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1. Check Direct CPE oO for BD MAX User manual Check Direct CPE for BD MAX Real time PCR kit for the detection of carbapenemase producing Enterobacteriaceae Version 1 2 Date of issue 01 10 2014 18 0082 Y 24 IFU 082 03 EU C IVD U S For Research Use Only Not for use in diagnostic procedures Contents NARMS WSS E EA E 2 Introduction and principle Of the method l s ssssssssssssesssssssseesresrsessssesresrsessnrseseesesseerse 2 Kit contents for 24 reactions sesessssesnsesseseseerensesenesensenrssureseesennsaresesensenrurennsessenenes 2 Materials required but not supplied with the kit ccc cececsseetsereeseeseeseeseeseeseeeees 2 Storage handling and stability sacasecaccuteaaindiededeeieatsheounmam ananedandecdadwenacatas 2 G00 laboratory Practices sensisse isie E E AEE E e 3 Sample preparation procedures cscceccsccescsscsscsscsscesecseeesseseeseesseeseeseesesseeceeceeseeseneaes 4 BD MAX operatio N sanesna i a a o i E E 4 RESUMES Interpretatio Messeren aaee ER R E EEEE EEEE iE 5 Frequently asked questions FAQ amp Troubleshooting ccccecescescessessessesteeseseeees 6 Lirntation Sen e E E E 7 Key to symbols SEd vracics cass diranssasudsnusncnsnelaiuslentin in eiadncenetsnenctianaaysseasuadualvadudedveadsemnetuemntell 7 Technical assistance handy ves teaica deena rncuueciedy asiieucomaaaet ibaa netuenaaevei totdrcabronutanbananaens 7 Appendix 1 creating the Check Direct CPE te2. 4 and 5 of this User Manual 10 ul target DNA 500 ul PCR grade water added to SB 1 The whole procedure was executed twice The 10 and 10 DNA copies were always detected regardless of the target DNA used Only at 100 copies added as input DNA for the test a difference in LoD was visible which is depicted in the table below KPC 100 3 out of 6 VIM 100 12 6 out of 6 OXA 48 100 12 6 out of 6 NDM 100 12 3 out of 6 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 designed to specifically identify the gene variants listed in the Table below A 100 sequence match with the primers and probes by in silico analysis was assumed to warrant reliable detection of each of the depicted variants Single mismatches with the primers and probes exist in some variants of which we expected that detection would not be compromised This was confirmed by testing such variants in comparison with variants which were 100 homologous Primers and Probes sequences were tested for potential homologies with genes from other organisms using all gene sequences present in the international gene bank on April 1 2014 GenBank NIH genetic sequence database using sequence comparison analysis No cross homology was found with other organisms for the selec
3. Max User manual Version 1 2 Issued 01 10 2014 o Check Direct CPE for BD MAX Waste pa ae Tips j e wn e 2 5 2 D ra o p x Lu Master Mix position Figure 1 DNA Unitized Reagent Strip setup 3 BD MAX instrument set up 3 1 Open the Run screen of the BD MAX System software v2 96A 3 2 In the Assay menu select Check Direct CPE see Appendix 1 if not specified 3 3 Enter the Sample Buffer Tube barcode using the barcode scanner you can also enter the barcode manually Start with position 1 of rack A 3 4 Place each of the Sample Buffer Tubes in their corresponding position in the BD MAX racks with septum cap 3 5 Enter the specimen or patient identification information into the work list Check that each specimen or patient information correspond to its specific Sample Buffer Tubes in the Rack 3 6 Load the Rack s into the BD MAX System Rack A is positioned on the left side of the instrument and Rack B on the right side 3 7 Load the BD MAX PCR cartridge s 3 8 Close the instrument door and select Start Run Results Interpretation Important points before starting For a detailed description on how to analyze data refer to BD MAX System User s manual Always visually inspect the amplification plot for each sample tested versus C values obtained with the software 1 Reported results The BD MAX software reports C values and amplification curves for each
4. 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 Dye amp quencher compounds in this product are sold under license from Biosearch Technologies Inc and protected by U S and world wide patents either issued or in application The license grant covers human in vitro diagnostic IVD applications Trademarks BD BD MAX are trademarks Becton Dickinson GmbH Check Points Health BV Tel 31 317 453 908 e e 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 BD Max User manual 7 Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Appendix 1 Creating the Check Direct CPE test program Important points before starting Refer to BD MAX System User s Manual for detailed instructions on how to operate the BD MAX System and software version 2 96A 1 Create a new Test select Create test and enter the following parameters e Test Name type Check Direct CPE e Extraction Type Select Exk DNA 1 Plasma Serum e Master Mix Format choose Type 1 BD MMK or MMK SPC and Dried Primers amp Probes e Channel detector Settings set Gain and Threshold with parameters presen
