User manual - Check
Contents
1. ccccccccccsssecessccsseccececeesseceeecesssseeeeceeseseasseesseseaueeeseeseseeeees 4 2 FOR USE ON THE ABI 75000 sccsconssonssessersecnecensessensconscensesscensenneceusersenseonacenssnsennecesserses 5 Date of Issue 05 09 2013 3 FOR USE ON THE LC 480 I amp ll ooo ccc ccccccccscccccccsseecessceusesseseeususecececeeeseuseseesseeeeuseeueeueecseaneaeess 5 4 FOR USE ON THE CFX96 ccc ceccssessssesccecececesescececeseanessscecececeeaeensseecceeeeauenseeectsaeegeeeeeeeeeas 5 5 FOR USE ON THE ROTOR GENE Q ssssseececcceeceaeeeeceeeseceessseececeseauenseneeeeeeseaeeesneeeeeeeeangeseees 5 DATA ANALYSIS iscscccccececcscsuseccccscscscensucccsveteccsussceconssepucestecdusdevesc uerswccdssevesasvecdusetescsssueucosssepee 6 18 0080 48 iFU 080 03 1 FOR USE ON THE ABI 7500 ssssesssesssessssesssecssesseessecsssessscssscsssesssecsuessuesssessuessnesseesseesseeeses 6 2 FOR USE ON THE LC480 I amp ll ccceccccscccccccsseecescesscecseeseuseeccesecsssseeesceuseeeeuseeuseceeeseaueaeess 6 3 FOR USE ONITHE CFXO6 iosi iei a a A i E Aa E 7 4 FOR USE ON THE ROTOR GENE Q nnessoesssesenresresserrerrssrsteresrssrsrsrerrresesssrereesssrreereerssererrenee 7 DATA INTERPRETATION 0 0sccccesssccvoscaccsessococcteeccnesssacesteaccacsesdesotsecoacnsnesasdssncserssenssnsccesesspee 8 For use on ABI 7500 FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING ssssccsssccsseccsseecsseecssecenee 8 Lig2. Issued 05 09 2013 8 5 Real time results show very low fluorescent signals in all samples and detector channels including the internal control signal Possible causes and troubleshooting e The CPE solution containing the fluorescent probes and primers is degraded Please check expiration date the number of thaw freezing cycles that the CPE solution tube has undergone and if the kit was stored correctly e The real time PCR system may be responsible for these results Please refer to instrument User s manual or contact your real time PCR instrument local representative 6 C C C values troubleshooting e Samples with very low Cr values and no amplification curves The manual threshold is too low Samples showing a C value lt 15 and no amplification curve are considered negative The observed Cy value is an artifact of the software analysis the threshold crosses the background noise of the curve e Invalid Run C values expected for the controls in Table 6 do not match the C values observed In the experiment Check that these differences are not due to the threshold being too high or too low If changing the threshold does not improve the results go to FAQ 3 to 6 7 The real time PCR instrument gives an error message Refer to the real time PCR instrument user manual or contact the local technical support of the real time PCR instrument company 8 I left Solutions CPE Internal control negative or positive control out of
3. 100 ul Manual 9 0078 Leaflet download from website Not critical Storage handling and stability Check Direct CPE reagents are shipped cooled The CPE PCR Mastermix should be stored at 4 C upon receipt All other reagents should be stored at 20 C upon receipt Please visually inspect the box upon initial opening to ensure that its contents are intact Check Direct CPE solution should not be exposed to more than 12 freeze thaw cycles Please contact the Check Points office at support check points com if you have any further questions Store kit reagents at indicated temperature until expiration date 2 Materials required but not supplied with the kit Supplies Equipment NucliSENS easyMAG Extraction kit bioM rieux France Swabs for rectal and perianal specimen collection and transport example Sigma transwabs Medical Wire amp Equipment UK or Eswab Copan Diagnostics US in Amies transport media Disposable laboratory powder free gloves Lab coat Pipettes amp disposable filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 96 well PCR clear plate for use on ABI7500 96 well PCR white plate for use on CFX96 and LightCycler 480 PCR plate seal PCR strip tubes and caps 0 1ml QIAGEN US For use on the LightCycler 480 the 4 Color Compensation Set Ref 18 0070 Check Points Health B V NL NucliSENS easyMAG Extraction platform bioM rieux Franc
