User manual - Check
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1. Check Direct CPE for BD User manual 0956 INTRODUCTION AND PRINCIPLE OF THE METHOD 2 KIT CONTENTS FOR 48 REACTIONS 2 STORAGE HANDLING AND STABILITY 2 Check Direct CPE for BD MAX MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT ssssecccccssssssseeeecceccessseseeeees 2 GOOD LABORATORY PRACTICES issisiccisccsssassvcsectcevessvassccestestussavasecacesasseesavacenceudesavaceeunassesses 2 Real time PCR kit for the detection of carbapenemase producing SPECIMEN AND CELL SUSPENSION PREPARATION 4 2 4842 4 MAX SAMPLE AND CONTROL PREPARATION 4 Enterobacteriaceae 1 SAMPLE PREPARATION ayant ous CEEA EEEE EEE EEA DEAE EAEE RERA oe 4 2 POSITIVE AND NEGATIVE CONTROL PREPARATION 00 4 BD MAX SYSTEM REAL TIME PCR sccsisecssccecesvevsasvececcosssesesaveccunsesssesiscccesstsesseesscecnessivenvereed 4 1 MULTIPLEX REAL TIME PCR QPCR 5 4 Version 1 3 2 QPCR MIX PREPARATION ccssssssesssssesssssescsccsessseccsesscssesesscsscsscscsucssescaucsecucsueeeancaceecaneasees 4 3 CREATING THE CHECK DIRECT CPE TEST 000 4 Date of Iss2. positive control solution was not added qPCR reaction mix CPE Solution with Primer Probe Diluent was not added to the assay Please repeat the test The BD MAX DNA SPC PCR mix was not added or may have expired Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 Check Direct CPE for BD Real time results show very low fluorescent signals in all samples and detector channels including the internal control signal Possible causes and troubleshooting e CPE solution containing the fluorescent probes and primers is degraded Please check expiration date the number of thaw freezing cycle that the CPE solution tube has undergo and if the kit has 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 The BD MAX System states an error or failure Refer to the BD MAX instrument user manual or contact your BD local representative All amplification curves show a spike at the same point in time A problem with the power supply may have occurred Verify that the system is correctly connected to its un interruptible power supply and repeat experiment have left Solutions CPE 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
3. fully guaranteed if these solutions were left out of 20 C 4 F for more than 24 hours Duplicate samples tested with Check Direct CPE test do not yield identical results values of identical samples may vary between individual reactions Large variations gt 2 values suggest pipetting errors or other differences between the duplicate samples 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 t should be noted that other rare carbapenemase gene families are not detected Check Direct CPE was developed for bacterial cell suspension The quality of the input DNA is an important factor for obtaining reliable results with Check Direct CPE DNA must be extracted from bacterial cell suspension validated with Check Direct CPE and described in this manual The assay has been tested extensively with purified DNA 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 represent
4. E 5 3 Enter the Sample Preparation reagent tube barcode using the barcode scanner you can also enter the barcode manually Start with position 1 of rack A 5 4 Place each of the Sample Buffer Tubes in their corresponding position in the BD MAX racks with septum cap 5 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 5 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 5 7 Load the BD MAX PCR cartridge s 5 8 Close the instrument door and select Start Run BD MAX Results 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 values obtained with the software 1 How to create and print data report 1 1 the main menu select Results and double click the run to be analyzed 1 2 Select the Run to analyze 1 3 In the Print tab select the box for Results Protocol Details and PCR 1 4 Select create report The report can be saved as pdf file format To print it select Print Report 2 Analysis The BD MAX software reports C values and amplification curves for each detector channel of each specimen tested in the following way e value
