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1. Check Direct CPE User manual Check Direct CPE for SmartCycler Real time PCR kit for the detection of carbapenemase producing Enterobacteriaceae Version 1 0 Date of issue 15 06 2014 18 0083 Yy as IFU 083 01 EU CE lIVD U S For Research Use Only Not for use in diagnostic procedures Contents Intended UNSC nrrnienieeeni a aa E EEE ERTS 2 Nauigale hic o EAEE E re E EA E an arerter inert mee 2 Principle ofthe method senienas e E E a 2 Kit contents for 48 reactions ciccxctswesavunsubdururdieddqtuiriendugudghoannsananeantantabnceniuwniwa tae asians 2 Materials required but not supplied with the kit ssssssssssssessessesssssssssesrsesssssseressessee 2 Storage handling and stability seiisecerias doxcsotiensinevanvstvaniadeueiieas eiduanvaunsuveaveavesvensaivenebecanes 3 Good laboratory practices siccscccntos cet aicescaicasseacasngenieateeuatunaeueeietewanmabuuisenshanemaaciueaes 3 Specimen collection and DNA extraction cccccesseseseeseessestesesceseesseeceeceecaeceeceeseeeaes 4 Real time PCR assay and SmartCycler Operation ccsescsseseeseessesseeseeseeeeececeeeeeees 4 Results Interpretation sziniccasaeancscaeaies matesadeatinsedeiaeanasavnanaeieeiucienoeenaneneualenaaaas 5 Frequently asked questions FAQ amp Troubleshooting ccccecsesescessessessesteeeeeeees 7 Key WS MINS Used ac gece sich coca ha act wre erpneene eae R e E a 7 Limitati NS e senna ence een mere ene errr nr eT error2. 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 pre PCR room and a PCR room e DNA extraction and preparation of the amplification reactions are 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 for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE Specimen collection and DNA extraction Crude DNA extraction from bacterial cells Important points before starting D
3. bacterial suspensions Pipettes amp disposable filter tips for volumes of 10 to Mini centrifuge with rotor for SmartCycler 25 ul Reaction Tubes 1000 ul cat no 900 0021 PCR grade water e g Milli Q or aqua bidest SmartCycler Tube Rack cat no 300 0276 1 5 ml tubes Eppendorf tubes e Vortex mixer 2 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE 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 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 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
4. een enrer ren re erm aren caer ra tr er een erent r er rr 8 Technical assistar Esset naa sph ou ae nari oi e a aeea ia aA Era 9 Appendix 1 Creating the Check Direct CPE protocol e sssssssessessssssessesrsssersessrneesesse 10 Appendix 2 Default settings for analysis of Check Direct CPE results seeeees 10 Appendix 3 Performance Character istics ccsccscscscesccescssesscesseesesaesaesseseesseseessaseaes 10 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 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 At present the test is validated for bacteria cultured from various 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 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 s
5. NA 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 Bacteria from selective chromogenic plates may also be used 2 Prepare a bacterial cell suspension of McFarland 0 5 1 0 or ODsoo 0 08 0 15 using PCR grade water e g Milli Q or bidest water 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 stored at 20 C for longer periods a 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 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 neg
6. anual 130 0 NA 5 410 3 60 lo 2 Cy3 Assay ON 5 40 Primary Curve Manual 130 0 NA 5 10 3 60 lo 3 TxR Assay ON 5 40 Primary Curve Manual 130 0 NA 5 10 3 60 lo 4 cy5 Assay ON 5 40 Primary Curve Manual 130 0 NA 5 10 3 60 lo Appendix 3 Performance Characteristics 1 Limit of Detection The analytical limit of detection LoD of Check Direct CPE was determined using the individual positive controls supplied with the test These positive controls contain the target DNA at 10 copies per ul Serial dilutions were made of each of the positive controls and 10 10 10 and 10 copies were added in quadruplicate a total of 64 reactions following the protocol as described on pages 4 and 5 of this User Manual The 10 10 and 10 DNA copies were always detected regardless of the target DNA used Only at 10 copies added as input DNA for the test a difference in LoD was visible which is depicted in the table A Table A Target Input DNA Success KPC 10 4 out of 4 VIM 10 4 out of 4 OXA 48 10 4 out of 4 NDM 10 2 out of 4 10 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE 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 pr
7. ative 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 SmartCycler 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 SmartCycler System Table 1 SmartCycler multiplex real time PCR setup eem a ett oe 2 3 4 Channel 1 Target KPC VIM NDM OXA 48 like 1 C 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 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 reaction mix for all the calculated reactions 2 3 Pipette 15 ul of qPCR reaction mix to each of the SmartCycler Reaction Tubes i e Smart Tubes 4 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE 2 4 Pipette 10 ul of test sample or con