4. Catalog number Batch code IFU number SOLUTION CPE CPE solution Internal control Temperature limitation Contains sufficient for lt n gt tests Consult instructions for use ONTROL Negative control CONTROL KPC KPC positive control ONTROL VIM _ VIM positive control NDM NDM positive control CONTROL OXA 48 OXA 48 positive control PCR Mastermix ICPE CPE PCR Mastermix Use before YYYY MM Manufacturer 10 Limitations Check Direct CPE is a DNA based real time PCR assay to detect the presence of the carbapenemase genes KPC NDM VIM and OXA 48 in Enterobacteriaceae The test detects the following carbapenemase gene variants blay m 6 8 34 0laoxa 48 162 163 181 232 244 245 Planpm1 8 and _ blaxpci 15 KPC NDM VIM and OXA 48 represent the clinically most prevalent carbapenemases in Enterobacteriaceae in most parts of the world However other rare carbapenemases may also be responsible for carbapenemase_ production in Enterobacteriaceae and these are not detected by Check Direct CPE Carbapenem resistance is caused by carbapenemase production but also by various other mechanisms A negative result with Check Direct CPE does not imply that the bacterium is not carbapenem resistant it implies that the bacterium is not likely to carry any of the carbapenemase gene variants of KPC NDM VIM and OXA 48 detected by Check Direct CPE Therefore the test result of Check Direct CPE should never be used as g
5. buffer Real time PCR assay Important points before starting e Preparation of the real time PCR reactions is carried out in the pre PCR room e Specific parameters and materials should be used for each type of real time PCR instrument use to perform the test e Targets are detected in the FAM VIC and TexasRed channels The internal control is detected in the Cy5 channel 1 Real time PCR reaction setup 1 Calculate the number of reactions Thaw reagents mix well spin down and keep on ice 2 Prepare the real time PCR qPCR reaction mix as described in Table 1 Multiply the CPE solution and the CPE PCR Mastermix by the right number of samples and include 10 surplus to ensure that you have enough qPCR reaction mix for all the calculated reactions Pipette 15 ul of qPCR reaction mix to the wells of the 96 well plate 4 Pipette 10 ul of each test sample to the wells of the 96 well plate previously filled with 15 ul of qPCR Mastermix see Table 2 5 Pipette 10 ul of positive control solution s KPC NDM VIM and or OXA 48 to the well of the 96 well plate previously filled with 15 ul of qPCR reaction mix Table 2 6 Pipette 10 ul of negative control solution O to the well of the 96 well plate previously filled with 15 ul of qPCR reaction mix Table 2 7 Seal the plate or close the tubes Mix the plate by tapping it on the bench and spin down briefly 8 Transfer the plate to the post PCR room 9 With
6. C 10 min 1 OFF standard 4 4 C s 3 95 C 15 sec Plate read 4 4 C s 4 60 C 60sec Optiesen nee 2 2 C s 3 or 10 min can be used for denaturation time depending on other assays to be run simultaneously with the the Check Direct CPE test 5 Data analysis Important points before starting e For a detailed description on how to operate real time PCR instruments and how to analyze data please refer to the real time PCR instrument instruction manual e Always visually inspect the amplification plot for each sample tested versus C values obtained with the software 1 For use on the ABI 7500 1 Analyze data using ROX as a passive reference 2 In the Options setting for each Target see Table 3 deselect the box for auto threshold and auto baseline as shown in Figure 1 red circle and select Reanalyze 3 Set the threshold manually in the Analysis settings window for each detector channel using the threshold values recommended in Table 5 4 Check amplification plots versus C values calculated by the software for each target Amplification Plot Plot Type An vs Cycle Graph Type Log Color Target C Save current settings as the default PPawBKE N Standard Curve Amplification Plot be Multicomponent Plot Raw Data Piot Threshold QC Summary Multiple Plots View Options l Target Check Direct CPE NOM VIM v Teena E Xeo 0000688 1A 6 8 on u 0 wp z u os gt oe Oo ou u Cycle ovens
