User manual User manual - Check
Contents
1. following the protocol as described on pages 4 and 5 of this User Manual Results are depicted in the Table below SBT refers to the BD MAX Sample Buffer Tube CFU per SBT CFU PCR Success Rate a goo y Co o J o 100 40 In silico Specificity The specificity of the Check Direct CPE Screen 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 selected primers and probes Carbapenemase gene Variants detected VIM OXA 48 like 48 162 163 181 204 232 244 245 247 370 Analytical Specificity The analytical specificity of the Check Di2. 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 CPE Screen reagent or CP MasterMix 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 Screen reagent tubes containing the fluorescent probes and primers may be degraded Please check expiration date and make sure that the CPE Screen 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 tested with Check Direct CPE Screen assay do not yield identical results Cr values of identical samples may vary between individual reactions Large variations gt 2 Cy values suggest pipetting erro
3. 5ul of CP MasterMix at the bottom of the tube making sure not to create air bubbles 2 2 d Click the Unitized Reagents Strips into their rack positions Check Direct CPE Screen for BD Max User manual 4 Version 1 0 Issued 09 09 2014 eS Check Direct e CPE Screen for sp max Extraction T Reagent JUK Sen ug Extraction Pipette Tips at Reagents Probe 175 pL Primer Master Mix 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 Screen 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 visu
4. 2014 Check Direct e CPE Screen for sp max 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 Table 3 Data interpretation guidelines for rectal swabs KPC VIM OXA NDM a l c 9 SPC Cz values Interpretation C values Note e YES means that a C value is observed and given in the results table e C values of rectal swabs may vary widely due to differences in fecal material and bacterial load of rectal swabs in transport medium 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 CPE Screen reagent or CP MasterMix 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
5. 30 630 665 680 715 Table B GardRail parameters Parameters Lysis Heat Time Lysis Temperature Sample Tip Height Sample Volume Wash Volume Neutralization Volume DNase Heat Time Table C Real time protocol parameters Step Name Profile Type Cycles Time s Temp C Detect Denaturation Amplification amp Detection 2 temperature 50 Table D Spectral cross talk parameters 475 520 530 565 585 630 630 665 680 715 arm oo 00 00 00 esos 00 oo oo 00 oms o0 oo oo aa Check Direct CPE Screen for BD Max User manual Version 1 0 Issued 09 09 2014 Check Direct e CPE Screen for sp max Appendix 2 Performance Characteristics Limit of Detection with Rectal Swabs The analytical limit of detection LoD of Check Direct CPE Screen for BD MAX was determined using rectal swabs spiked with well defined amounts of target bacteria E swab Amies transport medium Copan was sampled with approximately 10mg ml of human feces mimicking a typical rectal swab specimen Strains containing the target carbapenemase genes were grown o n and cell suspensions were prepared in Milli Q water with a density of 0 5 McFarland These cells suspensions were used to spike the artificial rectal swabs to create specimens with a well defined amount of fecal material and target bacteria A large collection of specimens created as described above were used to assess the analytical limit of detection LoD
6. Check Direct CPE Screen for sp max User manual Check Direct CPE Screen for BD MAX Real time PCR kit for the detection of carbapenemase producing Enterobacteriaceae Version 1 0 Date of issue 09 09 2014 18 0051 Y 24 51 01 EU C IVD U S For Research Use Only Not for use in diagnostic procedures Contents Mend WN a E S A A E tases 2 Introduction and principle of the method l esessssessesesssererrersrsrssssesrenrererrrsrsssseerereeresess 2 Kit contents for 24 reactions esessssssssesssssssssssesreressssssssssssrereresssesstsreeesresessssessrereeen 2 Materials required but not supplied with the kit sesesesesssrrssreesseseerererrrrresrsesrerreres 2 Storage handling and stability s ssssseseseseseesererrrsrsesseserrerrrsrssssesrenrererrrsessesesrrrrerenrese 2 GOOG laporatory i AC Ce Sire sacs dct rencrcec cae scrreveseace denah EE EE AaS EE NS EEEIEE 3 Sample preparation PROCE QUIS cs sssucststacicisnassccanuesducteacetaccditaanlacataamon tevntvandeesses seaavaliaates 4 BD PVA OID CN a COI seresa A EE A A E E E 4 ROS UES Merret AUIO IA e E E AAAA OA 5 Frequently asked questions FAQ amp Troubleshooting esssseesesseesesrrerrresrerrrrrrsns 6 LO ONS aree i E EE A T E E E E 7 KEE e CAO o E E AAA E AEE E A 7 Tecnicas SIS CAIN GS e E A E R 7 Appendix 1 Creating the Check Direct CPE Screen test program sesssscrrercsrerer 8 Appendix 2 Performance Characteristics ccccccccccsscssscescesssessss
