User manual - Check
Contents
1. Positive control ABI 7500 Esse s 25 3 29 3 26 3 24 3 30 3 Rotor Gene Positive control LC 480 I amp II 28 3 30 3 27 3 2633 3033 Negative sample extracted with IC FOR ALL N A N A N A N A 30 3 2 Results interpretation Table 9 If the run is valid interpret results as positive negative or invalid with the values obtained for the samples with the guidelines summarized in Table 9 Positive carbapenemase samples will show a C value in the FAM VIC and or Red TXR channel Positive carbapenemase samples will also show C value in the Cy5 channel if the target has not out competed the internal control IC for the resources during the reaction Negative carbapenemase samples will show no C value in the FAM VIC and Red TXR Channel In the Cy5 Channel a C value is expected at 30 33 e Samples with a FAM VIC and or Red TXR C undetermined or N A and with a Cy5 IC showing no value or a C 233 mean that the sample is invalid see FAQ and Troubleshooting 3 to7 Table 9 Data interpretation guidelines KPC NDM VIM 48 values values Interpretation YES YES or N A Positive sample 30 3 Negative sample N A N A or 233 Invalid If observed C values vary significantly from expected C values see FAQ and Troubleshooting section 8 Frequently asked questions FAQ amp Troubleshooting 1 May other specimen prepa2. Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale Adjust FAM green KPC Auto calc lated 1605150 0 02 threshold manually using the threshold bar on the amplification plot Blue FAM signal VIC yellow NDM VIM 0 001 Auto calculated 200 250 0 015 TXR orange OXA 48 0 006 Auto calculated 400 800 0 03 Cy5 red IC 0 003 Auto calculated 40 100 0 06 Check Direct CPE User manual Version 2 2 Issued 05 05 2014 7 4 For use on the Rotor Gene Q 1 Open the data file Open the raw channel page for each detector Select Options and Crop start cycles In the pop up window Remove data before cycle enter 10 and select OK 2 Select Analysis and the proper channel from 10 and press Show Then use the parameters indicated in Table 7 to analyze each detector channel 4 Setthe threshold manually for each detector channel using the value recommended in Table 6 5 Check amplification plots versus C values calculated by the software for each target in the Quant Results table figure 4 e Table 7 Rotor Gene Q analytical parameters Green KPC ON ON 1096 10 Yellow NDM VIM ON ON 6 10 Orange 48 ON ON 1096 10 Red IC ON ON 1596 10 S Quant Results Cycling 10 Green Page 1 Dynamic Tube lt Slope Correct Ignore First 2 Outlier Removal b Save Defaults E NES
3. e PCR grade water e g Milli Q e Device to measure McFarland OD oo e Follow proper pipette dispensing techniques to prevent aerosols culture e Eppendorf tubes safe lock Centrifuge for Eppendorf tubes e Wear clean disposable gloves and clean lab coats for the different steps of the test e Heating block for Eppendorf tubes e Change gloves whenever you suspect that they are contaminated 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 Direct CPE User manual Version 2 2 Issued 05 05 2014 3 Specimen collection and DNA extraction 1 Human specimen collection In order to obtain adequate specimen the procedure for specimen collection must be followed carefully with adequate swab material see section Materials required but not supplied with the kit 1 Collect perianal rectal specimen according to local guidelines and swabs manufacturer recommendations 2 Place swabs in their containers containing 1 ml of liquid transport medium 3 Labelthe containers 4 Refertothe swab manufacturer instruction for storage handling and stability 2 DNA extraction from perianal rectal swabs with NucliSENS easyMAG Important points before starting Check Points advices to validate your specimen collection and pr