7. the 20 C 4 F storage These reagents must be stored at 20 C 4 F for proper performance of the test The performance of the product cannot be fully guaranteed if these solutions were left out of 20 C 4 F for more than 24 hours 9 Duplicate samples tested with Check Direct CPE test did not yield identical results Cr Cp 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 10 Data Analysis and Interpretation If you encounter difficulties with the data analysis and interpretation please contact Check Points Technical Support at support check points com Check Direct CPE User manual Version 1 2 Issued 05 09 2013 se Check Direct CPE Performance Characteristics 1 Analytical sensitivity The analytical limit of detection LOD of Check Direct CPE real time PCR test was assessed for four carbapenemase genes associated with carbapenemase production in Enterobacteriaceae KPC NDM VIM and OXA 48 No quantified genomic standards for these markers were available at the time therefore the analytical sensitivity test was performed using plasmid having a 400 bp target sequence DNA fragment Thus the LOD of the Check Direct CPE real time PCR test was established using plasmid DNA directly in the PCR reaction mix To determine analytical sensitivity an end point dilution was used until the assa
8. 2 e qme Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale Adjust Adjust Scale Auto Scale Default Scale Linear Scale sst SALILL2SESRSFESSLTSELSLSKSRL BRE BBNR threshold manually using the threshold bar on the amplification plot Blue FAM signal Figure 4 Screen shot of the typical analytical window with the Rotor Gene Q software v2 1 0 Build 9 Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale in the Green channel Check Direct CPE User manual Version 1 2 Issued 05 09 2013 4 se Check Direct CPE Data interpretation Frequently asked questions FAQ amp Troubleshooting 1 Run validation Table 6 1 May other specimen preparation and DNA extraction methods be used with Check Check the positive and negative control amplification curves Valid run reports Direct CPE Check Direct CPE test has been optimized using specific swabs and transport medium in combination with the NucliSENS easyMAG extraction methods Check Points does not guarantee the performance of the test with methods other than those recommended in this manual e Noinstruments system failures during the run e Negative control with a C value of 30 3 in the Cy 5 detector channel and no Cy value in the other detector channel e Positive control C values as expected in Table 6 The exact C values of the positive controls vary depending on the qPCR inst
9. BR white Plate Setup View Edit Plate and Select Fluorophore as described in Table 3 5 For use on the Rotor Gene Q e Reaction Volume 25 ul e 72 well rotor selected e Gain settings Green 6 Yellow 4 Orange 3 Red 8 Check Direct CPE User manual Version 1 2 Issued 05 09 2013 Check Direct CPE Table 1 qPCR reaction mix setup Component Volume per reaction CPE PCR Mastermix 12 5 ul CPE Solution 2 5 ul Total volume 15 ul Table 2 Real time PCR sample setup Reaction Type Component Volume per reaction Test sample Sample DNA 10 ul Positive control KPC NDM VIM OXA 48 10 ul Negative control Negative sample or negative control provided O 10 ul Table 3 Real time PCR setup MS ABI7500 LC 480 I LC 480 II CFX96 Rotor Gene Q P 8 Detector Detector Detector Detector Detector Neer er KPC FAM FAM 483 533 FAM 465 510 FAM Green 510 5 VIC HEX Yellow555 VIC HEX NDM VIM VIC 523 568 533 580 VIC Yellow 55745 Pe TexasRed Red610 TexasRed OXA 48 TXR Red610 558 610 533 610 TXR Orange 610 5 Internal Control Cy5 IC Cy5 Cy5 615 670 618 660 Cy5 Red 660 10 Table 4 Real time protocol parameters DA Ramp Rate Mode ata Step Temperature Time Cycles Collection ABI 7500 CFX96 LC 480 Rotor Gene Q I amp I 1 50 C 2 min 1 OFF standard 4 4 C s 2 95
10. 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 11
11. Show 7 Threshold 7 Baseline Start Well W Target amp Baseline End Well W Target 4 C Save current settings as the default Figure 1 Screen shot of a typical analytical window on the ABI7500 with ABI7500 software v2 0 6 Amplification plot of positive and negative samples for the Check Direct CPE test logarithmic scale Red circle deselect the box of auto Threshold Auto baseline Check Direct CPE User manual Version 1 2 Issued 05 09 2013 Check Direct CPE 2 For use on the LC 480 I amp II 1 Select Abs Quant 2 Derivative Max analysis Figure 2 2 Analyze the data using the color compensation object application specific to the Check Direct CPE assay previously created Select Color Comp On In database select the CC object Refer to Check Points User Manual for the 4 Color Compensation set Ref 18 0070 3 Select the High Sensitivity option and Calculate C values for each filter combination For each filter combination always check amplification plot versus C values 5 In the results table check C values for each targets versus the Status given by the software red positive green negative blue call uncertain Validate the Status of the sample using the software by visually inspecting amplification curves Figure 2 Abs Quant 2nd Derivative Max for swabs Se a a a TT a Fe Poe SEa Quant results B Positive Negative MB Urcetain MB Standard La