5. ation or warranty that it is capable of correctly detecting the carbapenemase genes in all gram negative species subspecies or types or in all clinical sample source 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 Technical assistance support check points com 31 317 453 908 Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2013 Key to symbols used For In Vitro Diagnostic Use CONTRO Negative control Catalog number o KPC KPC positive control Batch code VIM VIM positive control IFU number CONTRO NDM NDM positive control CPE solution 48 OXA 48 positive control 54 Use before YYYY MM Manufacturer Consult instructions for use Temperature limitation Contains sufficient for lt gt tests Despite the utmost care in the development a
6. ble 5 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 ai 530 565 0 0 0 0 0 0 Excitation 585 630 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 3 2 Select Save Test 4 BD MAX Rack set up 4 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 Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 Check Direct CPE for BD 4 2 Prepare Unitized Reagents Strips 4 2 a Snap the appropriate DNA extraction Reagent tube white cap into positions 1 into the DNA Strip see Figure 1 4 2 b Snap in a DNA MMK SPC Master Mix tube green yellow cap into position 2 of the DNA strip see Figure 1 4 2 c Snap a Conical Tube into position 3 Figure 1 Pipette 12 5 ul of qPCR mix Table 2 into the Conical Tube in position 3 Avoid bubbles and make sure the liquid is at the bottom of the tube Pipette Tips Buffers 5 5 x lt Master Mix po Conical tube posi Figure 1 DNA Unitized Reagent Strip setup 5 BD MAX instrument set up 5 1 Open the Run screen of the BD MAX System software v2 92 5 2 In the Assay menu select Check Direct CP
7. down the solution to avoid contamination when opening the lid Avoid unnecessary freeze thawing of the kit content e Follow recommendations for storage handling and freeze thaw cycles to preserve the quality of the kit s reagents e Protect reagents from light to avoid photo bleaching of the dyes e Periodically verify the accuracy and precision of pipettes as well as correct functioning and calibration of the instruments Prevention of contaminations Use separate rooms a pre PCR room and a post PCR room Preparation of the amplification reactions are carried out in the pre PCR room e DNA extraction and real time PCR assays are carried out in the post PCR room e Never transfer items from the post PCR room to the pre PCR room To keep laboratory free of PCR product contamination 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 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 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 0 5 sodium hypochlorite solution Check Direct CPE for BD Max User manual 3 Version 1 3 Issued 24 01 2014 Specime
8. in channel 1 475 520 2 530 565 3 585 630 4 630 665 and a Cr value of 29 3 for the sample processing control SPC channel 5 680 715 Table 6 Criteria for a valid run with Check Direct CPE test C 475 520 530 565 C 630 665 Cr 680 715 Sample Type KPC VIM OXA 48 like NDM SPC Positive controls 32 3 30 3 29 3 3143 29 3 Negative sample 1 1 1 1 29 3 If observed CT values vary significantly from expected CT values see FAQ and Troubleshooting section 3 2 Results interpretation If the run has been validated interpret results as positive negative or invalid with the values obtained for the samples following the guidelines outlined below and summarized in Table 7 Positive carbapenemase samples will show value in channel 1 475 520 2 530 565 3 585 630 and or 4 630 665 Positive carbapenemase samples will also show value for the SPC in channel 5 680 715 Negative carbapenemase samples will show no Cy value in channel 1 475 520 2 530 565 3 585 630 and 4 630 665 In channel 5 680 715 value for the SPC is expected of 29 3 Samples with carbapenemase target of 1 and with a SPC showing Cy value gt 32 means that the assay is not valid and should be repeated see FAQ and Troubleshooting 3 to 6 Table 7 Data interpretation guidelines KPC VIM OXA NDM SPC values Interpretation C value
9. ive control KPC VIM OXA and 500 uL of PCR grade water into one Sample Buffer Tube Vortex for 10 seconds Positive controls may also be combined add 10 uL of each positive control to be tested and 500 uL of PCR grade water into one Sample Buffer Tube Vortex for 10 seconds e Negative control s Pipette 10 uL of the negative control solution O provided in the kit and 500 uL of PCR grade water into one Sample Preparation Reagents tube Vortex for 10 seconds Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 Check Direct CPE for BD BD MAX system real time PCR 1 Multiplex real time PCR qPCR setup Table 1 presents the multiplex qPCR 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 1 2 3 4 5 OXA 48 like NDM SPC Target KPC VIM SPC Sample Preparation Control 2 qPCR Mix preparation 2 1 Calculate the number of reactions for the specimen and controls maximum 24 samples in total in one run Thaw reagents mix well spin down and keep on ice 2 2 Prepare the qPCR reaction mix as described in Table 2 using the CPE solution provided in the Check Direct CPE kit and the BD diluent provided the BD DNA SPC reaction kit Multiply the CPE solution and the BD diluents solutions by the
