Conjugate dilution was not properly removed by washing steps 13 and 14 detection been Ai properly E be aia 4 i step Continue incubation with Staining Solution for 15 minutes and take the image as 3 1 y s a gt SAR Srey Cali COUE ATE OUIOT Wee Or F EOVEE PrO pEr described in the protocol If the image is too dark please refer to question 5 Please re re GUEREN a 9 2 The reaction mixture after completion of step B was not added to the AT Please The picture of the array is very dark a A step with new Aam i p wih he th The conjugate dilution detection step 12 was not properly removed by washing steps 9 3 Hybridisation Secale a 18n Pease i Y Dar ME ANEUNOMIXET 13 and 14 detection step Please replace the Staining Solution with Detection Buffer Salk areiete Was 20 E WOEN a Als ae nee dded h
10. much as possible Clean the lab benches and all equipment regularly with a 0 5 sodium hypochlorite solution Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 Protocol It is strongly recommended that the full protocol is read before using the test The protocol consists of the following steps 1 DNA recognition step A 2 DNA amplification step B 3 Detection step 1 DNA recognition step A Important points before starting e Step Ais completely carried out in the pre PCR room e To ensure the best results we recommend using purified nucleic acids isolated from colonies or cells grown in nutrient broth Methods should be used that purify total DNA i e genomic as well as plasmid DNA Generally such methods copurify DNA and RNA from bacterial cells A nucleic acids concentration of 5 50 ng ul should be used compensating for the excess of RNA generally present in DNA preparations using commercially available extraction methods Column based isolation methods are recommended e Store DNA extracts according to the manufacturer s protocol for the DNA extraction method used Procedure 1 Thaw all reagents i e Solution A DNA samples if kept at 20 C mix well and keep on ice First add 5 ul Solution A brown cap to every reaction tube of the strip supplied with the kit Next add 10 ul DNA solution of each sample NB use 1 100 part of the elution volume of the isola
11. test with the diluted DNA 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 Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 11 11 12 13 14 15 16 10 5 Reaction mixtures from step B step 7 of detection step were not all added to the AT 3 reaction mixtures should be added one or two may be omitted 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 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 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 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 su
12. 00005 5000008 S000006 Operator DE Figure 4 Sample information Figure 2 Array Selection Note d Enter the lot number of the kit in the appropriate field and the name of the e Additional remarks may be added per sample optional when sample references operator followed by pressing the Next Step button have been filled out Double click on one of the sample reference fields A pop up will appear see figure 5 allowing remarks to be added to individual or all samples by marking the box es of the sample s in question Hef 1 Check Points Ref 2 1 Array selection 2 Sample information 3 Detection step 3 Detection aly Figure 3 Hef 3 2 Sample i i ple information Example of filling out sample references in the software o Check MDR and assigning samples to the corresponding tubes a Please enter your sample codes in the table below Sample information Ref 1 5000004 S Aditional sample information 5000002 5000005 S Ref Fa S000003 S000006 1 2 3 4 5 6 amp 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 AMM MMMM MMMM MMMM wv BMH VWVMM MMMM Mw wv www Ref 3 CHV MV VV VV RP OVP ORO Additional information Select all Deselect all Figure 5 Pop up for additional sample information Ml H r H Ml m e Insert the sample codes in the screen Sample information as shown in figures 3 f Goto the Detection step screen see figure 6 by clicking the Next Step button
13. 64S SHV 238A DHA CTX M 9 group TEM 164H SHV 240K ACT MIR TEM 238S CMY II Comments Note e For support concerning results please send the result image along with the desired Figure 10 information to support check points com For this purpose double click on the Check the box to send pictures directly over the internet result s in question in the result summary window A pop up will appear see figure 9 in which your comments may be added to the file om sl Your email address Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 10 Frequently asked questions FAQ amp Troubleshooting The thermocycler states an error in step A or B Please contact Check Points Technical Support support check points com 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 have left Solutions A B and or C out of the 20 C 4 F 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 one Check MDR CT 101 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
14. User manual Check MDR CT101 Rapid Molecular Detection and Identification of Carbapenemases KPC and NDM ESBL CTX M TEM and SHV and AmpC CMY DHA FOX MOX ACC MIR and ACT 10 0020 72 CE IFU 020 01 Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 Key to symbols used For In Vitro Diagnostic Use Catalog number Batch code IFU Instructions for use number Use before YYYY MM li Consult instructions for use Contains sufficient for lt n gt tests Manufacturer Temperature limitation INTENDED USE siinissicsscnasantnsnnvasiaeasnessnaeswanseuasiasdesnssaiansnsnessisenteasanessassennesmiecensaessas 2 INTRODUCTION scsssnissenassctenestossinssnesenaeswansdunssansssuseniaeonsnesnisensesadsessansavassmiaeensoatsns 2 PRINCIPLE OF THE METHOD sigossissscsssicosnassssastassonnstandennsaosiusennea snes snambunseauisennnoasaes 3 KIT CONTENTS FOR 72 SAMPLES seiiccssiscessvetecessteanccusvesssccacectccuewieoensaeeanoceswecccters 3 SHELF LIFE STORAGE AND HANDLING ccecceccccecsccececcccececcecescecescecesessnees 4 MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT ssssssssesesssosssosssssssssssss 4 GOOD LABORATORY PRACTICES wiicsscscscssccsesuseecsacsnacodancsvecsnavanesesessuacsnsoesuseedseoaes 4 PROTOCOL isiccssusssiaesececsuaeseuasessveileestnaestnwosdonneneatassndsesdagedawsnessenessnaeseisonntensunoeeaes 5 1 DNA RECOGNITION STEP A srriccriirairiin i