12. al Samples Results Include Color Pos Name Cp Cone Standard Status Gl Sample 73 G2 Sample 74 G3 Sample 75 G2 Sample 76 GS Sample 77 Sample 78 Sample 79 ee 2 aah amet pe Figure 2 Screen shot of a typical analytical window on the LC 480 system I with the LC 480 software v1 5 Amplification plot of positive and negative samples for the Check Direct CPE test linear scale threshold is automatically calculated by the software Table 5 46 WMS Table 5 Recommended threshold settings Detector Target ABI 7500 LC 480 I amp II CFX 96 Rotor GeneQ FAM green KPC 0 002 Auto calculated 60 150 0 02 VIC yellow NDM VIM 0 001 Auto calculated 200 250 0 015 TXR orange OXA 48 0 006 Auto calculated 400 800 0 03 Cy5 red IC 0 003 Auto calculated 40 100 0 06 Check Direct CPE 3 For use on the CFX96 4 For use on the Rotor Gene Q 1 Open the Data file for Data Analysis In the Analysis Settings use the following 1 Open the data file Open the raw channel page for each detector Select Options and parameters Crop start cycles In the pop up window Remove data before cycle enter 15 and e Analysis Mode Fluorophore select OK e Baseline Setting Baseline Subtracted Curve Fit Apply Fluorescent Drift Correction 2 Then use the parameters indicated in Table 6 to analyze each detector channel e C4 Determination Single Threshold 3 Set the thr
13. ck Direct CPE assay and the internal control was detected in all samples The Check Direct CPE test showed 100 specificity based on the reference strains tested Table C Organisms Organisms Citrobacter freundii 5 Enterococcus faecalis 2 Campylobacter jejuni 2 Klebsiella oxytoca 1 Enterobacter aerogenes 1 Klebsiella pneumoniae 16 Enterococcus casseliflavus 1 Pseudomonas aeruginosa 2 Enterobacter cloacae 42 Staphylococcus aureus 2 Escherichia coli 51 Salmonella thypimurium 1 Pseudomonas mirabilis 3 Stenotrophomonas maltophilia 2 Serratia marcescens 1 3 Analytical Reactivity To evaluate the analytical reactivity a retrospective study was performed with 93 bacterial strains of 26 different gram negative species Table D The 93 bacterial strains tested with Check Direct CPE were previously identified carbapenemase positive with the micro array diagnostics test Check MDR CT103 Check Points Heatith Check Direct CPE User manual Version 1 2 Issued 05 09 2013 se Check Direct CPE Results All 93 bacterial strains were typed correctly for the targeted carbapenemase genes with the Check Direct CPE test Table D Check MDR CT103 Check Direct CPE 19 KPC KPC 16 NDM NDM VIM 1 NDM 0XA 48 NDM VIM 0XA 48 33 VIM NDM VIM 1 VIM OXA 48 NDM VIM 0XA 48 23 OXA 48 OXA 48 Key to symbols used INTERNAL CONTROL For In Vitro Diagnostic Use
14. cliSENS easyMAG automated DNA extraction procedure for perianal rectal swabs in transport medium Follow the protocol Generic 2 0 1 from the manufacturer for perianal rectal swabs 2 Use 200 ul of perianal rectal swab fluid from each specimen and add 5 ul of the internal control solution IC solution to each well of the easyMAG cartridge Start the DNA extraction using the Generic extraction protocol 3 DNA is eluted in 70 ul elution buffer 4 DNA extracts can be stored at 4 C for up to 6 months and at 20 C for a longer period of storage 5 Use the DNA solution directly and continue with the real time PCR assay or store as specified until use 3 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 Positive controls contains the internal control Check Direct CPE User manual Version 1 2 Issued 05 09 2013 Check Direct CPE e Negative control s Use the negative control O provided in the kit as a sample to validate the run The negative control contains the internal control We also recommend performing a DNA extraction as specified earlier with the internal control solution using a sample known to be negative for the test in use i e carbapenemase negative sample or elution
