User manual User manual - Check
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1. Check Direct CPE User manual Check Direct CPE Real time PCR kit for the detection of carbapenemase producing Enterobacteriaceae Version 2 3 Date of issue 01 09 2014 18 0080 Y as IFU 080 06 For use on ABI 7500 LC480 I amp Il CFX96 RotorGene Q EU CE U S For Research Use Only Not for use in diagnostic procedures Contents Intended Usern a A NE EE EE 2 anagalo Eea a PAE E E E aspera careneGeaunenalavnubraen 2 Principle OF the Method sssini 2 Kit contents for 48 reactions s sesssesessessesesrareseosenesrnresinsarisenrssennaresesessesrerenasenrerenne 2 Materials required but not supplied with the kit sssssssssssssessensrssssresssesrsesssseseesssesses 3 Storage handling and stability sce ces dea seesiectncsionesdins bist tacetweiwastedelacnderieaveaveandawcadeintaretenines 3 Good laboratory practices ccc cdcctietascciccicnscesassaimasaekieeciinenienieniaseneumneninanancseamaucs 4 Specimen collection and DNA extraction sicssisccscesatsaccaicsicetatencsicaned sciateacinananauneeaeenecees 5 Real time PCR assay and cycler operation scsssseeseescssessesceesseceeseececeeceeecsececesenes 6 Results Interpretatio Nimeen A A S 7 Frequently asked questions FAQ amp Troubleshooting cccccesessessessessessesteeteeeees 8 Key to symbols Used sredina a i a a a 9 MIVA ONS ceee RAEE A E A E E E E A 9 Technical assistante soesoenan eia AE E E KAA E EEE 10 Appendix 1 Check Direct CPE Test Program2. 2 2 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 X 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 Check Direct CPE User manual 19 Version 2 3 Issued 01 09 2014 Check Direct CPE Table X 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 3 Analytical Reactivity To evaluate the analytical reactivity a retrospective study was performed with 93 bacterial strains of 26 different gram negative species Table V The 93 bacterial strains tested with Check Direct CPE were previously identified carbapenemase positive with the micro array diagnostics test Check MDR Cy 103 Check Poin
3. VIM and OXA 48 in Enterobacteriaceae The test detects the following carbapenemase gene variants blayimie s 34 Dlaoxa 48 162 163 181 232 244 245 Dlanpm1 8 and blaxpcz 15 KPC NDM VIM and OXA 48 represent the clinically most prevalent carbapenemases in Enterobacteriaceae in most parts of the world However other rare carbapenemases may also be responsible for carbapenemase production in Enterobacteriaceae and these are 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 Diagnosti
4. These organisms are associated with high mortality rates and have the potential to spread widely The most common 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 NDM New Delhi metallo B lactamase or 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 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
5. PCR amplification 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 Components Mat No Descriptio Storage conditions CPE PCR Mastermix 9 0080 1 transparent tube and cap 630ul 4 C CPE solution 9 0071 1 brown tube purple inlay 140 ul Internal control 9 0077 1 tube red inlay 600 ul Negative control 9 0070 1 tube white inlay O 100 ul KPC positive control 9 0073 1 tube green inlay 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 100 ul User manual 9 0078 Leaflet download from website 2 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Materials required but not supplied with the kit Disposable laboratory powder free e Vortex mixer gloves Mini centrifuge Lab coat Thermo Block for
6. control target Table A4 displays in which detector channel each gene or target is detected Table A4 Multiplex real time PCR setup for the RotorGene Q cycler Detector Green Yellow Orange Red Channel 1 2 3 4 Target KPC VIM amp NDM OXA 48 like 1 C 1 C Internal Control The RotorGene Q needs to be programmed as outlined below e Reaction Volume 25 ul e 72 well rotor selected e Gain settings Green 6 Yellow 4 Orange 3 Red 8 Table B4 Real time protocol parameters for RotorGene Q Step Temperature Time el Data Collection Ramp Rate Mode T 50 C 2 min Standard 2 95 C 3 min 1 OFF Standard 3 95 C 15 sec Plate read 4 60 C 60 sec i Optics on sandara Denaturation time may be up to 10 minutes Data analysis 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 Select Analysis and the proper channel from 10 and press Show Then use the parameters indicated in Table D to analyze each detector channel Set the threshold manually for each detector channel using the value recommended in Table C4 Check amplification plots versus Cy values calculated by the software for each target in the Quant Results table See Figure below U ee Table C4 Treshold values Detector Target Treshold Green KPC 0 02 Yellow NDM VIM 0 015
