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1. be Check Direct ESBL o for BD MAX User manual Check Direct ESBL for BD MAX Real time PCR kit for the detection of ESBL producing Enterobacteriaceae Version 1 0 Date of issue 10 07 2014 18 0092 Y 24 IFU 092 01 EU C IVD U S For Research Use Only Not for use in diagnostic procedures Contents Intended USE aaa asec cglcces das a E S 2 Introduction and principle Of the method cc csecsecsececeeceeceseeseesseseesaesaeseeseeseeeeaeees 2 Kit contents for 24 reactions cucencanersnanrnnnesaviadivaaiextenndndndaiedseiishesteanauatacaiens 2 Materials required but not supplied with the kit ccc csseeteeceseeeseseeeeeseeseeeees 2 Storage handling and stability s sesnesenessessessesesssessrssssressresrressereseesnsesreresnsessrosseesens 3 Good laboratory practices ssessesssssssesseessrtessessseeseeresieestnresessstestnessestutesttensessestnenee sense 3 Sample preparation procedures sssssesssesssessesssesseseestsssestessssseestnsssessarestnesserenresnsessessnes 4 BD MAX Operati i osisssa in eiA EE EAE EA A EEE NEA EEA REEE 4 Res lts Interpretati Mss Seer rener e e E E ie rar AEREE i 5 Frequently asked questions FAQ amp Troubleshooting ccccescessescessessessesseeteeeees 6 Himatione a E E A GE 7 Key to symbols used ssosssssssssssessessrssssesssresrressestsrtsrtensesssesenseesteststsstesstestsestestntest tenses stenst 7 FSC Cell ASSIS CANN CE ssrnreirareuen irena a e2. it may never be excluded that other Gram negative bacteria or certain strains of the above species will yield poor results Check Direct ESBL cannot and does not make any representation or warranty that it is capable of correctly detecting the ESBL 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 ESBL 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 lt M EEE Technical assistance support check points com 31 317 453 908 Despite the utmost care in the development an
3. typing susceptibility testing and for further confirmatory identification Introduction and principle of the method The worldwide emergence and dissemination of extended spectrum B lactam resistance in 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 extended spectrum B lactam resistance in Enterobacteriaceae is the production of Extended Spectrum Beta Lactamases i e ESBL s Most Enterobacteriaceae producing ESBL s have resistance to all B lactam antibiotics except carbapenems Presently the vast majority of clinically relevant ESBL s are expressed from one of the following plasmid encoded gene families CTX M1 CTX M2 CTX M9 and SHV ESBL Check Direct ESBL is a multiplex real time PCR assay for detection of the CTX M1 CTX M2 CTX M9 and SHV ESBL gene families 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 The CTX M1 CTX M2 CTX M9 and SHV ESBL gene families have many gene variants and Check Direct ESBL has been designed to reliably detect all variants Check Direct ESBL for BD MAX employs five different fluorescent probes and enables detection and discrimination of the 4 ESBL gene families and the control target SPC that monitors DNA extraction and PCR amplificat
4. a a aa 7 Appendix 1 creating the Check Direct ESBL test program cccecscsecsecsesseseeeeeeenes 8 Appendix 2 performance characteristics ccccsecsecsecseceeceeceeceeceeeseesaesaesaesaseeseesees 9 Check Direct ESBL for BD Max User manual Version 1 0 Issued 10 07 2014 Se Check Direct ESBL for BD MAX Intended use Check Direct ESBL for BD MAX is a qualitative in vitro diagnostic test for the rapid detection of Extended Spectrum Beta Lactamase ESBL genes in Enterobacteriaceae The assay is performed on the BD MAX system using bacterial cell suspensions from clinical specimens of individuals at risk of colonization with ESBL producing Enterobacteriaceae Check Direct ESBL detects the presence of the ESBL gene families CTX M1 CTX M2 CTX M9 and SHV ESBL presently the primary cause of extended spectrum lactam resistance 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 ESBL for BD MAX can be used as an aid to identify prevent and control ESBL producing Enterobacteriaceae that colonize patients in healthcare settings Check Direct ESBL for BD MAX is not intended to diagnose infections with ESBL producing Enterobacteriaceae nor to guide or monitor treatment for these infections Parallel cultures are necessary to recover organisms for epidemiological