15. e Check Direct CPE User manual eet ee INTRODUCTION ssccccsssecccsssseccsesecccecceeccseseccceesecececsesececseseceesesececeseccucsesesecsesesecseseeasessess 2 PRINCIPLE OF THE METHOD isin cussccccccisccsassccccsossoucusacnaacusostousuoacnsntsonskovensacssosesastousesesneususectes 2 e KIT CONTENTS FOR 48 REACTIONS sssssssssssssesesesssssesesssssesssssesesesesesesnsssssssnseseseees 2 Check Dire ct CPE STORAGE HANDLING AND STABILITY ccsssccccsssccccsssccccsssecccessccceessecceeseecceessceeeeseseeeees 2 A f MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT ccsssscccssssecccesseeccessecceeseeeees 3 Real time PCR kit for the detection of carbapenemase GOOD LABORATORY PRACTICES sscccssssscccsssecccscsecccsesecccesesececseseceesessceeseseeeesessceesesseeens 3 prod ucing Enterobacteriaceae SPECIMEN COLLECTION AND DNA EXTRACTION ccccscssssssececcccccssssssececcccccssssseeeeeeeeeseeee 4 1 HUMAN SPECIMEN COLLECTION cccccccccesesesecececceeneseececececeeseneececsecsceeueneececessaeegeneeceeesagenes 4 2 DNA EXTRACTION FROM PERIANAL RECTAL SWABS WITH NUCLISENS EASYMAG 000006 4 3 POSITIVE AND NEGATIVE CONTROL PREPARATION sesesecececececeseseseeeeecececeseneeenececececeeeeeeeeeeece 4 REAL TIME PCR ASSAY siccccssciscccssccadasveccsssscccussuecdessvccucasuscacusivcccvscoccsasvesdsnssecdsssiscedsausecesstense 4 Version 1 2 1 REAL TIME PCR REACTION SETUP
16. e Real time PCR instruments ABI7500 Applied Biosystems US LightCycler 480 I amp II Roche CH CFX96 Bio Rad US Rotor gene Q 5plex QIAGEN US Rotor disc 72x0 1ml wells locking Ring 72 Well Rotor and loading block 72x0 1ml tubes QIAGEN US Vortex mixer mini centrifuge Plate centrifuge Good laboratory practices Recommendations for best results The test must be performed by adequately trained personnel Do not use reagents after their expiration date Before use thaw frozen reagents completely at room temperature and vortex briefly to obtain a homogeneous solution After vortexing briefly spin down the solution to avoid contamination when opening the cap Follow recommendations for storage handling and freeze thaw cycles 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 Check Direct CPE User manual Version 1 2 Issued 05 09 2013 se Check Direct CPE Prevention of contaminations Use separate rooms a pre PCR room and a post PCR room DNA extraction and preparation of the amplification reactions are carried out in the pre PCR room Incubation in the real time PCR thermocycler is carried out in the post PCR room Never transfer items from the post PCR room to the pre PCR room To keep laboratory fr
17. ed or may have expired Table 6 Criteria for a valid run with Check Direct CPE test OXA 48 Sample Type KPC NDM VIM IC i a deie values Instrument FAM green VIC yellow VIC yellow a Cys lt red 3 The real time results show no Cr Co Ca values for the positive control or AB175000 CEX OG 5 interpretation indicating that sample is invalid Possible causes and troubleshooting Positive control Rotar Gene Q 25 3 29 43 26 3 24 3 30 43 e The positive control solution was not added Repeat the test Basitive control LC 480 I amp II 28 3 30 3 27 3 26 3 30 3 e CPE Solution or CPE PCR Mastermix was not added to the assay Please repeat the test Negative sample N A N A N A N A 30 3 extracted with IC FORALL l e Reagent solutions are degraded or may have expired Table 7 Data interpretation guidelines KPC NDM VIM OXA 48 Ic Interpretation 4 Troubleshooting for invalid result Cr value Crvalues Repeat the test using the same DNA extract If invalid results persist repeat the test by Bis VES or N A Positive sample preparing a new DNA extract from the original specimen Alternatively repeat the test N A 30 3 Negative sample with a new DNA extraction from a newly collected specimen N A N A or 233 Invalid If observed Cr values vary significantly from expected C values see FAQ and Troubleshooting section Check Direct CPE User manual Version 1 2