7. ally 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 way e C value of 0 indicates that there was no Cy value calculated by the software Amplification curve of the sample showing a O 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 Cry 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 Check Direct CPE Screen 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 Screen test Cr 475 520 Cr 530 565 Cr 585 630 Cr 630 665 Cr 680 715 Sample Type KPC VIM OXA 48 like NDM SPC Check Direct CPE Screen for BD Max User manual 5 Version 1 0 Issued 09 09
8. egative control reactions for each Check Direct CPE Screen PCR run The positive control is supplied with the kit e Positive control Pipette 10 uL of the positive control into one Sample Buffer Tube Vortex for 10 seconds e Negative control Pipette 10 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 setup Detector 475 520 530 565 585 630 630 665 680 715 Channel SPC Sample Processing Control When the test is performed for the first time create the PCR test program Check Direct CPE Screen 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 Put the unitized Reagents Strips in their positions in the BD MAX rack Do not click in the Strips yet 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 6 Snap a CPE Screen reagent tube blue seal into position 3 of the DNA Strip see Figure 1 2 2 c Pierce the blue seal of the CPE Screen reagent tube in position 3 e g with a disposable pipette tip Next carefully dispense 12
9. esescescecsussessesseeeeseneeas 9 Check Direct CPE Screen for BD Max User manual 1 Version 1 0 Issued 09 09 2014 Check Direct e CPE Screen for sp max Intended use Check Direct CPE Screen for BD MAX is a qualitative in vitro diagnostic test for the rapid detection in rectal swabs of carbapenemase genes from Enterobacteriaceae Check Direct CPE Screen 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 Screen 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 Screen 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 among Enterobacteriaceae is a serious threat to public health These organisms are associated with high mortality rates and have the potential to sp
10. excess air 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 Screen for BD Max User manual 2 Version 1 0 Issued 09 09 2014 Check Direct CPE Screen for sp 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 The test must be performed by adequately trained personnel Do not use reagents after their expiration date Follow recommendations for storage and handling to preserve the quality of the kit s reagents Protect reagents from light to avoid photo bleaching of the dyes 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 Use pipettes with hydrophobic filter tips Make sure to always use a new pipette tip when adding solutions test samples and controls to a reaction tube to avoid contamination Follow proper pipette dispensing techniques to prevent aerosols Wear clean disposable gloves and clean lab coats for the different steps of the test Change glove
11. icted in the table below Number of Check MDR CT103 result Check Direct CPE Screen result strains tested NDM OXA 48 NDM OXA 48 VIM OXA 48 VIM OXA 48 10 Check Direct CPE Screen for BD Max User manual Version 1 0 Issued 09 09 2014
12. itions CPE Screen reagent tubes 9 0121 24 sealed tubes blue seal 4 C store in the dark CPE positive control 9 0061 1 tube purple cap CP Mastermix 9 0122 1 tube green cap 330 pl User Manual 9 0124 Leaflet download from website Materials required but not supplied with the kit Supplies Equipment BD MAX ExK DNA 1 Extraction Kit Ref 442818 BD MAX PCR Cartridges Ref 437519 i Real ti PCR i BD Disposable laboratory powder free gloves eal time PCR instrument MAX System software version 2 96A Vortex mixer Pipettes amp disposable filter tips for volumes of 10 and 25 ul PCR grade water e g Milli Q or aqua bidest Swabs and transport media compatible with rectal specimen collection Recommended swab collection device Copan ESwab Cat No 480CE Storage handling and stability The Check Direct CPE Screen 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