7. from the bacterial cell suspension is carried out in the microbiology room e DNA extraction from rectal and perianal swabs is carried out in the pre PCR room e Preparation of the amplification reactions is carried out in the pre PCR room e Incubation in the real time PCR thermocycler is carried out in the PCR room e Never transfer items from the PCR room to the pre 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 test samples and controls to wells of a 96 well plate Follow proper pipette dispensing techniques to prevent aerosols Wear clean disposable gloves and clean lab coats for the different steps of the test Change gloves whenever you suspect that they are contaminated Keep the tubes of all kit components and samples closed as much as possible Clean the lab benches and all equipment regularly with a 0 5 sodium hypochlorite solution Users are strongly advised to read the full protocol before starting the test Check Direct CPE is a very sensitive assay detecting down to 1 cfu per test Care should be taken to avoid contamination particularly when working with bacterial cell suspensions Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Specimen collection and DNA extraction Specimen collection of rectal and perianal swabs In
8. 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 Collect perianal rectal specimen according to local guidelines and swabs manufacturer recommendations Place swabs in their containers containing 1 ml of liquid transport medium Label the containers Refer to the swab manufacturer instruction for storage handling and stability Poe Ne DNA extraction from perianal rectal swabs with NucliSENS easyMAG Important points before starting Check Points advises to validate your specimen collection and processing 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 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 2 DNA is eluted in 70 ul elution buffer 3 DNA extracts can be stored at 4 C for up to 6 months and at 20 C for a longer period of storage 4 Use the DNA solution directly and continue with the real time PCR assay or store as specifi
9. 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 ensure 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 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Good laboratory practices Recommendations for best results e The test must be performed by adequately trained personnel 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 cap e Follow recommendations for storage handling and freeze thaw cycles to preserve the quality of the kit s reagents e Protect reagents from light to avoid photo bleaching of the dyes e Periodically verify the accuracy and precision of pipettes as well as correct functioning and calibration of the instruments Prevention of contaminations Use separate rooms a microbiology room a pre PCR room and a PCR room e Bacterial cell suspensions are prepared in the microbiology room e Crude DNA extraction
10. target Table C1 Treshold values Detector Target FAM green KPC 0 002 VIC yellow NDM VIM 0 001 TXR orange OXA 48 0 006 Cy5 red IC 0 003 11 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE ppa eE Amplification Plot AA Multiple Plots View Analysis Tab of a typical analytical window on the ABI7500 with ABI7500 software v2 0 6 Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale Red circle deselect the box of auto Threshold Auto baseline 12 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Appendix 1b LC480 program Important e Please refer to the LC480 instruction manual for a detailed description on how to operate the real time PCR instrument and how to analyze the data e Always visually inspect the amplification plot for each sample tested versus C values obtained with the software LC480 cycler program Check Direct CPE detects 4 carbapenemase genes and an internal DNA control target Table A2 displays in which detector channel each gene or target is detected Table A2 Multiplex real time PCR setup for the LC480 cycler system amp Il Detector FAM VIC Red Cy5 Channel 1 2 3 4 Target KPC VIM amp NDM OXA 48 like 1 C 1 C Internal Control To use the Check Direct CPE real time PCR kit on the LightCycle
11. 1 5 tubes General e Pipettes amp disposable filter tips for for DNA extraction from cells volumes of 1 to 1000 pl 1 5 ml tubes Eppendorf tubes ABI 7500 e 96 well PCR clear plate REF N8010560 e ABI 7500 Applied Biosystems US PCR plate seal Plate centrifuge LC 480 e LightCycler 480 multiwell plate 96 e LightCycler 480 I II Roche CH Product no 04729692001 Plate centrifuge e 4 Color Compensation Set Ref 18 0070 Real time j PCR Check Points Health B V NL instrament PCR plate seal CFX96 e 96 well PCR white plate e CFX96 Bio Rad US PCR plate seal Plate centrifuge Rotor PCR strip tubes and caps 0 1ml Rotor Gene Q 5plex Gene Q QIAGEN Germany 72 well rotor and locking ring Loading block 72x0 1ml tubes Rectal NucliSENS easyMAG Extraction kit NucliSENS easyMAG Extraction platform perianal bioM rieux France bioM rieux France swab e Swabs for specimen collection and transport e g Sigma transwabs Medical Wire amp Equipment UK or Eswab Copan Diagnostics Italy in Specimen Amies transport media Bacterial e PCR grade water Milli Q or aqua bidest Densitometer for bacterial suspensions culture e Eppendorf tubes safe lock e Centrifuge for Eppendorf tubes Check Direct Quick Extraction kit Check e Heating block for Eppendorf tubes Points Storage handling and stability Check Direct CPE reagents are