4. e Reagent solutions are degraded or may have expired Troubleshooting for invalid result Repeat the test using the same DNA extract If invalid results persist repeat the test by preparing a new DNA extract from the original specimen Alternatively repeat the test with a new DNA extraction from a newly collected specimen Check Direct CPE User manual Version 2 2 Issued 05 05 2014 10 e Check Direct CPE Real time results show very low fluorescent signals in all samples and detector channels including the internal control signal Possible causes and troubleshooting CPE solution containing the fluorescent probes and primers is degraded Please check expiration date the number of thaw freezing cycles that the CPE solution tube has undergone and if the kit was stored correctly real time PCR system may be responsible for these results Please refer to instrument User s manual or contact your real time PCR instrument local representative C C C values troubleshooting Samples with very low C values and no amplification curves The manual threshold is too low Samples showing value 15 and no amplification curve are considered negative The observed C value is an artifact of the software analysis the threshold crosses the background noise of the curve Invalid Run values expected for the controls in Table 6 do not match the values observed In the experiment Check that these diff
5. is a registered trademark of Medical Wire amp Equipment ESwab is a trademark of Copan Italia S p A ABI FAM VIC ROX are registered trademarks of Applera Corporation Cy5 is a registered trademark of Amersham Biosciences Ltd Texas Red is a registered trademarks of Molecular Probes Inc NucliSENS easyMAG is a registered trademark of bioM rieux LightCycler is a registered trademark of Roche Diagnostics Check Direct CPE User manual Version 2 2 Issued 05 05 2014 Check Points Health BV Binnenhaven 5 6709 PD Wageningen The Netherlands Check Points rapid molecular detection Tel 31 317 453 908 Fax 31 317 210 147 info check points com www check points com 12
6. Analytical Specificity The experimental specificity of the Check Direct CPE real time diagnostic test was determined by testing the cross reactivity with samples containing non target organisms 132 carbapenemase negative strains were used to test the specificity of the Check Direct CPE real time test see bacterial strains listed in Table C Results All isolates tested negative with the Check Direct CPE assay and the internal control was detected in all samples The Check Direct CPE test showed 100 specificity based on the reference strains tested Table C Organisms Organisms Citrobacter freundii 5 Enterococcus faecalis 2 Campylobacter jejuni 2 Klebsiella oxytoca 1 Enterobacter aerogenes 1 Klebsiella pneumoniae 16 Enterococcus casseliflavus 1 Pseudomonas aeruginosa 2 Enterobacter cloacae 42 Staphylococcus aureus 2 Escherichia coli 51 Salmonella thypimurium 1 Pseudomonas mirabilis 3 Stenotrophomonas maltophilia 2 Serratia marcescens 1 10 3 Analytical Reactivity To evaluate the analytical reactivity a retrospective study was performed with 93 bacterial strains of 26 different gram negative species Table D The 93 bacterial strains tested with Check Direct CPE were previously identified carbapenemase positive with the micro array diagnostics test Check MDR C 103 Check Points Heatlth Results All 93 bacterial strains were typed correctly for the targeted carbapenemas
7. accuracy and precision of pipettes as well as correct functioning Product no 04729692001 e Plate centrifuge and calibration of the instruments Real time e 4 Color Compensation Set Ref 18 0070 Check Points Health B V NL 2 PCR i A e PCR plate seal Prevention of contaminations CFX96 e 96 well PCR white plate e CFX96 Bio Rad US Use separate rooms a pre PCR room and a post PCR room PCR plate seal Plate centrifuge UE e DNA extraction and preparation of the amplification reactions are carried out in the Rotor PCR strip tubes and caps 0 1ml Rotor Gene Q 5plex re PCR room GeneQ QIAGEN US e 72 well rotor and locking ring nS i i e Loading block 72x0 1ml tubes ncubation in the real time PCR thermocycler is carried out in the post PCR room Rectal e 5 5 easyMAG Extraction kit 5 5 easyMAG Extraction e Never transfer items from the post PCR room to the pre PCR room perianal bioM rieux France platform bioM rieux France R swab e Swabs for specimen collection and To keep laboratory free of PCR product contamination transport e g Sigma transwabs Medical Wire amp Equipment UK or Usepipettes with hydrophobic filter tips l l l Specimen Eswab Copan Diagnostics US in e Make sure to always use a new pipette tip when adding solutions test samples and Amies transport media controls to wells of a 96 well plate Bacterial