10. n and cell suspension preparation 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 Prepare a bacterial cell suspension of McFarland 0 5 1 x 10 CFU ml in cell suspension buffer e g PBS or 10mM Tris pH8 0 or PCR grade water from bacterial cells grown on the agar plate Dilute the suspension 100 times to obtain a suspension of 1 x 10 CFU ml BD MAX sample and control preparation Important points before starting Refer to the BD MAX ExK DNA 1 kit user manual and BD MAX System User s Manual for detailed instructions 1 Sample preparation e After specimen collection transfer the bacterial cell suspensions to be analyzed in the sample preparation area e 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 Material required but not supplied with the kit e Close the Sample Buffer Tube with a septum cap and vortex 10 second at low speed 2 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 Test the relevant positive control s provided in the kit Pipette 10 uL of posit
11. nd 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 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 rapid molecular detection Tel 31 317 453 908 Fax 31 317 210 147 info check points com www check points com Check Points Health BV Binnenhaven 5 6709 PD Wageningen The Netherlands
12. of 0 indicates that there was no value calculated by the software Amplification curve of the sample showing a O C value must be checked manually e value of 1 indicates that no amplification process has occurred Check that there is no amplification curve for the sample with value of 1 on the graphical results e Any other C value should be interpreted in correlation with the amplification curve PCR Analysis tab and according to the interpretation method see Table 6 7 3 Interpretation 3 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 6 shows criteria for a valid real time Check Direct CPE run on the BD MAX System Expected values for the positive and the negative controls are given as an indication not a definitive result These values will depend on the threshold set by the software If the controls C values are not as expected refer to FAQ and Troubleshooting 3 Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 Check Direct CPE for BD Valid run reports e No BD System failures during the run e Positive controls with value for the carbapenemase targets and sample processing control SPC as depicted in Table 6 Negative control with a value of 1
13. ore all opened reagents at 20 C until expiration date Store in the dark 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 Materials required but not supplied with the kit Lab coat ul BD ExK DNA 1 Extraction Kit Ref 442818 BD DNA MMK SPC Master Ref 442829 BD MAX PCR Cartridges Ref 437519 Disposable laboratory powder free gloves Pipettes amp disposable filter tips for volumes of 1 to 1000 DNAase free 1 5 ml tubes 1 5 and or 2ml e PCR grade water e g Milli Q Real time PCR instrument System software version 2 92 Vortex mixer Mini centrifuge Check Direct CPE for BD Good laboratory practices Recommendations for best results Users are strongly advised to read the full protocol The quality of the results depends on strict compliance with the following good laboratory before starting the test practices especially concerning PCR practices e 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
