User manual - Check
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1. ove Check MDR Carba User Ma nN uqa Key to symbols used For In Vitro Diagnostic Use Catalog number Batch code IFU Instructions for use number Use before YYYY MM lil Consult instructions for use Check MDR Carba Rapid Molecular Detection of carbapenemase genes 14 0051 y 24 Temperature limitation Contains sufficient for lt n gt tests Manufacturer LightCycler 480 For use with the LightCycler 480 real time PCR system Roche CH in combination INTENDED USE soinnin EEE Aaa 2 with the QuantiTect Probe PCR Master Mix QIAGEN CA USA INTRODUCTION ecccocooooocococcoooocoocoooooooooooo00000000000000000000000000000000000000000000000000000000000000 2 PRINCIPLE OF THE METHOD sccccsscccccssccccsssccccessecccesscccessscccessscceeesseceeesccs 2 KIT CONTENTS FOR 24 REACTIONS cccesccccescccsscccessccccsscccesscccsssccessccessseees 2 SHELF LIFE STORAGE AND HANDLING cccssccccssscccessscceessscceessscceesssccesssscccees 2 MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT secccessccccesscceesseee 3 GOOD LABORATORY PRACTICES ssccccssscccsssscccessscccessscccesscccsssscecsesceeseseceeeess 3 PROTOCOL cssccccssscccesscccesssscccsssccccesscccessscccesssececessecceesseceeesseceessseceesssceesnecs 4 1 DNA EXTRACTION FROM BACTERIAL CELLS cccsccvicccocescccaeuceenaccueeaicenree leaves seasneseimavenees 4 2 DNA RECOGNITION STEP ccs bac siccsevascecces i aniser No EN2. gt 35 or no C and an IC Cy5 gt 35 no C the assay has not worked well and should be repeated with a new DNA extract of the bacterial culture Table 7 Criteria for a valid run with Check MDR real time assay Carba FAM Internal Control Cy5 Cp values Cp values No C or 2 IC internal control If Cp values are not as expected see FAQ Reaction Sample Type Negative sample extracted with IC Table 8 Data interpretation guidelines Carba FAM Internal Control Cy5 C values C values see FAQ Interpretation Frequently asked questions FAQ amp Troubleshooting How important is the amount of input cells for DNA extraction Check MDR Carba has been optimized using a well defined amount of cells A deviation of 20 in the amount of cells will have no major consequences Larger deviations will not give optimal results May other DNA extraction methods be used with Check MDR Carba Check MDR Carba test has been optimized using DNeasy Blood amp Tissue Kit QIAGEN CA USA NucliSENS easyMAG bioM rieux France and MagNA Pure system Roche CH extraction methods Check Points does not guarantee the performance of the test with extraction methods other than those recommended in this manual The thermocycler states an error in step A Please contact Check Points Technical Support support check points com During the step A the sample s have partly evaporated Reaction tubes may not have
3. 0 08 0 12 this may vary between spectrophotometers Add 2 5 ul of the Internal Control solution IC transparent cap to the cell suspension and start the DNA extraction DNA is eluted in 110 ul elution buffer DNA extracts can be stored at 20 C or 4 C for up to 6 months DNeasy Blood amp Tissue Kit QIAGEN CA USA DNA extraction procedure for bacterial cells manual extraction or QlAcube automated system Follow the manufacturer s protocol for gram negative bacteria Prepare 1 ml cell suspension of McFarland 1 2 1 8 or ODsoo of 0 16 0 24 this may vary between spectrophotometers and centrifuge at 14000 rpm for 10 minutes Discard the supernatant and add 5 ul of the IC solution transparent cap to the pellet Start the DNA extraction with the pelleted cells using either the manual procedure or the QlAcube automated procedure DNA is eluted in 200 ul elution buffer Store DNA extracts at 20 C or 4 C for up to 6 months Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 one Check MDR Carba MagNaA Pure system 32 samples Roche CH for DNA extraction procedure for clinical specimens Use 200 ul of cell suspension in PBS Phosphate buffered saline McFarland 0 5 1 0 or ODs 0 08 0 12 this may vary between spectrophotometers Add 2 5 ul of the IC solution transparent cap to the cell suspension and start the DNA extraction DNA is eluted in 100 ul elution buffer DNA extracts should b