8. not detected by Check Direct CPE Carbapenem resistance is caused by carbapenemase production but also by various other mechanisms A negative result with Check Direct CPE does not imply that the bacterium is not carbapenem resistant it implies that the bacterium is not likely to carry any of the carbapenemase gene variants of KPC NDM VIM and OXA 48 detected by Check Direct CPE Therefore the test result of Check Direct CPE should never be used as guidance for therapy The quality of the input DNA is an important factor for obtaining reliable results with Check Direct CPE DNA must be extracted from perianal or rectal swabs in transport medium using the NucliSENS easyMAG DNA extraction system bioM rieux FR Alternatively crude DNA extraction from bacterial cells may be used following the protocol specified in this manual Other DNA extraction systems have not been approved for use with Check Direct CPE yet The assay has been validated for both Sigma transwabs Medical Wire amp Equipment UK and Eswab Copan Diagnostics US in Amies transport media Other swab types are also expected to work well but this has not been validated yet The assay has been tested extensively with samples containing various 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
9. species will yield poor results Check Direct CPE cannot and does not make any representation or warranty that it is capable of correctly detecting KPC NDM VIM and OXA48 in all gram negative species subspecies or types or in all clinical sample sources 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 subtypes might not be detected The test reflects the state of knowledge of Check Points Health B V 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 performing DNA based molecular detection methods 11 Technical assistance support check points com 31 317 453 908 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 Direct CPE Notice to Purchaser This product is sold under license from PHRI Properties and may be used under PHRI Properties patent rights only for human in vitro diagnostics food testing veterinary testing or research Trademarks Sigma Transwab
10. the analytical sensitivity test was performed using plasmid having a 400 bp target sequence DNA fragment Thus the LOD of the Check Direct CPE real time PCR test was established using plasmid DNA directly in the PCR reaction mix To determine analytical sensitivity an end point dilution was used until the assay could no longer detect the target in question in more than 5 of the replicates Results See Table A Table A LOD copies PCR KPC 5 Target NDM 5 VIM 5 OXA 48 5 2 Specificity 2 1 In silico Specificity The specificity of the Check Direct CPE real time diagnostic test is ensured by the selection of the correct primers and probes as well as the selection of stringent reaction conditions Primers and probes sequences were validated with silico analysis Primers and Probes sequences were designed to specifically identify the gene variants listed in Table B Primers and Probes sequences were tested for potential homologies with all the gene sequences published by the international gene bank GenBank NIH genetic sequence database using sequence comparison analysis Results No cross homology was found with other organisms for designed primers and probes Check Direct CPE User manual Version 2 2 Issued 05 05 2014 e Check Direct CPE Table B Gene Variants KPC 1to15 NDM 1to8 VIM 1 to 6 8 to 37 OXA 48 162 163 181 204 232 244 245 2 2
11. ION SETUP 0044 5 Date of ISSue 05 05 2014 2 FOR 5 7500 22005000000 ERE ra EXE REPE par ap iai 5 3 FOR USE ON THE 1 48018 2 2 42000 000000 thin asset tn nsns 6 4 FOR USE N THE CEX9G E 6 5 FOR USE ON THE ROTOR GENE 6 18 0080 W 48 080 05 DATA ANALYSIS 6 1 FOR USE ON THE ABI 7500 6 2 FORUSEON THE EC 480 IRI oio hei i E ER ORE 7 3 F R USE Rpniss C n 7 4 FOR USE ON THE ROTOR GENE 8 For use on ABI 7500 DATA INTERPRETATION o ssscssssssssssssssscesssscsssssccssssecssssecssssecessseessssecessuecassueeessucesssseesssseessase 8 Light Cycler 480 I amp II FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING 9 TM PERFORMANCE 5 5 8 9 CFX96 KEY TO SYMBOLS USED sssesssssssssscsssscsssscsssscsssscsssscessscsssscsssscsssucessscsssucsssucsssucssssesssecesseeess 11 yos 11 Rotor Gene Q TECHNICAL ASSISTANCE scsssssssssssssssssssssss