14. recognition and amplification of target sequences by PCR and the simultaneous detection of the accumulation of PCR amplification products by fluorescent DNA probes Four molecular beacon probes labelled with four different dyes are used for the fluorescent detection and the discrimination of the carbapenemase markers For each of the 4 carbapenemase genes KPC VIM OXA 48 and NDM 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 all gene variants are reliably detected Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 Kit contents for 48 reactions Components Mat No CPE solution 9 0072 Description 1 brown tube blue inlay 550 ul Check Direct CPE for BD MAX Storage conditions Negative control 9 0070 1 tube white inlay O 100 ul KPC positive control 9 0073 1 tube green inlay 100 ul VIM positive control 9 0074 1 tube yellow inlay 100 ul 20 C store in the dark OXA 48 positive control 9 0076 1 tube orange inlay 100 ul NDM positive control 9 0075 1 tube gold inlay 100 pl User Manual 9 0079 Leaflet download from website Not critical Storage handling and stability The Check Direct CPE kit is shipped cooled and should be stored immediately at 20 C upon receipt St
15. s YES YES Positive sample 1 29 3 Negative sample 1 1 or gt 32 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 values or interpretation indicating that the sample is invalid Possible causes and troubleshooting e PCR reaction has been inhibited by exogenous or endogenous substances Please repeat sample testing e sample tested with Check Direct CPE is negative and the internal control was not amplified e DNA extraction failed since the SPC was not detected e CPE Solution or The BD DNA MMK SPC PCR mix was not added to the assay Please repeat the test e BD DNA MMK SPC PCR mix may have expired e error in liquid handling has occurred check unitized reagent strips and microfluidic 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 3 Real time results show no values for the positive control or interpretation indicating that sample is invalid Possible causes and troubleshooting
16. s at risk of colonization with carbapenemase producing Enterobacteriaceae Check Direct CPE is not intended to diagnose infections with carbapenemase producing Enterobacteriaceae nor to guide or monitor treatment for these infections Parallel cultures are necessary to recover organisms for epidemiological typing susceptibility testing and for further confirmatory identification Introduction 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 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 New Delhi metallo B lactamase or 48 Oxacillinase 48 Moreover CPE often have other non B 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
17. total number of reactions calculated plus 10 surplus to ensure that you have enough qPCR reaction mix for all reactions Table 2 qPCR reaction mix Component Volume per reaction strip BD Primer and Probe Diluent 12 5 2 ul CPE solution 10 5 ul Total volume for 1 strip 12 5 ul 3 Creating the Check Direct CPE test 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 v2 92 Skip step 3 if a PCR test program has already been created for the Check Direct CPE test 3 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 Master Mix Format choose Type 2 MMK SPC Probes Primers Add Manually e Channel detector Settings set Gain and Threshold with parameters presented Table 3 GardRail select Default Test details enter the PCR profile see Table 4 Spectral Cross Talk tab enter parameters presented in Table 5 Table 3 Gain parameters Detector Gain Threshold 475 520 40 100 530 565 80 150 585 630 30 200 630 665 80 300 680 715 40 300 Table 4 Real time protocol parameters Step Name Profile Type Cycles Time s Detect Denaturation Hold 1 600 98 NO Step Name Profile Type Cycles Time s Temp C Detect Amplification amp Detection 2 temperature 45 15 98 NO 62 60 YES Ta
18. ue 24 01 2014 4 BD MAX RACK SET UP cones geass 5 5 MAX INSTRUMENT 5 20 2220 2 0 0 00 1 000000000000000 0 8 5 BOMAN M RESULTS 6 y 1 HOW TO CREATE AND PRINT DATA REPORT 6 18 0081 48 081 04 PANTAN 6 2 6 FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING 7 LIMITATIONS ovcsscccsescicecscesscsiveceicececeasiiaesctceaceedsveevscecucexuveesnveceuccasssevisavuecseuevessvcccdueesddevssdoed 8 TECHNICAL ASSISTANCE Ss scccicccssesiccccesciessepesacecsvecisssevaceeecaseassesevaceuscsvssssesseeasccssvesiicoavacsesssves 8 KEY TO SYMBOLS USED eecccscscccescctecncoseiesscoccautesstc cicccceastedsectvecdecsansedcsveecceecnunsesvecdetecsuneetessed 8 IVD Check Direct CPE for BD Max User manual Version 1 3 Issued 24 01 2014 1 Intended use Check Direct CPE is a qualitative in vitro diagnostic test for the rapid detection of carbapenemase genes KPC VIM OXA 48 and NDM presently the primary cause of carbapenemase production in Enterobacteriaceae The assay is performed on the BD system using bacterial cell suspension from clinical specimen of individual
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