5. invalid with the C values obtained for the samples following the guidelines summarized in Table 3 Invalid runs should be retested Ct values obtained with bacterial cells will generally be in a specific Ct window for each target because of the well defined amount of cells used as input material for the test Note however that Ct values may differ significantly between individual strains Table 3 specifies the upper limit of this Cy window a higher C value suggests contamination of the sample or a strain that is not pure Therefore this will be regarded as an Invalid result Table 3 Data interpretation guidelines for bacterial cells 33 27 lt 26 lt 32 29 3 Positive 1 1 1 1 29 3 Negative gt 33 gt 27 gt 26 gt 32 29 3 Invalid 1 or YES 1 or YES 1 or YES 1 or YES lt 26 or gt 32 Invalid 1 or YES 1 or YES 1 or YES 1 or YES 1 Invalid Frequently asked questions FAQ amp Troubleshooting Refer to the troubleshooting section of the BD MAX System User s Manual for additional information 1 Real time results show no C values or interpretation indicates that the sample is invalid Possible causes and troubleshooting e The PCR reaction has been inhibited by exogenous or endogenous substances Please repeat sample testing When still inhibited a lower amount of input sample may improve the results e The DNA extraction failed since the SPC was not detected e The BD DN
6. tested with Check Direct CPE test do not yield identical results C values of identical samples may vary between individual reactions Large variations gt 2 Cy values suggest pipetting errors or other differences between the duplicate samples Check Direct CPE for BD Max User manual 6 Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Limitations Check Direct CPE uses a range of specific DNA markers to detect the presence of the carbapenemase genes KPC NDM OXA 48 and VIM which currently represent the clinically most prevalent carbapenemases The test detects all presently known variants of KPC NDM OXA 48 and VIM except VIM 7 a rare variant only found in Pseudomonas aeruginosa It should be noted that other rare carbapenemase gene families are not detected The quality of the input DNA is an important factor for obtaining reliable results with Check Direct CPE For cell suspensions the correct cell densities are an important factor to obtain reliable results and the procedure described in this manual must be strictly followed The assay has been tested extensively with DNA purified from gram negative bacteria such as Escherichia Salmonella Klebsiella Enterobacter Citrobacter and Pseudomonas with excellent results However it may never be excluded that other Gram negative bacteria or certain strains of the above species will yield poor results Check Direct CPE cannot and does not make any representation or
7. warranty that it is capable of correctly detecting the carbapenemase genes in all gram negative species subspecies or types or in all clinical samples 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 The presence of multiple bacterial species in a sample may hamper the interpretation of the test As with other diagnostic assays the results of this test may only be interpreted in combination with additional laboratory and clinical data available to the responsible person Use of this assay is limited to appropriately qualified personnel well trained in performing DNA based molecular detection methods Key to symbols used ___symbol___ Definition Control CPE control For In Vitro Diagnostic Use Catalog number Batch code IFU number Use before YYYY MM Consult instructions for use Manufacturer Temperature limitation Contains sufficient for lt n gt tests Ke woelle E al 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
8. A MMK may have expired e An error in liquid handling has occurred check unitized reagent strips and PCR cartridge to determine where liquid handling problem has occurred example air bubble in the cartridge and re run the sample If the problem persists contact your local BD representative 2 Troubleshooting for invalid results For Invalid results Repeat test with the original specimen by preparing a new Sample Buffer Tube Alternatively test newly collected specimen or use a lower amount of specimen 3 Real time results show no Cy values for the positive control or interpretation indicating that sample is invalid Possible causes and troubleshooting e The positive control solution was not added e The BD DNA MMK may have expired e Air bubbles have occurred in the PCR reaction chamber of the positive control 4 Real time results show very low fluorescent signals in all samples and detector channels including the SPC signal Possible causes and troubleshooting e The CPE reagent tubes containing the fluorescent probes and primers may be degraded Please check expiration date and make sure that the CPE tubes have been stored correctly e The BD MAX System can be responsible for these results Please refer to BD MAX User s manual or contact your BD local representative 5 The BD MAX System states an error or failure Refer to the BD MAX instrument user manual or contact your BD local representative 6 Duplicate samples
9. 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 multiplex real time PCR assay for detection of the KPC OXA 48 NDM and VIM carbapenemase genes The 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 For KPC VIM OXA 48 and NDM many gene variants exist and Check Direct CPE has been designed to reliably detect all variants Check Direct CPE for BD MAX employs five different fluorescent probes and enables detection and discrimination of the 4 carbapenemase genes and the control target SPC that monitors DNA extraction and PCR amplificatio