12. T NEG NTC 4 Quantitation Analysis Cycling A from 10 Green Page 1 NEG NEG NEG g 5 3 5 z NEG NTC NEG NEG NEG NTC NEG NTC NEG NTC NEG NEG NTC x NEG NTC Cycle NEG Adjust Scale Auto Scale Default Scale Linear Scale Figure 4 Screen shot of typical analytical window with the Rotor Gene Q software v2 1 0 Build 9 Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale in the Green channel Check Direct CPE User manual Version 2 2 Issued 05 05 2014 e Check Direct CPE Data interpretation 1 Run validation Table 8 Check the positive and negative control amplification curves Valid run reports e Noinstruments system failures during the run e Negative control with a C value of 30 3 in the Cy 5 detector channel and no C value in the other detector channel e Positive control C values as expected in Table 8 The exact C values of the positive controls vary depending on the qPCR instruments used and the threshold settings Table 8 Criteria for a valid run with Check Direct CPE test N B Combined positive controls in a single reaction will show a higher value This deviation may be up to 4 5 Sample Type Instrument Expected C values Red TXR orange FAM green yellow VIC yellow
13. cation were successful Five molecular beacon probes labeled with 4 different dyes are used to detect the various carbapenemases and the control DNA Check Direct CPE discriminates between KPC NDM VIM and OXA 48 For each of the 4 carbapenemase genes KPC OXA 48 NDM and VIM many gene variants exist PCR primers and fluorescent probes of Check Direct CPE are selected to target homologous gene segments of these carbapenemase genes and in this way gene variants are reliably detected Kit contents for 48 reactions Storage conditions Components Mat No Description CPE PCR Mastermix 9 0080 1 transparent tube and cap 630ul 4 C CPE solution 9 0071 1 brown tube purple inlay 6 140 ul Internal control 9 0077 1 tube red inlay 6 600 ul Negative control 9 0070 1 tube white inlay 100 ul KPC positive control 9 0073 1 tube green inlay 6 100 ul 20 C store in the dark NDM positive control 9 0075 1 tube gold inlay 100 ul VIM positive control 9 0074 1 tube yellow inlay 100 ul OXA 48 positive control 9 0076 1 tube orange inlay 9 100 ul User manual 9 0078 Leaflet download from website Storage handling and stability Check Direct CPE reagents are shipped cooled The CPE PCR Mastermix should be stored at 4 C upon receipt All other reagents should be stored at 20 C upon receipt Please visually inspect the box upon initial opening to ensu
14. detected the FAM and Texas Red channels The internal control is detected in the Cy5 channel Check Direct CPE 1 Real time PCR reaction setup Table 3 Example of combining of positive controls 1 Calculate the number of reactions Thaw reagents mix well spin down and keep on ice Component Volume p r reaction 2 Prepare the real time PCR qPCR reaction mix as described in Table 1 Multiply the CPE Positive control KPC 2 5 ul solution and the CPE PCR Mastermix by the right number of samples and include 10 Positive control NDM 2 5 ul surplus to ensure that you have enough qPCR reaction mix for all the calculated Positive control VIM 2 5 ul 2 t reactions For ABI 7500 1 480 and CFX96 96 well PCR plates are used for Rotor Positive control OXA 48 6 2 5 ul Gene Q PCR strip tubes TOS m 3 Pipette 15 ul of qPCR reaction mix to the wells or tubes M 4 According to Table 2 pipette 10 ul to each pre filled well tube of either e Test sample Table 4 Real time PCR setup e Positive control solution KPC 6 VIM or OXA 48 Positive Reaction mes bined by addi I vol f each ABI 7500 LC9480 I LC 4801 CFX96 RotorGeneQ controls may also be combined by adding equal volumes of eac control up to a E Mme po ee volume of 10 ul per reaction see Table 3 NB It is recommended to prepare a stock solu
15. e e Check Direct CPE User manual buntelsljei 2 PRINCIPLE OF THE METHOD v eS eue anno RN 2 Ch ec k Di rect C P E KIT CONTENTS FOR 48 REACTIONS 4 4 nennen ener ener nnne nnne 2 STORAGE HANDLING AND 5 44 4 044 022 22 2 MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT sssseccccccsssssseeeeececceessseeenees 3 Real time PCR kit for the detection of carbapenemase GOOD LABORATORY PRACTICES erae nau repre enn aeo as au o e eue en Vasa eus eoe een va se ua pw 3 prod ucing Enterobacteriaceae SPECIMEN COLLECTION AND DNA EXTRACTION ccccccccccccesscececsceseeeeeseseeesesesesseeseeesesesenens 4 1 HUMAN SPECIMEN rini nh inihi ihi i anas 4 2 DNA EXTRACTION FROM PERIANAL RECTAL SWABS WITH NUCLISENS EASYMAG 4 3 CRUDE DNA EXTRACTION FROM BACTERIAL 15 2 2 7 1 1 7 74 1 426000000000 4 4 POSITIVE AND NEGATIVE CONTROL PREPARATION 4 Version 2 2 REAL TIME PCR ASSAY 4 1 REAL TIME PCR REACT