4. A carbapenemase first identified in K pneumoniae isolates in North America and now found in many countries worldwide in various other enterobacterial species including E coli 2 NDM New Delhi Metallo B lactamase NDM first reported in 2009 in Sweden in both K pneumoniae and E coli nowadays regularly found in various European countries and quite prevalent in India and Pakistan 3 OXA 48 carbapenemase a class D B lactamase originating from Shewanella species and regularly found in Europe North Africa and Turkey in fermenting Enterobacteriaceae like E coli and Klebsiella and 4 VIM amp IMP carbapenemases metallo B lactamases first reported in Pseudomonas aeroginosa VIM 1 in Verona in 1997 and an IMP 1 in Japan in 1990 and since then transferred to E coli and K pneumoniae and many other Enterobacteriaceae Many gene variants of VIM and IMP exist of which the DNA sequences may differ significantly and therefore VIM and IMP should be regarded as two separate gene families of homologous gene variants Conventional methods for detection of B lactamases rely on phenotypic identification which is time consuming frequently inconclusive and not applicable to all species Check MDR Carba is a rapid molecular test for the detection of the most prevalent carbapenemase genes Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 one Check MDR Carba Principle of the method Check MDR Carba is based on specific molecul
5. NIENTE EUNIS E EN NINEN 4 3 REAL TIME PROBE AMPLIFICATION STEP B cceccscccccccscccccccecesceacecescesceacesesceacenessence 5 DATA ANALYSIS AND INTERPRETATION ccccssscccessccccesscccessscccesssccecesscceeesces 6 gO BF ANE A S Rng ne ee ey ee ee eee 6 2 By 8 Nigel 2t 224 9 Fe ee ce ee ee ene ent eee N eee ene 6 C FREQUENTLY ASKED QUESTIONS FAQ amp TROUBLESHOOTING sscccceseeceeeeees 7 LIMITATIONS cccccscccccsscccccssccccessccccesssccesssccceesseceesssccecesseceesssecesssseceesssnceeeenecs 8 IFU 051 01 Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 1 Intended use Check MDR Carba is designed for the rapid molecular detection of the clinically important carbapenemase genes in gram negative bacteria KPC NDM OXA 48 VIM and IMP Check MDR Carba generates definitive results within 4 5 hours compared to the 24 hours necessary for analysis with conventional phenotypic methods Introduction In the last decade various carbapenemases have been reported which cause elevated or complete resistance against carbapenem antibiotics when produced by bacteria Strains carrying genes encoding such enzymes are generally resistant to all B lactam antibiotics They often contain additional B lactamase genes and genes for resistance against quinolones and aminoglycosides The carbapenemase genes detected with Check MDR Carba include 1 KPC Klebsiella Pneumoniae Carbapenemase an Ambler class
6. Version 1 1 Issued 25 Sept 2012 one Check MDR Carba Prevention of contaminations PCR produces a very high quantity of DNA amplification products amplicons even from minute quantities of starting material Check MDR Carba may therefore yield unreliable results if samples become contaminated with amplicons from previous amplification reactions prior to the PCR Preventive measures to minimize the risk of amplicon contamination must be taken Please read carefully and follow the instructions outlined below Use separate rooms a pre PCR room and a post PCR room Sample preparation DNA recognition step A and preparation of the amplification step step B is carried out in the pre PCR room Incubation in the real time PCR thermocycler of step B is carried out in the post PCR room Never bring the reaction products of step B to the pre PCR room Never prepare the ligation and or amplification steps in the post PCR 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 or samples to a reaction tube to avoid contamination Follow proper pipette dispensing techniques to prevent aerosols Use separate equipment pipettes thermocyclers sample holders lab coats gloves disposables and reagents that are assigned to these rooms Never transfer items from the post PCR room to the pre PCR room Wear a clean lab co
7. with the kit Post PCR Thermocycler Vortex mixer Equipment D i e Real Time PCR instrument Mini centrifuge PCR plate spinner QuantiTect Probe PCR Master Mix DNA extraction procedure Disposable laboratory powder free gloves Pipettes amp disposable filter tips for Supplies volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 10 ml tubes for large volumes 96 well PCR plate PCR plate seal PCR tubes strips contact your local representative for specifications see Protocol section 1 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 e The test must be performed by adequately trained personnel e Spinning down for a few seconds is done in the various steps to ensure that all material is collected at the bottom of the tubes 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 down the solution to avoid contamination when opening the lid Avoid unnecessary freeze thawing of the kit content e Periodically verify the accuracy and precision of pipettes as well as correct functioning of the instruments e Keep Solution R yellow cap in the dark to avoid photo bleaching of the dyes Check MDR CARBA User manual