10. detector channel of each specimen tested in the following way e C value of 0 indicates that there was no Cy value calculated by the software Amplification curve of the sample showing a 0 C value must be checked manually e C value of 1 indicates that no valid amplification process has occurred Check that there is no amplification curve for the sample with a C value of 1 on the graphical results e Any other Cr value should be interpreted in correlation with the amplification curve PCR Analysis tab and according to the interpretation method outlined in Tables 2 and 3 2 Interpretation 2 1 Run validation Verify that the real time PCR run is valid before data interpretation of the results Check that there is no report of BD MAX System failure Check the positive and negative control amplification curves Table 2 shows criteria for a valid real time Check Direct CPE run on the BD MAX System If the C values of the controls are not as expected refer to FAQ and Troubleshooting 3 Table 2 Criteria for a valid run with Check Direct CPE test Cr 475 520 C530 C 585 630 C 630 665 Cr 680 715 Samnpletype vI OXA 48 like NDM SPC Positive controls 3243 30 3 Negative sample 1 1 Check Direct CPE for BD Max User manual 5 Version 1 2 Issued 01 10 2014 s0 Check Direct CPE for BD MAX 2 2 Results interpretation If the run has been validated interpret results as positive negative or
11. es that were previously identified carbapenemase positive with the Check Points micro array diagnostics test Check MDR CT103 Check Points Health All 93 bacterial strains were typed correctly for the targeted carbapenemase genes with the Check Direct CPE test Results are depicted in the table below Number of Check MDR CT103 result Check Direct CPE result strains tested 19 KPC KPC 16 NDM NDM 33 VIM VIM 23 OXA 48 OXA 48 1 NDM OXA 48 NDM OXA 48 1 VIM OXA 48 VIM OXA 48 Check Direct CPE for BD Max User manual 10 Version 1 2 Issued 01 10 2014
12. in the pouches prior to sealing Please contact the Check Points office at support check points com if you have any further questions Check Direct CPE for BD Max User manual 2 Version 1 2 Issued 01 10 2014 e Check Direct CPE for BD MAX Good laboratory practices Recommendations for best results The quality of the results depends on strict compliance with the following good laboratory practices especially concerning PCR practices e The test must be performed by adequately trained personnel e Do not use reagents after their expiration date e Follow recommendations for storage and handling 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 sample preparation room and a PCR room with the BD MAX system Never transfer items from the PCR room to the sample preparation 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 a reaction tube to avoid contamination e Follow proper pipette dispensing techniques to prevent aerosols e Wear clean disposable gloves and clean lab coats for the different steps of the test e Change glo
13. n Kit contents for 24 reactions Components Mat No Description Storage conditions CPE reagent tubes 9 0062 24 sealed tubes purple seal 4 C store in the dark CPE positive control 9 0061 1 tube purple cap 4 C User Manual 9 0079 Leaflet download from website Not critical Materials required but not supplied with the kit Equipment BD MAX ExK DNA 1 Extraction Kit Ref 442818 Real time PCR instrument BD BD MAX DNA MMK Master Mix Ref 442848 MAX System software version BD MAX PCR Cartridges Ref 437519 2 96A Disposable laboratory powder free gloves Lab coat Densitometer suitable for Pipettes amp disposable filter tips for volumes of 10 to 1000 pl bacterial suspensions PCR grade water e g Milli Q or aqua bidest Vortex mixer Storage handling and stability The Check Direct CPE kit is shipped at ambient temperature and should be stored at 4 C upon receipt Please visually inspect the product upon initial opening to ensure that its contents are intact Do not use this product if the packaging is damaged upon arrival and do not use reagents if their protective pouches are open or broken upon arrival Do not use reagents if desiccant is not present or broken inside and do not remove desiccant from protective pouches Store all opened reagents at 4 C until expiration date Store in the dark Close protective pouches promptly with the zip seal after each use Remove any excess air
14. perform positive and negative control reactions for each Check Direct CPE PCR run The positive control is supplied with the kit e Positive control Pipette 10 uL of the positive control and 500 uL of PCR grade water into one Sample Buffer Tube Vortex for 10 seconds e Negative control Pipette 500 uL of PCR grade water into one Sample Buffer Tube Vortex for 10 seconds BD MAX 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 BD MAX System Table 1 Multiplex qPCR A Detector 475 520 530 565 585 630 630 665 680 715 Channel Target KPC VIM OXA 48 like SPC SPC Sample Processing Control When the test is performed for the first time create the PCR test program Check Direct CPE as described in Appendix 1 2 BD MAX Rack set up 2 1 Load the BD MAX system racks with the number of DNA Unitized Reagents Strips necessary for the number of samples to test Gently tap each strip to make sure all liquids are at the bottom of their container 2 2 Prepare Unitized Reagents Strips 2 2 a Snap a DNA extraction BD Exk 1 Reagent tube white seal into position 1 of the DNA Strip see Figure 1 2 2 b Snap a DNA MMK Master Mix tube green yellow seal into position 2 of the DNA Strip see Figure 1 2 2 c Snap a CPE reagent tube blue purple seal into position 3 of the DNA Strip see Figure 1 Check Direct CPE for BD