16. e genes with the Check Direct CPE test Table D 103 Check Direct CPE 19 KPC KPC 16 NDM NDM VIM 1 48 48 33 VIM NDM VIM 1 VIM OXA 48 NDM VIM OXA 48 23 OXA 48 OXA 48 Key to symbols used LOT Fu SOLUTION CPE For In Vitro Diagnostic Use Catalog number Batch code IFU number INTERNAL CONTROL CPE solution Internal control Temperature limitation Contains sufficient for n tests Consult instructions for use Check Direct CPE User manual Version 2 2 Issued 05 05 2014 ONTROL ONTROL KPC ONTROL VIM VIM positive control ONTROL NDM ONTROL OXA 48 Negative control KPC positive control NDM positive control OXA 48 positive control CR Mastermix CPE PCR Mastermix ud Manufacturer e Check Direct CPE Limitations Check Direct CPE is a DNA based real time PCR assay to detect the presence of the carbapenemase genes KPC NDM VIM and OXA 48 in Enterobacteriaceae The test detects the following carbapenemase gene variants 1 162 163 181 232 244 245 and blaxpci 15 VIM and OXA 48 represent the clinically most prevalent carbapenemases in Enterobacteriaceae in most parts of the world However other rare carbapenemases may also responsible for carbapenemase production Enterobacteriaceae and these are
17. eline Subtracted Curve Fit Apply Fluorescent Drift Correction C Determination Single Threshold Baseline Method Auto Calculated iod 180900 D OE EE 1535 Settings Export Tools Bh plate Setup 5 Biosearch v Fuorophore x VE Data it Gone o l Ena roe nie Domen custom Daa vew ac F gt 1385 1235 1085 9352 Include Color Pos Name El Sample i ES Sample E4 Sample ES Sample E6 Sample 7 Sample ES Sample Fl Sample F2 Sample Sample F4 Sample FS Sample F6 Sample F7 Sample Fe Sample Gi Sample G2 Sample G3 Sample G4 Sample G5 Sample Standard Curve EARRA RAR SE S 8 S SEIS LISTES STETIT a 3A H a 5 Log Concentration D FAM EED CPE eA Figure 2 Screen shot of a typical analytical window on the LC 480 system I with the 1 480 software v1 5 FAM Amplification plot of positive and negative samples for the Check Direct CPE test linear scale threshold is i automatically calculated by the software Table 6 FAM Scan Mode All Channels Plate BR White Analysis Mode Baseline Subtrected Curve Fit Table 6 Recommended threshold settings Detector Target 7500 1 480 I amp II CFX96 Rotor Gene Q Figure 3 Screen shot of the typical analytical window on the CFX96 with the CFX96 software v2 0
18. erences are not due to the threshold being too high or too low If changing the threshold does not improve the results go to FAQ 3 to 6 The real time PCR instrument gives an error message Refer to the real time PCR instrument user manual or contact the local technical support of the real time PCR instrument company I left Solutions CPE Internal control negative or positive control out of the 20 C 47 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 more than 24 hours Duplicate samples tested with Check Direct CPE test did not yield identical results Cp C values of identical samples may vary between individual reactions Large variations gt 2 values suggest pipetting errors or other differences between the duplicate samples Data Analysis and Interpretation If you encounter difficulties with the data analysis and interpretation please contact Check Points Technical Support at support check points com Performance Characteristics 1 Analytical sensitivity The analytical limit of detection LOD of Check Direct CPE real time PCR test was assessed for four carbapenemase genes associated with carbapenemase production in Enterobacteriaceae KPC NDM VIM and OXA 48 No quantified genomic standards for these markers were available at the time therefore
19. gs Green 6 Yellow 4 Orange 3 Red 8 Check Direct CPE User manual Version 2 2 Issued 05 05 2014 Check Direct Data analysis Important points before starting For a detailed description on how to operate real time PCR instruments and how to analyze data please refer to the real time PCR instrument instruction manual Always visually inspect the amplification plot for each sample tested versus C values obtained with the software 1 For use on the ABI 7500 bd Multicomponent Plot Raw Data Plot Summary Multiple Plots View Analyze data using ROX as a passive reference In the Options setting for each Target see Table 4 deselect the box for auto threshold and auto baseline as shown in Figure 1 red circle and select Reanalyze Set the threshold values in the analysis settings window for each detector channel to the values specified in Table 6 Check amplification plots versus C values calculated by the software for each target Amplification Plot Plot Settings Plot Type ARn vs Cycle Graph Type Log Color Target Save current settings as the default e Amplification Plot Standard Curve leas Show 77 Threshold 7 Baseline Start Well Targeta Baseline End Well W Target 4 Save current settings as the default Figure 1 Screen shot of a typical analytical window on the ABI7500 with ABI7500 software v2 0 6 Amplification plot of positi