8. A Please repeat the test e Solution R and or TaqMan Universal PCR MasterMix was not added to the assay Please repeat the test e QuantiTect Probe PCR Master Mix may have expired What does it mean if the real time results show no Cp values for the positive control or interpretation concluded that sample is inconclusive Such results may have multiple explanations e The positive control solution was not added to its reaction tube in step A e Solution P and or Solution A was not added in step A Please repeat the test e Solution R or TaqMan Universal PCR MasterMix was not added to the assay Please repeat the test e QuantiTect Probe PCR Master Mix may have expired Duplicate DNA samples tested with Check MDR Carba test do not yield identical results Cy values of identical samples may vary slightly between individual reactions Larger variations gt 2 C values suggest pipetting errors or other differences between the duplicate samples May the assay be interrupted after step A and continued at a later time Reaction mixtures from Step A can be kept at hold at 4 C for up to 2 hours For further inquiries please contact Check Points Technical Support support check points com Limitations Check MDR Carba uses a range of specific DNA markers to detect the presence of the carbapenemase genes KPC NDM OXA 48 VIM and IMP which currently represent the clinically most prevalent carbapenemases The test detects all prese
9. ar recognition of target sequences by DNA probe ligation followed by real time PCR detection The recognition step uses two specific oligonucleotides ligation probes that are joined by a DNA ligase only when they match perfectly with the target DNA In addition to target specific sequences the DNA probes contain two universal primer binding sites as well as a DNA segment that is complementary to a molecular beacon Subsequent real time amplification with universal primers is used to detect the connected probes and their accumulation is visualized by fluorescence of a molecular beacon Kit contents for 24 reactions Components Mat No Description Storage conditions Internal control 9 0054 1 tube transparent cap 500 ul Positive control Carba 9 0056 1 tube white cap O 500 ul Manual 9 0058 Leaflet download from website Volume provided for 25 reactions which takes into account the preparation of excess master mix Shelf life Storage and Handling The components of the kit must be stored at 20 C Solution R has to be stored in the dark Reagents stored at the appropriate storage conditions can be used until the expiration date Please visually inspect the box upon initial opening to ensure that its content is intact Do not use when damaged Please contact the Check Points office at support check points com if you have any questions or in case shipping has taken more than 2 days Materials required but not supplied
10. at and clean gloves during all steps of the test Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or during sample preparation 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 Protocol It is strongly recommended that the full protocol is read before using the test The protocol consists of the following steps 1 DNA extraction from bacterial cells 2 DNA recognition step A 3 Real time probe amplification step B 1 DNA extraction from bacterial cells Important points before starting DNA extraction is completely carried out in the pre PCR room Make sure to always use a new pipette tip when adding solutions or samples to a reaction tube to avoid contamination Clinical specimens should first be incubated on nutrient agar plates Use bacterial cells from these agar plates for DNA extraction Typical growth media include blood agar MacConkey agar and Tryptic Soy agar Procedure 1 Check MDR Carba has been validated with the following extraction methods for bacterial cells NucliSENS easyMAG bioM rieux France automated DNA extraction procedure for bacterial cells Follow the manufacturer s protocol for bacterial cells and use 200 ul cell suspension of McFarland 0 5 1 0 or ODs