5. 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 Cy 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 Cy 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 ESBL run on the BD MAX System If the Cy values of the controls are not as expected refer to FAQ and Troubleshooting 3 Table 2 Criteria for a valid run with Check Direct ESBL test mente Cr 475 520 C 530 C 585 630 C 630 665 Cr 680 715 ideal CTX M1 CTX SHV ESBL CTX M9 SPC Positive controls 33 3 30 3 Negative sample 1 1 2 2 Results interpretation If the run has been validated interpret results as positive negative or invalid with the C values obtained for the samples following the guidelines summarized in Table 3 Invalid runs should be retested Check Direct ESBL for BD Max User
6. 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 ESBL for BD Max User manual 3 Version 1 0 Issued 10 07 2014 Se Check Direct ESBL 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 Milli Q water or aqua bidest may be used 3 Pipette 500 of Milli Q water or aqua bidest and 10 uL of the bacterial cell suspension 1 x 10 CFU 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 4 Close the Sample Buffer Tube with a septum cap and vortex 10 second at low speed 5 Transfer the Sample Buffer Tube with the bacterial cell suspensions to be analyzed to the PCR room Preparation of control reactions To validate the run perform positive and negative control reactions for each Che
7. able 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 selected primers and probes ESBL gene Variants detected 1 3 10 12 15 22 23 28 30 32 34 36 37 42 52 55 57 58 60 62 66 68 69 71 72 88 96 101 107 109 114 116 117 132 CTX M1 group 133 136 CTX M2 group 2 4 7 20 31 35 43 44 56 59 76 77 95 124 131 CTX M9 group 9 13 14 16 19 21 24 27 38 45 51 55 57 64 65 67 81 93 102 104 105 106 110 111 112 113 121 122 123 126 134 2 2a 3 4 5 7 9 10 12 15 20 23 30 34 39 45 46 55 64 66 86 90 105 106 123 124 128 129 134 141 152 153 152 160 SHV ESBL 163 165 Check Direct ESBL for BD Max User manual 9 Version 1 0 Issued 10 07 2014
8. ck Direct ESBL PCR run The positive control is supplied with the kit e Positive control Pipette 10 uL of the positive control and 500 uL of Milli Q water or aqua bidest into one Sample Buffer Tube Vortex for 10 seconds e Negative control Pipette 500 uL of Milli Q water or aqua bidest 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 EE Detector 475 520 Ez 585 630 630 665 680 715 5 SPC Channel 4 Target CTX M1 CTX M2 SHV ESBL CTX M9 SPC Sample Processing Control When the test is performed for the first time create the PCR test program Check Direct ESBL as described in Appendix 1 Pipette Tips TE UL Buffers Extraction position Master Mix position Conical tube position Figure 1 DNA Unitized Reagent Strip setup Check Direct ESBL for BD Max User manual Version 1 0 Issued 10 07 2014 Se Check Direct ESBL for BD MAX 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 positio
9. concentrations were tested i e 10 10 10 10 10 1 DNA copies ul Samples were tested on the BD MAX system in the PCR only mode First Check Direct ESBL Snap tubes were rehydrated with 12 5ul of a solution containing Tris EDTA buffer MMK PCR diluent and SPC target DNA Next 12 5 ul of target DNA solution was added Subsequently the resulting 25ul of each reaction mix this mix was transferred to the MMK tube to rehydrate the master mix Finally 12ul of each solution was manually transferred into the individuals well of a BD MAX PCR cartridge The cartridge was then placed into the BD MAX instrument and the PCR only protocol was started using the Check Direct ESBL test program The assays with 10 10 10 10 DNA copies ul were always positive The assays with 10 and 1 DNA copies ul reached the true LoD of the test results are presented in the table below Target Input DNA copi s DNA copies PCR Successrate CTXM 1 12 5 2 4 out of 4 CTXM 2 12 5 2 3 out of 4 CTXM 3 15 12 5 2 2 out of 2 CTXM 10 125 20 2 out of 2 CTXM 9 12 5 2 2 out of 2 SHV 2 12 5 2 2 out of 2 SHV 5 12 12 5 2 2 out of 2 In silico Specificity The specificity of the Check Direct ESBL 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 t
10. d 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 ESBL 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 ESBL for BD Max User manual 7 Version 1 0 Issued 10 07 2014 so Check Direct ESBL for BD MAX Appendix 1 Creating the Check Direct ESBL 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 ESBL e Extraction Type Select Exk DNA 1 Plas