18. ee 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 wells of a 96 well plate Follow proper pipette dispensing techniques to prevent aerosols Wear clean disposable gloves and clean lab coats for the different steps of the test Change gloves whenever you suspect that they are contaminated Keep the tubes of all kit components and samples closed as much as possible Clean the lab benches and all equipment regularly with a 0 5 sodium hypochlorite solution Users are strongly advised to read the full protocol before starting the test Specimen collection and DNA extraction 1 Human specimen collection In order to obtain adequate specimen the procedure for specimen collection must be followed carefully with adequate swab material see section Materials required but not supplied with the kit 1 Collect perianal rectal specimen according to local guidelines and swabs manufacturer recommendations 2 Place swabs in their containers containing 1 ml of liquid transport medium 3 Label the containers 4 Refer to the swab manufacturer instruction for storage handling and stability 2 DNA extraction from perianal rectal swabs with NucliSENS easyMAG Important points before starting DNA extraction is carried out in the pre PCR room Procedure 1 Check Direct CPE has been validated with the Nu
19. eshold manually for each detector channel using the value recommended in e Baseline Method Auto Calculated Table 5 e Cycle to Analyze 10 45 4 Check amplification plots versus Ct values calculated by the software for each target in e Baseline Threshold User defined to be set per fluorophore the Quant Results table 2 Set the threshold manually for each detector channel using the threshold values recommended in Table 5 Table 6 Rotor Gene Q analytical parameters 3 Check amplification plots in the Quantification Tab versus C values calculated by the Detector TARGET Vo ee center Eliminare Tube Correct Removal Cycles software for all targets in the results table Green KPC ON ON 10 15 Yellow NDM VIM ON on e a View Settings Export Tools B naa aa one f mare n je Caton Data vew A E Orange OXA 48 ON ON 10 15 Red IC on on 15 s X Quantitation Analysis Cycling A from 15 Green 3 amp Ignore First E Outlier Removal bd Save Defaults NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC l NEG NTC NEG wig NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC NEG NTC tii 36 66 36 48 5A Figure 3 Screen shot of the typical analytical window on the CFX96 with the CFX96 software v2 0
20. heck Direct CPE assay is based on specific recognition and amplification of target sequences by PCR and the simultaneous detection of the accumulation of PCR amplification products by fluorescent DNA probes A control DNA molecule the internal control is added to the clinical specimen prior to DNA extraction to monitor that DNA extraction and PCR amplification were successful Five molecular beacon probes labeled with 4 different dyes are used to detect the various carbapenemases and the control DNA Check Direct CPE discriminates between KPC NDM VIM and OXA 48 For each of the 4 carbapenemase genes KPC OXA 48 NDM and VIM many gene variants exist PCR primers and fluorescent probes of Check Direct CPE are selected to target homologous gene segments of these carbapenemase genes and in this way gene variants are reliably detected Kit contents for 48 reactions Components Mat No Description Storage conditions CPE PCR Mastermix 9 0080 1 transparent tube and cap 630ul 4 C CPE solution 9 0071 1 brown tube purple inlay 140 pl Internal control 9 0077 1 tube red inlay 300 ul Negative control 9 0070 1 tube white inlay O 100 ul KPC positive control 9 0073 1 tube green inlay 100 pl 20 C store in the dark NDM positive control 9 0075 1 tube gold inlay 100 ul VIM positive control 9 0074 1 tube yellow inlay 100 ul OXA 48 positive control 9 0076 1 tube orange inlay
21. ht Cycler 480 I amp II PERFORMANCE CHARACTERISTICS ccccssssccccsssecccsssecccecsecccesecccecsecececsesecacsesececseeeeeceeeess 9 KEY TO SYMBOLS USED is ciseciscccsecccanssacdscscescssssavecassccccsswsasucncscasvaasacdsucasansssoccusiescassussdcseies 10 CFX96 LEAWAUTGA TAOS cee ecstatic carts ateash sal eestinctenae alos heat ces cat ooveenctes eral neioatescenel sane 11 Rotor Gene Q TECHNICAL ASSISTA NO E r ccscecisacasecceecdsvcecaneseccsaceccdenenacucdsdescausecousussecencesusacksesessasecoueuse 11 CE IVD Check Direct CPE User manual Version 1 2 Issued 05 09 2013 1 Intended use Check Direct CPE is a qualitative in vitro diagnostic test for the rapid detection of carbapenemase genes directly from perianal and rectal swabs 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 is performed on an automated real time PCR instrument using perianal rectal swabs from individuals at risk of colonization with carbapenemase producing Enterobacteriaceae Check Direct CPE can be used as an aid to identify prevent and control carbapenemase producing Enterobacteriaceae that colonize patients in healthcare settings Check Direct CPE is not intended to diagnose infections with carbapenemase producing Enterobacteriaceae nor to guide or monitor treatment for these infections Parallel cultures are necessary to recover