13. logies 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 ee Binnenhaven 5 Fax 31 317 210 147 a 6709 PD Wageningen info check points com The Netherlands www check points com Check Points Check Direct CPE Screen for BD Max User manual Version 1 0 Issued 09 09 2014 Check Direct eo CPE Screen for sp max Appendix 1 Creating the Check Direct CPE Screen 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 Screen e Extraction Type Select Exk DNA 1 Plasma Serum e Master Mix Format choose Type 3 Liquid MM with Primers and Probes e Channel detector Settings set Gain and Threshold with parameters specified in Table A e GardRail select User and enter values specified in Table B Only the Sample Volume differs from the default settings e Test details enter the PCR profile as outlined in Table C e Spectral Cross Talk tab enter parameters specified in Table D 2 Select Save Test Table A Gain parameters Detector Threshold 475 520 530 565 585 6
14. r 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 o g eenn a 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 Screen 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 Techno
15. read 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 and OXA 48 like variants Moreover CPE often have other non f lactam resistance determinants resulting in multidrug and pandrug resistant isolates Check Direct CPE Screen for BD MAX 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 Screen has been designed to reliably detect all variants Check Direct CPE Screen 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 amplification Kit contents for 24 reactions Components Mat No Description Storage cond
16. rect CPE Screen 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 Screen real time test An overview of these strains is outlined in the table below All isolates tested negative with the Check Direct CPE Screen assay and the internal control was reliably detected in all samples The Check Direct CPE Screen for BD MAX assay showed 100 specificity based on the reference strains tested Check Direct CPE Screen for BD Max User manual Version 1 0 Issued 09 09 2014 o Check Direct CPE Screen for sp max f Strains Species Campylobacter jejuni Citrobacter freundii Enterobacter aerogenes Enterobacter cloacae Enterococcus casseliflavus Enterococcus faecalis Escherichia coli Klebsiella oxytoca Klebsiella pneumoniae Pseudomonas aeruginosa Salmonella typhimurium Pseudomonas mirabilis Staphylococcus aureus Serratia marcescens Stenotrophomonas maltophilia Analytical Inclusivity A retrospective study was performed with 93 bacterial strains of 13 different gram negative species that were previously identified carbapenemase positive with the Check Points microarray 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 Screen assay Results are dep
17. rs or other differences between the duplicate samples Check Direct CPE Screen for BD Max User manual 6 Version 1 0 Issued 09 09 2014 Check Direct e CPE Screen for sp max Limitations Check Direct CPE Screen for BD MAX 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 test is only intended to be used with rectal swabs in transport medium as input material The quality of the input DNA is an important factor for obtaining reliable results with Check Direct CPE Screen for BD MAX DNA must be extracted from rectal swabs using the devices and procedures described in this manual 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 Screen cannot and does not make any representation or warranty that it is capable of correctly detecting the carbapenemase genes in all gram negative species subspecies o
18. s 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 Please read the full protocol before starting the test Check Direct CPE Screen for BD Max User manual 3 Version 1 0 Issued 09 09 2014 Check Direct e CPE Screen for sp MAX Sample preparation procedures Test preparation for rectal swabs Note The procedure for specimen collection and storage must be followed carefully using adequate specimen collection devices see section Materials required but not supplied with the kit Rectal swabs will contain varying amounts of fecal material depending on the procedure for specimen collection Check Points advices to validate your specimen collection and processing method with Check Direct CPE Screen prior to routine use of the test Collect rectal specimen according to local guidelines and swab manufacturer recommendations Transfer the swabs to the tubes containing liquid transport medium Transfer rectal swab samples to be analyzed to the PCR room or store until further use Mix each tube with rectal specimen briefly and pipette 25 ul of the transport medium into one DNA Sample Buffer Tube SB 1 5 Close the Sample Buffer Tube with a septum cap and vortex 10 seconds at low speed a oe Preparation of control reactions To validate the run perform positive and n
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