20. ocessing method with Check Direct CPE prior to routine use of the test Procedure 1 Check Direct CPE has been validated with the NucliSENS easyMAG automated DNA extraction procedure for perianal rectal swabs in transport medium For perianal swabs follow the Generic Protocol for rectal swabs follow the Specific A Protocol 2 Use 200 ul of perianal rectal swab fluid from each specimen and add 5 ul of the internal control solution IC solution to each well of the easyMAG cartridge Start the DNA extraction using the Generic extraction protocol DNA is eluted in 70 ul elution buffer 4 DNA extracts can be stored at 4 C for up to 6 months and at 20 C for a longer period of storage 5 Use the DNA solution directly and continue with the real time PCR assay or store as specified until use W 3 Crude DNA extraction from bacterial cells Important points before starting DNA extraction is carried out in the pre PCR room Procedure 1 Inoculate nutrient agar plates with the clinical samples or the bacterial strains to be tested and incubate overnight at 37 C Typical growth media include blood agar MacConkey agar and Tryptic Soy agar Check Direct CPE User manual Version 2 2 Issued 05 05 2014 e Check Direct CPE 2 Prepare a bacterial cell suspension of McFarland 0 5 1 0 or ODs 0 08 0 15 using PCR grade water e g Milli Q water 3 For each cell suspension transfer 200 ul to a 1 5 ml Eppendorf t
21. ommon cause of carbapenem resistance in Enterobacteriaceae is the expression of carbapenemases i e Carbapenemase Producing Enterobacteriaceae or CPE CPE have elevated or complete resistance to carbapenems and most other B lactam antibiotics Presently the vast majority of CPE are associated with the presence of one of the following plasmid encoded carbapenemases KPC Klebsiella pneumoniae carbapenemase VIM Verona integron encoded metallo B lactamase New Delhi metallo B lactamase OXA 48 Oxacillinase 48 Moreover CPE often have other non B lactam resistance determinants resulting in multidrug and pandrug resistant isolates Patients usually carry CPE by colonization of the colon Therefore rectal swabs provide a proper specimen to assess carriage of CPE and perianal swabs may be used as a non invasive alternative Check Direct CPE is a rapid real time PCR test for the detection and discrimination of KPC NDM VIM and OXA 48 in rectal and perianal specimens Check Direct CPE User manual Version 2 2 Issued 05 05 2014 e Check Direct CPE Principle of the method Check Direct CPE assay is based on specific recognition and amplification of target sequences by PCR and the simultaneous detection of the accumulation of PCR amplification products by fluorescent DNA probes A control DNA molecule the internal control is added to the clinical specimen prior to DNA extraction to monitor that DNA extraction and PCR amplifi
22. ration and DNA extraction methods be used with Check Direct CPE Check Direct CPE test has been optimized using specific swabs and transport medium in combination with the NucliSENS easyMAG extraction methods The crude DNA extraction method from bacterial cells specified in this User Manual may also be used Check Points does not guarantee the performance of the test with methods other than those recommended in this manual The real time results show no values or interpretation indicating that the sample is invalid Possible causes and troubleshooting e sample DNA was not added to the assay e sample DNA tested with Check Direct CPE is negative and the internal control was not added prior to DNA extraction Please repeat the DNA extraction e The DNA extraction failed since the internal control was not detected Please repeat the DNA extraction e sample DNA contains contaminants inhibiting the reactions Please repeat the DNA extraction e Solution or CPE PCR Mastermix was not added to the assay Please repeat the test Reagent solutions are degraded or may have expired The real time results show no C C C values for the positive control or interpretation indicating that sample is invalid Possible causes and troubleshooting e positive control solution was not added Repeat the test e Solution or PCR Mastermix was not added to the assay Please repeat the test