22. organisms for epidemiological typing susceptibility testing and for further confirmatory identification Introduction The worldwide emergence and dissemination of carbapenem resistance among Enterobacteriaceae is a serious threat to public health These organisms are associated with high mortality rates and have the potential to spread widely The most common cause of carbapenem resistance in Enterobacteriaceae is the expression of carbapenemases i e Carbapenemase Producing Enterobacteriaceae or CPE CPE have elevated or complete resistance to carbapenems and most other 6 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 6 lactam resistance determinants resulting in multidrug and pandrug resistant isolates Patients usually carry CPE by colonization of the colon Therefore rectal swabs provide a proper specimen to assess carriage of CPE and perianal swabs may be used as a non invasive alternative Check Direct CPE is a rapid real time PCR test for the detection and discrimination of KPC NDM VIM and OXA 48 in rectal and perianal specimens Check Direct CPE User manual Version 1 2 Issued 05 09 2013 se Check Direct CPE Principle of the method C
23. out delay place the plate into the real time PCR instrument and start the run with the parameters corresponding to the real time PCR instrument in use see next points When the run is completed discard the plate according to local regulations w 4 10 Following the manufacturer s instructions for each real time PCR instruments start and set up the run using the parameters specified in Table 3 and 4 and in the text below 2 For use on the ABI 7500 Run mode Standard 7500 e Reaction volume 25 ul e ROX passive reference dye Included in the qPCR Buffer e TaqMan Reagents e Experiment Quantitation Standard Curve e Standard ramp speed Targets Reporter Dyes and Quencher setup see Table 3 3 For use on the LC 480 I amp II To use the Check Direct CPE real time PCR kit on the LightCycler 480 system I amp II a 4 color compensation object is required and generated by using the 4 Color Compensation Set supplied by Check Points Health B V Ref 18 0070 e Detection format LC 480 I Multi Color Hydrolysis Probe e Detection format LC 480 II 4 color FAM VIC HEX RED610 CY5 e Filter Combination Selection see Table 3 e Reaction Volume 25 ul e Analysis mode Quantification for the amplification step e Integration time mode Dynamic e Color compensation object or file required 4 For use on the CFX96 Sample Volume 25 ul Temperature Control Mode Calculated Scan Mode All channel Plate type
24. ruments used and the threshold settings 2 Results interpretation Table 7 2 If the run is valid interpret results as positive negative or invalid with the Cr values obtained for the samples with the guidelines summarized in Table 7 e Positive carbapenemase samples will show a Cy value in the FAM VIC and or Red TXR channel Positive carbapenemase samples will also show a Cy value in the Cy5 channel if the target has not out competed the internal control IC for the resources during the reaction e Negative carbapenemase samples will show no C value in the FAM VIC and Red TXR Channel In the Cy5 Channel a C value is expected at 30 3 The real time results show no C C C values or interpretation indicating that the sample is invalid Possible causes and troubleshooting e The sample DNA was not added to the assay e The sample DNA tested with Check Direct CPE is negative and the internal control was not added prior to DNA extraction Please repeat the DNA extraction e The DNA extraction failed since the internal control was not detected Please repeat the DNA extraction e The sample DNA contains contaminants inhibiting the reactions Please repeat the DNA extraction PRE ME E E we ane Winva Gre e CPE Solution or CPE PCR Mastermix was not added to the assay Please repeat the IC showing no C value or a Cy 233 mean that the sample is invalid see FAQ and tst T leshooti 7 i i ED peouneet e Reagent solutions are degrad