15. ber of CP Array Tubes ATs for detection approximately 10 minutes before the end of step B One AT is required for every 3 tube strip Remove the ATs from their package s and place them in the thermomixer 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 a e Be careful when removing or adding liquids with the pipette to or from the AT 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 6 program of the PCR thermocycler see Appendix 1 Briefly spin down the reaction mixture Transfer 10 ul reaction mixture from each tube of one strip to the corresponding AT in total 30 ul per AT The total volume of the AT will be 330 ul The lid of the AT should be labelled for reference Note e Samples may contain a white colored precipitate This is due to denaturation of
16. break management 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 first 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 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 rod 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 Aeromonas hydrophilia see table 1 Generally plasmid encoded AmpC genes give rise to significantly increased B lactamase production compar
17. e Check MDR CT 101 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 CT101 Check Points rapid molecular detection Tel 31 317 453 908 Fax 31 317 210 147 info check points com www check points com Check Points Health BV Binnenhaven 5 6709 PD Wageningen The Netherlands 14
18. ed 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 Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 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 in a species not being M morganii will indicate the presence of plasmid mediated AmpC Table 1 AmpC genes and the species from which they originate AmpC gene Origin of chromosomal gene CMY II Citrobacter freundii FOX Aeromonas caviae ACT MIR Enterobacter cloacae amp asburiae 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 leaving very few therapeutic options The carbapenemase genes tested for with Check MDR CT101 are briefly described in the following text
19. es 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 Check MDR CT101 requires DNA purified from a colony or bacterial culture Clinical soecimens cannot be tested directly The assay has been tested extensively with purified DNA from gram negative bacteria such as Escherichia Salmonella Klebsiella Enterobacter Citrobacter and Pseudomonas 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 Furthermore although TEM SHV and CTX M are the most prevalent ESBL genes various minor ESBL genes exist However these are not frequently found in clinical settings For more details see T Naas L Poirel and P Nordmann Clin Microbiol Infect 2008 14 Suppl 1 42 52 Check MDR CT101 cannot and does not make any representation or warranty that it is capable of correctly detecting the AmpC ESBL KPC and NDM 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
20. icet E EEEE N 5 2 DNA AMPLIFICATION STEP B cceiwsssscansversvesedauswercenweraboassnsteaeneescotmeisaumereousrnanenan 6 Se DETECHON TE Porna E E A 6 FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING csesceessees 11 APPENDIX 1 civacscasonsscsuasonsesnnsssuasvensainesnecsesiessenedunsobssesudeseusensnesnieedsnoeiacsutesanenseans 13 PPPENDIAX 2 ssicsscinsdateeiacovuessvedstucsnydeteaecvdesendssuessuabesanveensssdmestudeduessuestuaeseiessnneisess 13 UMMA TIONS sescusioccnedasteciessiecokanssvesivestwessedapheessiimesstne nanesiesttnestodenbesywnssesesenasecens 14 1 Intended use Check MDR CT101 is designed for the rapid molecular detection of a wide range of clinically important B lactamase genes in gram negative bacteria Check MDR CT101 detects the TEM SHV and CTX M extended spectrum B lactamase ESBL genes AmpC B lactamase genes CMY DHA FOX MOX ACC MIR and ACT the Klebsiella Pheumoniae Carbapenemase gene KPC and the New Delhi Metallo B lactamase gene NDM In most cases the presence of the genes indicates the expression of extended or full spectrum B lactamase activity For TEM and SHV ESBL and non ESBL variants exist and the test employs highly specific DNA markers to allow distinction between these variants Check MDR CT101 generates definitive results within the same working day compared to the 24 hours necessary for analysis with conventional phenotypic methods Information on gene type yields extra information for out
21. lt ___ cPTube 25850003 000007 S000008 S000009 DHA ACT MIR DHA ACT MIR DHA ACT MIR lt lt lt lt lt lt Options 7 Send picture directly over internet connection In case assistance is needed please double click on sample name to send picture to Check Points Figure 8 Summary of the results 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 Table 2 6 lactamase genes reported by the Check Points software Support may also be requested at a later stage when the program has been closed For this NDM CTX M 1 group TEM wt SHV wt CMY I MOX purpose the Send image to Check Points pop up see figure 10 may also be accessed from KPC CTX M 2 group TEM 104K SHV 2385 ACC 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 lmages 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 CTX M 8 amp 25 group TEM 1