23. re that its contents are intact The CPE solution should not be exposed to more than 12 freeze thaw cycles Please contact the Check Points office at support check points com if you have any further questions Store kit reagents at indicated temperature until expiration date Materials required but not supplied with the kit e Check Direct CPE Good laboratory practices Recommendations for best results Supplies Equipment e Disposable laboratory powder free e Vortex mixer The test must be performed by adequately trained personnel gloves Mini centrifuge Do not use reagents after their expiration date e Lab coat Before use thaw frozen reagents completely at room temperature and vortex briefly to Genera Pipettes amp disposable filter tips for 5 obtain a homogeneous solution After vortexing briefly spin down the solution to avoid volumes of 1 to 1000 ul e 1 5 ml tubes Eppendorf tubes contamination when opening the cap e Follow recommendations for storage handling and freeze thaw cycles to preserve the ABI 7500 e 96 well PCR clear plate e A ABI 7500 Applied Biosystems US li f the kit REF N8010560 Plate centrifuge quality of the kit s reagents e PCR plate seal e Protect reagents from light to avoid photo bleaching of the dyes LC 480 LightCycler 480 multiwell plate96 LightCycler 480 I II Roche CH e Periodically verify the
24. sitive control KPC 9 VIM and or OXA 48 9 10 ul Negative control Negative sample or negative control provided O 10 ul Check Direct CPE User manual Version 2 2 Issued 05 05 2014 5 2 For use on the ABI 7500 e mode Standard 7500 96 wells Experiment Quantitation Standard Curve e TaqMan Reagents e Standard ramp speed e Reaction volume 25 ul ROX passive reference dye Included in the qPCR Buffer Targets Reporter Dyes setup see Table 4 Quencher standard NFQ MGB 3 For use on the LC 480 I amp II To use the Check Direct CPE real time PCR kit on the LightCycler 480 system I amp II a 4 color compensation object is required and generated by using the 4 Color Compensation Set supplied by Check Points Health B V Ref 18 0070 e Detection format Check Points 4 color set see 4 Color Compensation Set User Manual V 1 1 or higher Reaction Volume 25 ul e For the 45 cycle amplification step select Quantification in the Analysis Mode tab In the Acquisition mode tab select none for 95 C and single for 60 C e Color compensation object or file required A For use on the CFX96 Sample Volume 25 ul Temperature Control Mode Calculated Scan Mode All channel Plate type BR white Plate Setup View Edit Plate and Select Fluorophore as described in Table 4 5 For use on the Rotor Gene Q e Reaction Volume 25 ul 72 well rotor selected Gain settin
25. sssssscsssscssssssscssscsssssssessucssscsssessessssssssesseessessees 12 4 3 IVD Check Direct CPE User manual Version 2 2 Issued 05 05 2014 1 Intended use Check Direct CPE is a qualitative vitro diagnostic test for the rapid detection of carbapenemase genes in Enterobacteriaceae from clinical specimens Perianal or rectal swabs may be used directly as well as bacteria cultured from other clinical specimens Check Direct CPE detects the presence of the carbapenemase genes KPC NDM VIM and OXA 48 presently the primary cause of carbapenemase production in Enterobacteriaceae The assay involves the extraction of DNA from rectal perianal swabs or bacterial cells followed by real time PCR using the reagents provided with the kit Check Direct CPE can be used as an aid to identify prevent and control carbapenemase producing Enterobacteriaceae that colonize patients in healthcare settings Check Direct CPE is not intended to diagnose infections with carbapenemase producing Enterobacteriaceae nor to guide or monitor treatment for these infections Parallel cultures are necessary to recover organisms for epidemiological typing susceptibility testing and for further confirmatory identification Introduction The worldwide emergence and dissemination of carbapenem resistance among Enterobacteriaceae is a serious threat to public health These organisms are associated with high mortality rates and have the potential to spread widely The most c