12. Orange OXA 48 0 03 Red IC 0 06 17 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Table D Rotor Gene Q analytical parameters E Green KPC ON ON 10 10 Yellow NDM VIM ON ON 6 10 Orange OXA 48 ON ON 10 10 Red IC ON ON 15 10 W Quantitation Analysis Cycling A from 10 Green Page 1 fo 8 Dynamic Tube 8 Slope Correct Ignore First E Outlier Removal fed Save Defaults NEG NTC NEG INTC NEG NTC NEG NTC NEG NTC 21 01 2172 21 96 21 99 _NEGINTC NEG INTC NEG NTC NEG INTC NEG INTO NEG NTC f 72 NEG NTC NEG NTC NEG NTC NEG NTC Adjust Scale Auto Scale Default Scale Linear Scale Figure 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 18 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Appendix 2 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 O
13. XA 48 No quantified genomic standards for these markers were available at the time therefore 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 Z Table Z 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 in silico analysis Primers and Probes sequences were designed to specifically identify the gene variants listed in Table Y 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 Table Y Gene Variants KPC 1to15 NDM 1to8 VIM 1 to 6 8 to 38 OXA 48 162 163 181 204 232 244 245 247 370
14. by the software red positive green negative blue call uncertain Validate the Status of the sample using the software by visually inspecting amplification curves See Figure P Table C2 Recommended threshold settings Detector Target LC 480 I amp II FAM KPC Auto calculated VIC NDM VIM Auto calculated RED OXA 48 Auto calculated Cy5 IC Auto calculated 13 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Amplification Curves Uncertain Standard C Samples 8 Fluorescence 465 510 El Sample 49 F Sampie 50 E3 Sample 51 E4 Sample 52 ES Sample 53 E6 Sample 54 E7 Sample 55 E8 Sample 56 F1 Sample 61 F2 Sample 62 F3 Sample 63 F4 Sample 64 FS Sample 65 F6 Sample 66 F7 Sample 67 Fe Sample 68 Gi Sample 73 G2 Sample 74 G3 Sample 75 G4 Sample 76 GS Sample 77 EE me J am Screen shot of a typical analytical window on the LC 480 system I with the LC 480 software v1 5 Amplification plot of positive and negative samples for the Check Direct CPE test linear scale threshold is automatically calculated by the software Table C2 14 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Appendix 1c CFX96 program Important e Please refer to the CFX96 instruction manual for a detailed description on how to operate the real time PCR instrument a
15. cs 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 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 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
16. ction e Always visually inspect the amplification plots to verify the results Table 4 Data interpretation guidelines for perianal rectal swabs KPC NDM VIM OXA 48 IC C values Interpretation C values YES 30 3 Positive sample N A 30 3 Negative sample N A N A or 233 Invalid If observed Cr values vary significantly from expected C values see FAQ and Troubleshooting section N A represent a negative test result The specific denotation may vary between different systems Table 5 Data interpretation guidelines for crude DNA extracts from bacterial cells KPC NDM VIM OXA 48 C values IC C values Interpretation lt 31 YES or N A Positive sample N A 30 3 Negative sample gt 31 YES or N A Invalid N A N A or 233 Invalid If observed Cr values vary significantly from expected C values see FAQ and Troubleshooting section N A represents a negative test result The specific denotation may vary between different systems Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Frequently asked questions FAQ amp Troubleshooting 1 May other specimen preparation 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 s
17. de blood agar MacConkey agar and Tryptic Soy agar 2 Isolate each strain to be tested according to the User Manual of the Check Points quick extraction kit 3 Use the supernatant immediately or store at 4 C and use within 24 hours Alternatively store at 20 C for longer periods 5 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 Check Direct CPE User manual 5 Version 2 3 Issued 01 09 2014 Check Direct CPE e 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 2 4 of the Real Time PCR preparation for further details e 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 i e carbapenemase negative sample or elution buffer Real time PCR assay and cycler operation 1 Multiplex real time PCR setup Table 1 presents the multiplex real time PCR setup with the targets detected in each detector channel of the various cyclers Details for each real time PCR cycler a