22. ol and take image immediately If the image is still too dark please repeat detection step Boe AEP DE MIX was MOK DES are dl PLOPENI OF WAS NOL AANE to meds Deer ate eee repeat the test 10 The software indicates Reaction not OK please contact Check Points Software indicates Background intensity too high See question 5 The software 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 The software indicates Reference spots not found The software did not find the reference spots on the AT Causes may be 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
23. ontains 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 12 Appendix 1 Step A Step Temperature Time Cycles 95 C 30sec 24x Step B Step Temperature Time Cycles es ee fre rao froin ec Step Melt Step Temperature Time Cycles Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 Appendix 2 Pipetting scheme for BC mix Ke ioe 25 31 3 43 49 28 S 61 67 69 15 21 2 33 39 45 51 63 on Check MDR CT 101 Pipetting scheme for coniugare dilution Gon idaste ul a ae ae 13 Limitations Check MDR CT101 uses a range of specific DNA markers to identify the presence or absence of ESBL genes AmpC genes and KPC and NDM carbapenemase genes The test detects the presence of the ESBL genes TEM SHV CTX M and it also detects Single Nucleotide Polymorphisms SNPs corresponding to amino acid positions 104 164 and 238 in TEM and 238 and 240 in SHV 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 CT101 also detects the AmpC gen
24. pettes amp disposable preferable filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 10 ml tubes Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 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 e 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 e Do not 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 CT101 may therefore yield 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
25. re 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 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 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 Duplicate DNA samples tested with Check MDR CT101 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 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 Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 17 18 one Check MDR CT 101 weeks For further inquiries please contact Check Points Technical Support support check points com 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 The AT image c
26. reliable results from Check MDR CT101 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 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 database 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 Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 on
27. tion and add up to 10 ul with MQ water or 0 1 TE buffer Please write down the sample reference for each tube of the strip Close the tubes and spin down briefly using the mini centrifuge to collect both sample and Solution A at the bottom of the tubes Mix well by tapping against each strip the solution should have a uniform blue color Spin down briefly again using the mini centrifuge Place the strip s in the thermocycler and run the CP step A progam total sample volume 18 ul The program will run for approximately 2 5 hours The step A program is outlined in Appendix 1 Note e The reaction tubes supplied with the kit are prefilled with a small amount of blue colored reagent Proper mixing of this reagent Solution A and the sample is crucial for optimal performance e When closing the tubes of the strip 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 1 if the settings of the thermocycler are lost 2 DNA amplification step B Important point before starting e The preparation of the reaction mix for step B is carried out in the pre PCR room Procedure 1 2 Briefly spin down the reaction tubes when step A has been completed For amplification 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 Sol
28. tions at position G238S A and E240K covering the clinically most relevant TEM and SHV ESBLs Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 one Check MDR CT 101 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 yield a definitive and objective assay result Kit contents for 72 samples Components Mat No Description Storage conditions Box Room Temperature Detection Buffer 9 0007 1 bottle 80 ml Blocking
29. ution C red cap from the freezer Prepare the required amount of BC mix using the pipetting scheme in Appendix 2 First add the required amount of Solution B to the tube Then dispense Solution C in Solution B by pipetting up and down 3 times Mix very well by vortexing and spin down briefly Add 30 ul of the freshly prepared BC mix to each sample in the strip s Close the tubes mix by tapping each strip and spin down briefly Transfer the strips to the post PCR room Place the strip 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 1 Briefly spin down the reaction mixture after step B has been completed Leave the reaction tubes in the PCR thermocycler at 4 C for a maximum period of two hours until step 5 of the detection step has been completed Alternatively store the reaction mixtures at 20 C for a maximum period of two weeks Check MDR CT101 User manual Version 2 1 Issued 1 October 2012 e Check MDR CT 101 3 Detection step Dd ypo chec E Figure 1 A the CP Array Tube AT and B the Check Points Tube Reader Important points before starting e The detection step is carried out in the post PCR room e The ArrayTube DNA microarray platform see figure 1 is sold under license from Alere Technologies GmbH Procedure 1 Start preparing the required num
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