11. iled since the SPC was not detected e The BD DNA 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 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 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 ESBL reagent tubes containing the fluorescent probes and primers may be degraded Please check expiration date and make sure that the ESBL 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 y
12. ion Kit contents for 24 reactions Components Mat No Description Storage conditions ESBL reagent tubes 9 0065 24 sealed tubes red seal 4 C store in the dark ESBL positive control 9 0064 1 tube red cap 4 C User Manual 9 0109 Leaflet download from website Not critical Materials required but not supplied with the kit Equipment Real time PCR instrument BD MAX System software version 2 96A BD MAX ExK DNA 1 Extraction Kit Ref 442818 BD MAX DNA MMK Master Mix Ref 442848 BD MAX PCR Cartridges Ref 437519 Disposable laboratory powder free gloves Lab coat Densitometer suitable for Pipettes amp disposable filter tips for volumes of 10 to 1000 ul bacterial suspensions Milli Q water or aqua bidest Vortex mixer Check Direct ESBL for BD Max User manual Version 1 0 Issued 10 07 2014 Se Check Direct ESBL for BD MAX Storage handling and stability The Check Direct ESBL 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 da
13. ma 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 presented 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 80 100 530 565 80 100 585 630 30 200 630 665 80 100 680 715 30 150 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 1 6 0 0 0 0 0 0 530 565 1 2 1 2 0 0 0 0 Excitation 585 630 0 0 0 0 10 7 0 0 Channel 630 665 0 0 0 0 6 3 5 6 680 715 0 0 0 0 0 0 7 8 Check Direct ESBL for BD Max User manual 8 Version 1 0 Issued 10 07 2014 so Check Direct ESBL for BD MAX Appendix 2 Performance Characteristics Limit of Detection with control DNA targets The analytical limit of detection LoD of Check Direct CPE was determined using linearized control plasmids containing target sequences for CTX M1 CTX M3 amp M15 CTX M10 CTX M2 CTX M9 SHV 2 and SHV 5 amp 12 Of each target DNA 6
14. manual 5 Version 1 0 Issued 10 07 2014 Se Check Direct ESBL for BD MAX Ct values of bacterial cells will be in a specific Ct window for each target because of the well defined amount of cells used as input material for the test However Ct values will differ between individual strains Ct values out of this Ct window are regarded as invalid and should be retested A higher Ct value above the Ct window suggest contamination of the sample or a strain that is not pure A lower Ct value suggests an aberrant amplification plot Please check the amplification plot in such a case to confirm this Table 3 Data interpretation guidelines for bacterial cells er en Len ee ee lt 28 lt 28 lt 30 lt 29 29 3 Positive 1 1 1 1 2943 Negative gt 28 gt 28 gt 30 gt 29 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 Cy 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 fa
15. n 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 ESBL reagent tube red seal into position 3 of the DNA Strip see Figure 1 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 ESBL 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 detector channel of each specimen tested in the following
16. our BD local representative 6 Duplicate samples tested with Check Direct ESBL 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 ESBL for BD Max User manual 6 Version 1 0 Issued 10 07 2014 so Check Direct ESBL for BD MAX Limitations Check Direct ESBL uses a range of specific DNA markers to detect the presence of the CTX M1 CTX M2 CTX M9 and SHV ESBL genes which currently represent the clinically most prevalent ESBL s The test detects almost all clinically relevant variants of CTX M1 CTX M2 CTX M9 and SHV ESBL It should be noted that some more rare variants of CTX M1 CTX M2 CTX M9 and SHV ESBL are not detected and that other ESBL gene families like e g TEM GES VEB and PER are also not detected Bacterial cell suspensions should be used as input material for the test The quality of the input DNA is an important factor for obtaining reliable results with Check Direct ESBL 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
17. te Store in the dark Close protective pouches promptly with the zip seal after each use Remove any access air in the pouches prior to sealing Please contact the Check Points office at support check points com if you have any further questions 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 gloves whenever
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