18. e set per fluorophore 2 Set the threshold manually for each detector channel using the threshold values recommended in Table C3 3 Check amplification plots in the Quantification Tab See Figure below versus C values calculated by the software for all targets in the results table Table C3 Treshold values Detector Target Treshold FAM KPC 60 150 VIC NDM VIM 200 250 TXR OXA 48 400 800 Cy5 IC 40 100 Check Direct CPE User manual 15 Version 2 3 Issued 01 09 2014 Check Direct CPE el j 003 Completed E cerned Al Chomna Scan Mede All Channels ype BR White ue a a E Figure Screen shot of the typical analytical window on the CFX96 with the CFX96 software v2 0 Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale Adjust threshold manually using the threshold bar on the amplification plot Blue FAM signal 16 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Appendix 1d RotorGene Q program Important e Please refer to the RotorGene Q instruction manual for a detailed description on how to operate the real time PCR instrument and how to analyze the data e Always visually inspect the amplification plot for each sample tested versus Cy values obtained with the software RotorGene Q program Check Direct CPE detects 4 carbapenemase genes and an internal DNA
19. ed until use Crude DNA extraction from bacterial cell suspensions Important points before starting DNA extraction is carried out in the microbiology room Use Milli Q or aqua bidest Do not use the saline water routinely used for preparing cell suspensions 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 2 Prepare a bacterial cell suspension of McFarland 0 5 1 0 or OD6o 0 08 0 15 using PCR grade water Milli Q water or aqua bidest 3 For each cell suspension transfer 200 ul to a 1 5 ml Eppendorf tube preferably safe lock and add 10 ul of the internal control solution IC solution Mix briefly 4 Heat the tubes at 98 C for 10 minutes After incubation vortex the tubes vigorously for 30 seconds 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 Alternatively store at 20 C for longer periods I Crude DNA extraction from bacterial cells using the Check Points quick extraction kit Important points before starting DNA extraction is carried out in the microbiology 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 inclu
20. gnals in all samples and detector channels including the internal control signal Possible causes and troubleshooting e The 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 e The 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 6 C C C values troubleshooting e Samples with very low Cr values and no amplification curves The manual threshold is too low Samples showing a Cy value lt 15 and no amplification curve are considered negative The observed Cy value is an artifact of the software analysis the threshold crosses the background noise of the curve e Invalid Run Cr values expected for the controls in Table 6 do not match the Cr values observed In the experiment Check that these differences 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 7 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 8 left Solutions CPE Internal control negative or positive control out of the 20 C 4 F storage These reagents must be stored at 20 C 4 F fo
21. ix 12 5 ul CPE Solution 2 5 ul qPCR mix 15 04 gt 15 ul Sample 10 ul Reaction volume 25 pl 3 Cycler Operation 3 1 Insert the plates or tubes into the real time PCR instrument 3 2 Specify the run parameters for each cycler as described in Appendix 1 3 3 Enter the sample D s if required For most systems this may also be done after the run 3 4 Click the Start Run button 3 5 When the run is completed discard the plates or tubes according to local regulations 6 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Results interpretation 1 Run validation Table 3 Check the positive and negative control amplification curves Valid run reports e No instruments 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 3 The exact C values of the positive controls vary depending on the qPCR instruments used and the threshold settings Table 3 Criteria for a valid run with Check Direct CPE test N B Combined positive controls in a single reaction will show a higher Cr value This deviation may be up to 4 Cy s KPC AM green 25 3 28 3 OXA 48 Red TXR orange NDM VIM Sample Type VIC yellow VIC yellow Expected C values ABI 7500 CFX96 Rotor Gene Q LC 480 I amp II Posi