26. tion of the combined positive controls Store this stock solution at 20 C KPC FAM mcs 5 FAM Green 51045 Negative control solution O 5 Sealthe 96 well plate or close the PCR strip tubes Mix the plate by tapping it on the NDM VIM VIC cd Tom We Yellow 35745 bench and spin down briefly 25 6 Transfer the plate or tubes to the post PCR room OXA 48 bc TNAM m TXR CE 7 Without delay place the plate or tubes into the real time PCR instrument and start the run following the manufacturer s instructions and using the parameters specified in 2 Cy5 in ea cys Red 660410 Tables 4 and 5 Instrument specific settings are further specified in the subsequent paragraphs 2 5 of this section 8 When the run is completed discard the plate or tubes according to local regulations Table 5 Real time protocol parameters Ramp Rate Mode Table 1 qPCR reaction mix setup Step Temperature Cycles Data Collection 7500 CFX96 LC 480 Component Volume per reaction Rotor Gene Q I amp II CPE PCR Mastermix 12 5 yl 1 50 C 2 min 1 OFF Standard 4 4 C s CPE Solution 2 5 ul 2 95 C 10 min 1 OFF Standard 4 4 C s 3 95 C 15 sec Plate read 4 4 C s Total vol 15 ul 4 60 C 60 sec 48 Optics Standard 2 2 C s Denaturation time should be at least 3 minutes Table 2 Real time PCR sample setup Reaction Type Component Volume per reaction Test sample Sample DNA 10 ul Po
27. ube preferably safe lock and add 10 ul of the internal control solution IC solution 6 Mix briefly 4 Heat the tubes at 98 C for 10 minutes After incubation vortex the tubes vigorously for 30 seconds 5 Centrifuge the tubes in an Eppendorf centrifuge for 2 minutes at maximum speed 6 Use the supernatant immediately or store at 4 C and use within 24 hours 4 Positive and negative control preparation To validate the run perform positive and negative control reactions for each Check Direct CPE PCR run The positive and negative controls are supplied with the kit Positive control s One positive control per target is provided with the kit Each positive control contains the internal control These controls may be used individually or combined Refer to step 4 of the Real Time PCR reaction setup for further details Negative control s Use the negative control O provided in the kit as a sample to validate the run The negative control contains the internal control We also recommend performing a DNA extraction as specified earlier with the internal control solution using a sample known to be negative for the test in use carbapenemase negative sample or elution buffer Real time PCR assay Important points before starting Preparation of the real time PCR reactions is carried out in the pre PCR room e Specific parameters and materials should be used for each type of real time PCR instrument e Targets are
28. ve and negative samples for the Check Direct CPE test logarithmic scale Red circle deselect the box of auto Threshold Auto baseline Check Direct 2 For use on the LC 480 I amp II 3 For use on the CFX96 1 Select Abs Quant 2 Derivative Max in the Analysis tab 1 Open the Data file for Data Analysis In the Analysis Settings use the following 2 Analyze the data using the color compensation object application specific to the parameters Check Direct CPE assay previously created Select Color Comp On In database select the CC object Refer to Check Points User Manual for the 4 Color Compensation set Ref 18 0070 Select the High Confidence option and Calculate C values for each filter combination For each filter combination always check amplification plot versus C values Cycle to Analyze 10 45 5 n the results table check C values for each targets versus the Status given by the Baseline Threshold User defined to be set per fluorophore software red positive green negative blue call uncertain Validate the Status of 2 Set the threshold manually for each detector channel using the threshold values the sample using the software by visually inspecting amplification curves Figure 2 recommended in Table 6 3 Check amplification plots in the Quantification Tab figure 3 versus values calculated by the software for all targets in the results table Analysis Mode Fluorophore Baseline Setting Bas
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