11. been closed properly Please restart the procedure from step A have left Solutions A P R Internal control 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 fully guaranteed if these solutions were left out of 20 C 4 F for more than 24 hours May I change the C threshold when analyzing my real time PCR data The Check MDR Carba assay was developed using the automatic Abs Quant 2nd Derivative Max calculation method from the LightCycler 480 instrument software release 1 5 0 We therefore strongly advise to use this method for data analysis Please refer to the LightCycler 480 instrument operator s manual for more information Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 10 11 one Check MDR Carba What does it mean if the real time results show no Cp values or interpretation concluded that the sample failed Such results may have multiple explanations e The sample DNA was not added to the assay in step A e The sample DNA tested with Check MDR Carba is negative and the internal control was not added prior to DNA extraction e The DNA extraction failed since the internal control was not detected e The sample DNA contains contaminants inhibiting the reactions Please repeat the DNA extraction e Solution P and or Solution A was not added in step
12. e stored at 4 C only for up to 6 months Use the DNA solution directly and continue with step A or store as specified until use 2 DNA recognition step A Important points before starting Step A is completely carried out in the pre PCR room Make sure to always use a new pipette tip when adding solutions or samples to a reaction tube to avoid contamination It is advised to perform a positive and negative control reaction The positive control white cap O is supplied with the kit for the negative control we recommend to perform a DNA extraction as specified earlier with IC solution for a sample known to be negative for the test in use i e Carba negative sample elution buffer clean water Use a thermocycler located in the pre PCR room to perform the step A reaction Use PCR tubes that are suitable for the type of thermocycler used Procedure 1 Determine the number of reactions Thaw all reagents i e Solution A DNA samples If kept at 20 C and positive control mix well and keep on ice 2 Prepare the ligation reaction mix as described in table 1 and include 10 surplus to ensure that you have enough ligation reaction mix Mix well and spin down 3 To each tube add 7 5 ul of ligation reaction mix and 10 ul of sample DNA or controls see table 2 4 Close the tubes mix well by tapping or vortexing and spin down briefly The solutions should have a uniform blue color 5 Place the tube s in the thermocycle
13. group 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
14. ion Solution R yellow cap 10 ul QuantiTect Probe PCR mastermix 15 ul Total volume of master mix 25 ul Table 5 Filter combination Target Detector ra 4895330 Internal Control Cy5 615 610nm Table 6 Real time cycling parameters Step Temperature Time Cycles Acquisitionmode Ramprate Denaturation gt osc 10min 1 None ACH Amplification 40 cooing are Hold Bone Data analysis and interpretation LC 480 real time PCR instrument For a detailed description on how to operate the LightCycler 480 instrument and how to analyze your data please refer to the LightCycler 480 instrument operator s manual 1 Data analysis 1 In the Analysis Overview use Analysis modules Abs Quant 2nd Derivative Max Abs Quant 2nd Der for all samples This process is described in the LightCycler 480 instrument operator s manual 2 Check the Amplification plot Figures 1 shows typical amplification plots of Carbapenemase positive and negative samples Amplification Curves Amplification Curves luorescence 615 670 F eo e Figure 1 Typical real time amplification plots of A FAM signal for carbapenemase positive in red and negative in green samples B Cy5 signal for carbapenemase negative and positive samples in red The internal control curve Cy5 may be absent or at a higher Cp in positive samples Software release 1 5 0 2 Data interpretation Interpret results positive negative or inconcl
15. ntly known variants of KPC NDM OXA 48 and VIM except VIM 7 a rare variant only found in Pseudomonas aeruginosa IMP is the most diverse carbapenemase gene family and the test detects the clinically most important IMP variants in Enterobacteriaceae including IMP 1 3 4 5 6 7 8 10 and 13 It should be noted that other rare carbapenemase gene families are not detected Check MDR Carba requires DNA purified from a colony or bacterial culture Clinical specimens cannot be tested directly The quality of the input DNA is an important factor for obtaining reliable results with Check MDR Carba DNA must be extracted from cultured bacteria using the extraction methods validated with Check MDR Carba and described in this manual page 4 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 MDR Carba 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 or type or in any 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 pos