22. nd how to analyze the data e Always visually inspect the amplification plot for each sample tested versus Cy values obtained with the software CFX96 cycler program Check Direct CPE detects 4 carbapenemase genes and an internal DNA control target Table A3 displays in which detector channel each gene or target is detected Table A3 Multiplex real time PCR setup for the CFX96 Detector FAM VIC Texas Red Cy5 Channel 1 2 3 4 Target KPC VIM amp NDM OXA 48 like 1 C C Internal Control The CFX96 cycler needs to be programmed as outlined below e Sample Volume 25 pl e Temperature Control Mode Calculated e Scan Mode All channel e Plate type BR white e Plate Setup View Edit Plate and Select Fluorophore as indicated in Table A3 Table B3 Real time protocol parameters for CFX96 Step Temperature Time cytes Data Collection Ramp Rate Mode 1 OFF 1 50 C 2 min Standard 2 95 C 3 min 1 OFF Standard 3 95 C 15 sec Plate read 4 60 C 60 sec 4 gt Optics on Standard Denaturation time may be up to 10 minutes Data analysis 1 Open the Data file for Data Analysis In the Analysis Settings use the following parameters Analysis Mode Fluorophore Baseline Setting Baseline Subtracted Curve Fit Apply Fluorescent Drift Correction C Determination Single Threshold Baseline Method Auto Calculated Cycle to Analyze 10 45 Baseline Threshold User defined to b
23. ogram Check Direct CPE detects 4 carbapenemase genes and an internal DNA control target Table A1 displays in which detector channel each gene or target is detected Table A1 Multiplex real time PCR setup for the ABI7500 cycler Detector FAM VIC Texas Red Cy5 Channel 1 2 3 4 Target KPC VIM amp NDM OXA 48 like 1 C C Internal Control The ABI7500 cycler needs to be programmed as outlined below e Run mode Standard 7500 96 wells Experiment Quantitation Standard Curve TaqMan Reagents Standard ramp speed Reaction volume 25 ul ROX passive reference dye Included in the qPCR Buffer e Targets Reporter Dyes setup see Table A Quencher standard NFQ MGB Table B1 Real time protocol parameters for ABI7500 Step Temperature Time cytes Data Collection Ramp Rate Mode 1 OFF 1 50 C 2 min Standard 2 95 C 3 min 1 OFF Standard 3 95 C 15 sec Plate read 4 60 C 60 sec a Optics on standard Denaturation time may be up to 10 minutes Data analysis 1 Analyze data using ROX as a passive reference 2 In the Options setting deselect the box for auto threshold and auto baseline for each Target as shown in the Analysis Tab depicted below and select Reanalyze 3 Set the threshold values in the analysis settings window for each detector channel to the values specified in Table C1 4 Check amplification plots versus C values calculated by the software for each
24. 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 is a registered trademark of Medical Wire amp Equipment Copan 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 Points Health BV Tel 31 317 453 908 e e Binnenhaven 5 Fax 31 317 210 147 6709 PD Wageningen info check points com The Netherlands www check points com Check Points 10 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Appendix 1a ABI 7500 program Important e Please refer to the ABI7500 instruction manual for a detailed description on how to operate the real time PCR instrument and how to analyze the data e Always visually inspect the amplification plot for each sample tested versus Cy values obtained with the software ABI 7500 cycler pr
25. pecified 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 2 The real time results show no C C C values or interpretation indicating that the sample is invalid Possible causes and troubleshooting e The sample DNA was not added to the assay e The 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 The DNA extraction failed since the internal control was not detected Please repeat the DNA extraction The sample DNA contains contaminants inhibiting the reactions Please repeat the DNA extraction CPE Solution or CPE PCR Mastermix was not added to the assay Please repeat the test Reagent solutions are degraded or may have expired 3 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 The positive control solution was not added Repeat the test e CPE Solution or CPE PCR Mastermix was not added to the assay Please repeat the test e Reagent solutions are degraded or may have expired 4 Troubleshooting for invalid result 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 5 Real time results show very low fluorescent si
26. r 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 e 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 Table B2 Real time protocol parameters for LC480 Ramp Rate Mode Step Temperature Cycles Data Collection LC 480 I amp II 1 50 C 2 min 1 OFF 4 4 C s 2 95 C 3 min 1 OFF 4 4 C s 3 95 C 15 sec Plate read A 2 4 60 C 60 sec i Optics on FACIS 22 Cs Denaturation time may be up to 10 minutes Data analysis 1 Select Abs Quant 2 Derivative Max in the Analysis tab 2 Analyze the data using the color compensation object application specific to the 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 5 Inthe results table check C values for each targets versus the Status given