25. says 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 Check Direct CPE User manual Version 1 2 Issued 05 09 2013 Technical assistance support check points com 31 317 453 908 Despite the utmost care in the development and preparation of the protocol Check Points cannot take any responsibility for errors omissions and or future changes herein Literature Citation When describing a procedure for publication using this product please refer to it as the Check Direct CPE Notice to Purchaser This product is sold under license from PHRI Properties and may be used under PHRI Properties patent rights only for human in vitro diagnostics food testing veterinary testing or research Trademarks TaqMan is a registered trademarks of Roche Molecular Systems Inc Sigma transwabs is a registered trademarks of Medical Wire amp Equipment Eswab is a registered trademarks of Copan Italia S p A ABI FAM VIC ROX are registered trademarks of Applera Corporation or its subsidiaries in the US and or certain other countries Red610 is a registered trademark of Biosearch Technologies Inc Cy5 is a registered trademark of Amersham Biosciences Ltd NucliSENS easyMAG is a registered trademark of bioM rieux Check
26. uidance for therapy The quality of the input DNA is an important factor for obtaining reliable results with Check Direct CPE DNA must be extracted from perianal or rectal swabs in transport medium using the NucliSENS easyMAG DNA extraction system bioM rieux FR Other DNA extraction systems have not been approved for use with Check Direct CPE yet The assay has been validated for both Sigma transwabs Medical Wire amp Equipment UK and Eswab Copan Diagnostics US in Amies transport media Other swab types are also expected to work well but this has not been validated yet The assay has been tested extensively with samples containing various gram negative bacteria such as Escherichia Salmonella Klebsiella Enterobacter Citrobacter and Pseudomonas with excellent results However it may never be excluded that other Gram negative bacteria or certain strains of the above species will yield poor results Check Direct CPE cannot and does not make any representation or warranty that it is capable of correctly detecting KPC NDM VIM and OXA48 in all gram negative species subspecies or types or in all clinical sample sources Results may need to be confirmed by additional methodologies in specific cases e g for regulatory samples Due to the high variability of bacterial genomes it is possible that certain subtypes might not be detected The test reflects the state of knowledge of Check Points Health B V As with other diagnostic as
27. y could no longer detect the target in question in more than 5 of the replicates Results See Table A Table A LOD copies PCR KPC 5 Target NDM 5 VIM 5 OXA 48 5 2 Specificity 2 1 In silico Specificity The specificity of the Check Direct CPE real time diagnostic test is ensured by the selection of the correct primers and probes as well as the selection of stringent reaction conditions Primers and probes sequences were validated with in silico analysis Primers and Probes sequences were designed to specifically identify the gene variants listed in Table B Primers and Probes sequences were tested for potential homologies with all the gene sequences published by the international gene bank GenBank NIH genetic sequence database using sequence comparison analysis Results No cross homology was found with other organisms for designed primers and probes 9 Table B Gene Variants KPC 1to15 NDM 1to8 VIM 1 to 6 8 to 37 OXA 48 162 163 181 204 232 244 245 2 2 Analytical Specificity The experimental specificity of the Check Direct CPE real time diagnostic test was determined by testing the cross reactivity with samples containing non target organisms 132 carbapenemase negative strains were used to test the specificity of the Check Direct CPE real time test see bacterial strains listed in Table C Results All isolates tested negative with the Che
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