16. r and run the Probe ligation program see table 3 total sample volume 18 ul Table 1 Ligation reaction mix Component Volume per reaction Solution P Carba green cap 2 5 ul Solution A purple cap 5 ul Total volume of ligation reaction mix 7 5 ul Table 2 Ligation reaction setup Reaction Type Component Volume per reaction Test sample Sample DNA 10 ul Positive control Carbapenemase positive white cap O 10 ul Negative control Negative sample extracted with the IC 10 ul Table 3 Probe ligation cycling parameters e It is recommended to mix Solution P and Solution A first in a separate tube for x number of samples including controls and 10 surplus and then add 7 5 ul of this mix to each reaction tubes Prepare this mix shortly before step A is started and use immediately Do not store this mix and dispose after use e Close the PCR tube s carefully excessive pressure may distort the cap and lead the sample evaporation during step A 3 Real time probe amplification step B Important points before starting The preparation of the reaction mix for step B is carried out in the pre PCR room Step B was developed for the real time LightCycler 480 LC 480 instrument Roche CH therefore Roche PCR 96 well plates and corresponding adhesive seals should be used Roche CH Step B requires the QuantiTect Probe PCR Master Mix QIAGEN CA USA Procedure 1 Briefly spin down the reaction mixtures from s
17. sible 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 Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 one Check MDR Carba 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 MDR Carba Notice to Purchaser Molecular beacons are licensed from the Public health Research Institute of the City of New York PHRI under PHRI s patents Trademarks Nuclisens and easyMAG and are registered trademarks of bioM rieux SA DNeasy and QlAcube are registered trademarks of the QIAGEN group MAGNA PURE is a trademark of Roche FAM is a trademark of Applera Corporation Cy5 is a registered trademark of GE Healthcare LightCycler is a registered trademark of Roche Diagnostics QuantiTect is a registered trademark of the QIAGEN
18. tep A 2 Take Solution R yellow cap from the freezer Thaw completely at room temperature protecting from exposure to light Mix well and spin down briefly 3 Prepare the real time PCR qPCR reaction mix as described in table 4 and include 10 surplus to ensure that you have enough qPCR reaction mix Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 one Check MDR Carba Add 25 ul of qPCR master mix and 5 ul of each step A reaction to each well of the 96 well plate Use a new pipette tip for each step A reaction sample added Seal the plate mix by tapping the plate on the bench and spin down briefly using a centrifuge with 96 well plate adapters Transfer the plate to the post PCR room Following the manufacturer s instructions start and set up the run of the real time PCR LightCycler 480 instrument using the following parameters see also table 5 and 6 e Detection format Multi Color Hydrolysis probe e Reaction Volume 30 ul e Nocolor compensation needed e Analysis mode quantification for the amplification step e Integration time mode Dynamic e Analysis modules Abs Quant 2nd Derivative Max for All Samples Abs Quant 2nd Der Without delay place the plate or strips into the real time PCR instrument and start the run with the cycling conditions presented in table 6 When the run is completed discard the plate according to local regulations Table 4 gPCR reaction mix Component Volume per react
19. usive with the Cp values obtained for the samples following the guidelines outlined below and summarized in table 7 and 8 1 The relevant columns in the sample results table are Position Name Reporter Cp and status All other columns may be ignored or removed from the table 2 Verify that the real time PCR run is valid before data analysis and interpretation of the results Table 7 shows criteria for a valid real time run with Check MDR Carba test Check MDR CARBA User manual Version 1 1 Issued 25 Sept 2012 one Check MDR Carba Positive carbapenemase samples will show a lower FAM C value than the Internal Control IC Cy5 signal Positive Carbapenemase samples will be typed positive with an expected C value lt 31 Negative carbapenemase samples will show a higher FAM C value than the IC Cy5 signal Negative carbapenemase samples will show a C value gt 35 In the results table the status will display gt Late C call high uncertainty or detector call uncertain Late C call or No C value corresponding to a negative call For negative carbapenemase samples the IC Cy5 signal is expected at 33 2 Samples with a FAM C between 32 and 35 and a C for the IC Cy5 at 33 2 are considered inconclusive Please repeat the whole procedure with a new DNA extract of the bacterial culture If still inconclusive the sample can be regarded neither positive nor negative In samples with a FAM C
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