27. r 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 9 Duplicate samples tested with Check Direct CPE test did not yield identical results Cr Cp C values of identical samples may vary between individual reactions Large variations gt 2 Cr values suggest pipetting errors or other differences between the duplicate samples 10 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 Check Direct CPE User manual 8 Version 2 3 Issued 01 09 2014 Check Direct CPE Key to symbols used For In Vitro Diagnostic Use Negative control Catalog number CONTROL KPC KPC positive control Batch code CONTROL VIM VIM positive control IFU IFU number CONTROL NDM NDM positive control 2 Use before YYYY MM OXA 48 OXA 48 positive control pH Consult instructions for use INTERNAL CONTROL Internal control ual Manufacturer ISOLUTION CPE CPE solution F Temperature limitation PCR Mastermix CPE CPE PCR Mastermix Yy Contains sufficient for lt n gt tests Check Direct CPE User manual Version 2 3 Issued 01 09 2014 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
28. re described in Appendix 1 Table 1 Multiplex real time PCR setup for the various cyclers Detector FAM Green VIC Yellow Texas Red 610 Cy5 670 Channel 1 2 3 4 Target KPC VIM NDM OXA 48 like Le 1 C Internal Control When the test is performed for the first time create the PCR test program Check Direct CPE as described in Appendix 1 2 Real time PCR preparations 2 1 Calculate the number of reactions Thaw reagents mix well spin down and keep on ice 2 2 Prepare the real time PCR qPCR mix as described in Table 2 Multiply the CPE solution and the CPE PCR Mastermix by the right number of samples and include 10 surplus to ensure that you have enough qPCR reaction mix for all the calculated reactions For ABI 7500 LC 480 and CFX96 96 well PCR plates are used for Rotor Gene Q PCR strip tubes 2 3 Pipette 15 ul of qPCR mix to the wells or tubes 2 4 Pipette 10 ul of test sample or control sample to each pre filled well or tube The 4 positive controls may also be combined into a single mixed positive control by adding equal volumes of each control up to a volume of 10 ul per reaction i e 4 x 2 5 ul of each control See Table 2 2 5 Seal the 96 well plate or close the PCR strip tubes Mix the plate by tapping it on the bench and spin down briefly 2 6 Transfer the plate or tubes to the PCR room Table 2 real time PCR reaction mix setup Component qPCR mix CPE PCR Masterm
29. s csssssssssccceeesssssssneeeeceeesssessenanaes 11 Appendix 2 Performance Characteristics cccccseesssssecceeeceeesenneaeeeeeeesseesenaaaeeeeees 19 Check Direct CPE User manual Version 2 3 Issued 01 09 2014 Check Direct CPE Intended use Check Direct CPE is a qualitative in 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
30. tive control Positive control 30 3 27 3 Negative sample extracted with IC FORAN N A N A N A N A 2 Results interpretation Tables 4 amp 5 e Ifthe run is valid interpret results as positive negative or invalid using the C values specified in Tables 4 amp 5 Positive carbapenemase samples will show a Cy value in the FAM VIC and or Red TXR channel Negative carbapenemase samples will show no Cy value in the FAM VIC and Red TXR Channel In the Cy5 Channel a Cy value is expected at 30 3 e Samples with a negative C value N A or undetermined for FAM VIC or Red TXR and with a negative or 233 Cr value for Cy5 IC indicate that the reaction is inhibited and therefore that the sample is invalid See Tables 4 amp 5 and FAQ and Troubleshooting e For positive rectal perianal swabs the bacterial load may vary significantly Therefore any C value for FAM VIC or Red TXR may indicate a positive sample e For cell suspensions the bacterial load is well defined Therefore the C value will be within a specific range Cr values out of this range cannot be trusted and the test should be repeated A typical error is a high C value due to contamination when working with cell suspensions Please check the maximum C value for a true positive sample in Table 5 Positive carbapenemase samples will also show a C value in the Cy5 channel if the target has not out competed the internal control IC during the rea
31. ts Heatlth Results All 93 bacterial strains were typed correctly for the targeted carbapenemase genes with the Check Direct CPE test Table V Check MDR C 103 Check Direct CPE 19 KPC KPC 16 NDM NDM VIM 1 NDM 0OXA 48 NDM VIM OXA 48 33 VIM NDM VIM 1 VIM OXA 48 NDM VIM OXA 48 23 OXA 48 OXA 48 20 Check Direct CPE User manual Version 2 3 Issued 01 09 2014
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