15. st program cccscssesseeseeseeeseeseeseeeees 8 Appendix 2 performance Characteristics cccsecsccesceecsssssseseeesecseessessessessessessasees 9 Check Direct CPE for BD Max User manual Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Intended use Check Direct CPE for BD MAX is a qualitative in vitro diagnostic test for the rapid detection of carbapenemase genes in Enterobacteriaceae The test is intended to be used for bacteria cultured from 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 uses the BD MAX system for extraction of DNA and subsequent real time PCR employing the reagents provided combined with universal reagents and disposables for the BD MAX system Check Direct CPE for BD MAX can be used as an aid to identify prevent and control carbapenemase producing Enterobacteriaceae that colonize patients in healthcare settings Check Direct CPE for BD MAX 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 and principle of the method The worldwide emergence and dissemination of carbapenem resistance
16. ted in Table A e GardRail select Default e Test details enter the PCR profile see Table B e Spectral Cross Talk tab enter parameters presented in Table C 2 Select Save Test Table A Gain parameters Detector Gain Threshold 475 520 40 100 530 565 80 150 585 630 30 150 630 665 80 150 680 715 40 300 Table B Real time protocol parameters Step Name Profile Type Cycles Time s Temp C Detect Denaturation Hold 1 600 98 NO 15 98 NO Amplification amp Detection 2 temperature 45 62 60 YES Table C Spectral cross talk parameters False Receiving Channel 475 520 530 565 585 630 630 665 680 715 475 520 0 0 0 0 0 0 0 0 530 565 0 0 0 0 0 0 0 0 Excitation 585 630 0 0 0 0 7 4 0 0 Channel 630 665 0 0 0 0 0 0 0 0 680 715 0 0 0 0 0 0 4 4 Check Direct CPE for BD Max User manual 8 Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Appendix 2 Performance Characteristics 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 and 10 copies were added in triplicate to a sample buffer tube a total of 36 reactions processed in 2 BD MAX runs following the protocol as described on pages
17. ted primers and probes carpenemasegene Variants deteeted KPC 1 17 NDM 1 10 VIM 1 6 amp 8 38 OXA 48 like 48 162 163 181 204 232 244 245 247 370 Analytical Specificity The analytical specificity of the Check Direct CPE real time diagnostic test was determined by testing the cross reactivity with samples containing a high amount of non target organisms 132 carbapenemase negative strains were used to test the specificity of the Check Direct CPE real time test An overview of these strains is outlined in the table below All isolates tested negative with the Check Direct CPE assay and the internal control was reliably detected in all samples The Check Direct CPE test showed 100 specificity based on the reference strains tested Check Direct CPE for BD Max User manual 9 Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Suede Strains tested Campylobacter jejuni 2 Citrobacter freundii 5 Enterobacter aerogenes 1 Enterobacter cloacae 42 Enterococcus casseliflavus 1 Enterococcus faecalis 2 Escherichia coli 51 Klebsiella oxytoca 1 Klebsiella pneumoniae 16 Pseudomonas aeruginosa 2 Salmonella typhimurium 1 Pseudomonas mirabilis 3 Staphylococcus aureus 2 Serratia marcescens 1 Stenotrophomonas maltophilia 2 Analytical Inclusivity A retrospective study was performed with 93 bacterial strains of 13 different gram negative speci
18. ves whenever you suspect that they are contaminated e Keep the tubes of all kit components and samples closed as much as possible e Clean the lab benches and all equipment regularly with a 0 5 sodium hypochlorite solution Please read the full protocol before starting the test Check Direct CPE for BD Max User manual 3 Version 1 2 Issued 01 10 2014 so Check Direct CPE for BD MAX Sample preparation procedures Test preparation for bacteria from culture 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 x 10 CFU ml from the bacterial cells grown on the agar plate Cell suspension buffer e g PBS or 10mM Tris HCl pH8 0 or PCR grade water may be used Dilute this suspension 100 times in cell suspension buffer or PCR grade water to obtain a suspension of 1 x 10 CFU ml Transfer the bacterial cell suspensions to be analyzed to the PCR room 4 Pipette 500 uL of the bacterial cell suspension 1 x 10 CFU ml into one DNA Sample Buffer Tube SB 1 supplied by BD with the DNA extraction kit refer to Materials required but not supplied with the kit 5 Close the Sample Buffer Tube with a septum cap and vortex 10 second at low speed ba Preparation of control reactions To validate the run
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