8. ciences Ltd Texas Red is a registered trademarks of Molecular Probes Inc SmartCycler is a registered trademark of Cepheid Inc Check Points Health BV Tel 31 317 453 908 R Binnenhaven 5 Fax 31 317 210 147 o 6709 PD Wageningen info check points com e The Netherlands www check points com Check Points Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE Appendix 1 Create the Check Direct CPE protocol Important points before starting Refer to SmartCycler System User s Manual for detailed instructions on how to operate the SmartCycler System and software version 2 0d Open the SmartCycler software and click Define Protocols Click New Protocol bottom left Enter Name Check Direct CPE Enter the parameters for the Check Direct CPE protocol as outlined in Figure A below When ready click Save Protocol ot te Stage 3 Repeat 45 times 2 Temperature Cycle hi DegiSec Tem Secs NA 95 0 15 Off 60 0 60 NA On C Advance to Next Stage Figure A Real time protocol parameters Appendix 2 Default settings for analysis of Check Direct CPE results Ch Dye Usage Bkgnd Bkgnd Bkgnd Care mates ier Setting Manual Thresh Auto Thresh Auto Min les Max Valid Min Valid Max Boxe eee Sub g Cycle Max Cycle Fluor Units SD s Cycle Cycle Cycle Cycle Cyc 1 FAM Assay ON 5 40 Primary Curve M
9. 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 D The 93 bacterial strains tested with Check Direct CPE were previously identified carbapenemase positive with the micro array diagnostics test Check MDR CT103 Check Points Heatlth Results All 93 bacterial strains were typed correctly for the targeted carbapenemase genes with the Check Direct CPE test Table D 19 KPC KPC 16 NDM NDM VIM 1 NDM 0XA 48 NDM VIM OXA 48 33 VIM NDM VIM 1 VIM OXA 48 NDM VIM OXA 48 23 OXA 48 OXA 48 11 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014
10. ct 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 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 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 5 Real time results show very low fluorescent signals 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 stor
11. ed 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 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 C values expected for the controls in Table 6 do not match the C 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 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 9 Duplicate samples tested with Check Direct CPE test did not yield identical results C values of identical samples may vary between individual reactions Large var
12. iations gt 2 C 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 for SmartCycler User manual Version 1 0 Issued 15 06 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 VIM and OXA 48 in Enterobacteriaceae The test detects the following carbapenemase gene variants VIM1 6 amp 8 38 OXA 48 162 163 181 232 244 245 247 370 NDM1 10 and KPC1 17 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 imp
13. in the Sample ID column 3 6 Click the Start Run button 3 7 When the run is completed discard the Smart Tubes according to local regulations Results interpretation Important points before starting For a detailed description on how to analyze data refer to SmartCycler System User s manual Default settings should be used for analysis of Check Direct CPE results Refer to Appendix 2 Always visually inspect the amplification plot for each sample tested versus C values obtained with the software 1 Reported results The SmartCycler software reports Result C value and amplification curves for each detector channel of each specimen tested Results are reported in the Result table of the Views window in the following way e NEG indicates no amplification was detected for that detector channel i e FAM Cy3 TxR and Cy5 C value of NEG result 0 00 Amplification curves of samples showing a NEG result and 0 00 C value must be checked visually For this purpose click on the FAM Cy3 TxR or Cy5 Tab in de Views window e POS indicates that amplification was detected for that detector channel i e FAM Cy3 TxR and Cy5 POS result will indicate a certain C value Amplification curves of samples showing a POS result should also be confirmed visually and checked for abnormalities For example see Figure 1 e C values should be interpreted in correlation with the amplification curves and accordi
14. ng to the interpretation method outlined in Tables 3 and 4 2 Interpretation 2 1 Run validation Check the positive and negative control amplification curves Valid runs e No instruments system failures during the run e Positive and Negative Controls are within the C values specified in Table 3 If the C values of the controls are not as expected refer to FAQ and Troubleshooting 3 Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE Table 3 Criteria for a valid run with Check Direct CPE test Controls KPC ND VIM OXA 48 IC Expected C values FAM Cy3 Cy3 TxR Cy5 Positive controls 30 3 3143 29 3 27 3 35 3 Separate Positive controls 33 43 30 43 30 3 30 3 34 3 Mixed Negative control 0 00 0 00 0 00 0 00 35 3 2 2 Results interpretation If the run is valid interpret results as positive negative or invalid with the C values obtained for the samples with the guidelines summarized in Table 4 e Positive carbapenemase samples will show a Cr value in the FAM Cy3 and or TxR channel Positive carbapenemase samples will also show a Cy value in the Cy5 channel if the target has not out competed the internal control IC during the reaction Always visually inspect the amplification plot to verify the result See Figure 1 as an example e Negative carbapenemase samples will show no C value in the FAM Cy3 and TxR Channel In the Cy5 Channel a C
15. obes sequences were validated with in 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 Table B Gene varans KPC 1to17 NDM 1to 10 VIM 1 to 6 8 to 38 OXA 48 162 163 181 204 232 244 245 247 370 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 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 e organis 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
16. ortant factor for obtaining reliable results with Check Direct CPE This User Manual describes the use of crude DNA extracts from bacterial cells This will only work properly using pure water i e MilliQ or aqua bidest and the specified amount of cells Other buffers or saline solutions will yield poor results Other DNA extraction methods have not been tested with Check Direct CPE for SmartCycler and may yield poor results 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 OXA 48 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 i
17. s limited to appropriately qualified personnel well trained in performing DNA based molecular detection methods Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 2014 Check Direct CPE Key to symbols used For In Vitro Diagnostic Use Negative control Catalog number CONTROL KPC KPC positive control LOT Batch code CONTROL VIM VIM positive control IFU IFU number CONTROL NDM NDM positive control 2 Use before YYYY MM CONTROL OXA 48 OXA 48 positive control pH Consult instructions for use INTERNAL CONTROL Internal control wal Manufacturer ISOLUTION CPE CPE solution y Temperature limitation PCR Mastermix CPE CPE PCR Mastermix Yy Contains sufficient for lt n gt tests 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 FAM is a registered trademarks of Applera Corporation Cy3 and Cy5 are registered trademarks of Amersham Bios
18. trol sample to each pre filled Smart 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 2 5 Close the Smart Tubes and centrifuge for minimum 7 seconds Check that no air bubbles have accumulated in the optical part of the tubes see SmartCycler Il Operator Manual Place the tubes in the SmartCycler Tube Rack and transfer the tubes to the PCR room Table 2 real time PCR reaction mix setup Component NOL pe reaction CPE PCR Mastermix 12 5 ul CPE Solution 2 5 ul Total volume 15 ul 3 SmartCycler Operation 3 1 Insert the Smart Tubes into the required PCR Sites reaction chambers of the SmartCycler Press the tubes firmly and close each PCR Site 3 2 Click Create Run and specify Run Name If needed add remarks in Notes 3 3 Select the Dye Set FCTC25 3 4 Click the Add Remove Sites button A new window Select Protocols and Sites will be displayed in which you need to specify the Protocol and the number of PCR Sites for the test procedure Select Check Direct CPE as the Protocol See Appendix 1 if not specified Highlight the Sites in use for the test procedure and press the right arrow When the Protocol and Sites have been selected click OK 3 5 Enter the sample I D s for every site
19. usceptibility 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 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 f lactam resistance determinants resulting in multidrug and pandrug resistant isolates Check Direct CPE is a rapid real time PCR test for the detection and discrimination of KPC NDM VIM and OXA 48 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 amplification
20. value is expected at 35 3 e Samples with a FAM Cy3 and TxR C value of 0 00 NEG and with a Cy5 Cy value of 0 00 or gt 38 indicate that the sample run is invalid see FAQ and Troubleshooting 2 to 6 Table 4 Data interpretation guidelines KPC NDM VIM OXA 48 IC A Interpretation C values C values YES YES or 0 00 Positive sample 0 00 35 3 Negative sample 0 00 0 00 or gt 38 Invalid If observed Cr values vary significantly from expected C values see FAQ and Troubleshooting section Figure 1 Screenshot of KPC positive sample run in triplicate on the SmartCycler system Check Direct CPE for SmartCycler User manual Version 1 0 Issued 15 06 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 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 2 The real time results show no 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 Dire
21. 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 660ul 4 C CPE solution 9 0081 1 brown tube blue inlay 140 ul Internal Control IC 9 0107 1 tube red inlay 600 ul Negative control 9 0100 1 tube white inlay O 100 ul KPC positive control 9 0083 1 tube green inlay 100 ul 20 C store in the dark NDM positive control 9 0085 1 tube gold inlay 100 ul VIM positive control 9 0084 1 tube yellow inlay 100 ul OXA 48 positive control 9 0086 1 tube orange inlay 100 ul User manual 9 0088 Leaflet download from website Materials required but not supplied with the kit SmartCycler 25 ul Reaction Tubes cat no 900 0003 or e Real time PCR instrument Cepheid SmartCycler 2 0 software 900 0022 version 2 0d e Disposable laboratory powder free gloves Lab coat Densitometer suitable for
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