Sample & Assay Technologies mericon™ Plant and Animal
Contents
1. Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 15 mericon Chicken Kit The mericon Chicken Kit is designed for the detection of chicken matter in food and in animal feed Limit of detection The mericon Chicken Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Chicken Kit exhibits high specificity for chicken No cross reactivity was observed with other animal species Table 7 using 50 ng of the tested DNA Table 7 Results from cross reactivity experiment Genetic construct Genetic construct GMO or species Result GMO or species Result Chicken Deer Turkey Cod a Goose Water buffalo Duck Salmon K Ostrich Rabbit A Cattle Corn te Pig Soy T Sheep Rice Goat Wheat Horse Potato Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens 16 mericon Plant and Animal Identification Handbook 04 2013 mericon Turkey Kit The mericon Turkey Kit is designed for the detection of turkey matter in food and in animal feed Limit of detection The mericon Turkey Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Turkey Kit exhibits high specificity for turkey No cross reactivity was observed with other animal species2. Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens 20 mericon Plant and Animal Identification Handbook 04 2013 mericon Horse Kit The mericon Horse Kit is designed for the detection of horse matter in food and in animal feed Limit of detection The mericon Horse Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Horse Kit exhibits high specificity for horse No cross reactivity was observed with other animal species Table 12 using 50 ng of the tested DNA Table 12 Results from cross reactivity experiment Species Result Species Result Horse Water buffalo Donkey Goat Cattle Salmon Deer Rabbit Turkey Chicken Goose Soy Duck Rice Pig Wheat Sheep Potato Cod Corn Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 21 Equipment and Reagents to Be Supplied by User When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information consult the appropriate safety data sheets SDSs available from the product supplier M Nucleic acid isolation kit We recommend the DNeasy mericon Food Kit see Ordering Information E Pipets adjustable E Sterile pipet tips with filters M
3. Fax 800 18817 Technical 800 18712 Singapore Orders 65 67775366 Fax 65 67785177 Technical 65 67775366 Spain Orders 91 630 7050 Fax 91 630 5145 Technical 91 630 7050 Sweden Orders 020 790282 Fax 020 790582 Technical 020 798328 Switzerland Orders 055 254 22 11 Fax 055 254 22 13 Technical 055 254 22 12 UK Orders 01293 422 911 Fax 01293 422 922 Technical 01293 422 999 6 amp 6 amp USA Orders 800 426 8157 Fax 800 718 2056 Technical 800 DNA PREP 800 362 7737 QIAGEN awe Sample amp Assay Technologies
4. N p 25 30 35 40 5 10 15 25 30 35 40 5 10 15 20 20 Cycle Cycle Figure 3 The PCR is inhibited There is no amplification of the three samples in the green channel left or the internal control in the yellow channel right All curves lie along the baseline and do not exceed a preset threshold Table 20 Summary of possible outcomes Amplification of Amplification of internal control sample Result Sample is positive Sample is negative PCR failed Partial inhibition of the PCR due to the presence of detectable but tolerable concentrations of inhibitors in the samples is typically indicated by a shift of the internal control to higher cycle thresholds C values As a guideline the uninhibited internal control should give a cycle threshold value ranging between 28 and 32 A cycle threshold value above 33 indicates inhibition In the event of PCR inhibition dilute the extracted samples 1 10 with RNase free water and repeat the test If DNA template concentration in the PCR reaction is very high a shift of the Internal Control to lower cycle values may occur which does not influence its sensitivity toward PCR inhibitors or amplification of the target DNA i E mericon Plant and Animal Identification Handbook 04 2013 33 Troubleshooting Guide This troubleshooting guide may be helpful in solving any problems that may arise For more information see al
5. Table 8 using 50 ng of the tested DNA Table 8 Results from cross reactivity experiment Species Result Species Result Turkey Deer m Chicken Cod Goose Water buffalo Duck Salmon Ostrich Rabbit Cattle Corn Pig Soy Sheep Rice Goat Wheat Horse Potato Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 17 mericon Ruminants Kit The mericon Ruminants Kit is designed for the detection of ruminant matter in food and in animal feed Limit of detection The mericon Ruminants Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Ruminants Kit exhibits high specificity for ruminants No cross reactivity was observed with other animal species Table 9 using 50 ng of the tested DNA Table 9 Results from cross reactivity experiment Species Result Species Result Cattle Water buffalo Sheep Goat Deer Salmon Turkey Rabbit Chicken Corn Goose Soy Duck Rice Pig Wheat Horse Potato Cod z gt Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens 18 mericon Plant and Animal Identification Handbook 04 2013 mericon Sheep Kit The mericon Sheep Kit is designed for the detection
6. For updated license terms see www qiagen com NOTICE TO PURCHASER LIMITED LICENSE The purchase price of this product includes a limited non transferable license under some or all of US Patents Nos 5 210 015 5 487 972 5 476 774 5 219 727 and 5 804 375 or corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd and US Patents Nos 5 538 848 5 723 591 5 876 930 6 030 787 and 6 258 569 and corresponding patent claims outside the United States owned by Applera Corporation to use only this amount of product to practice the 5 nuclease process solely in the Food Testing Applications Field including reporting results of purchaser s activities for a fee or other commercial consideration and also for the purchaser s own internal research No right under any other patent claims such as apparatus or system claims in US Patent No 6 814 934 and no right to use this product for any other purpose is hereby granted expressly by implication or by estoppel Further information on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Center Drive Foster City California 94404 USA License Probe manufactured by Integrated DNA Technologies Inc under license from Applied Biosystems License Probe incorporates IDT s lowa Black quencher technology and is accompanied by a limited research use only license from IDT 2012 2013 QIA
7. Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay GMO Screen CTP2 CP4EPSPS Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay GMO Screen bar Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay Mon 810 Corn Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Quant GMO Detection Assays mericon Quant Roundup Ready Soy 48 For 48 reactions PCR Assay Quant RR Soy PCR Assay Ref System Quant RR Soy Quant Control DNA Quant RR Soy Standard DNA Quant RR Soy Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution Larger kit sizes available please inquire mericon Plant and Animal Identification Handbook 04 2013 Cat no 291113 291023 291053 291063 291073 291514 39 Product mericon Quant MON 810 48 Contents Cat no For 48 reactions PCR Assay Quant 291524 MON 810 PCR Assay Ref System Quant MON 810 Quant Control DNA Quant MON 810 Standard DNA Quant MON 810 Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon
8. Pathogen Detection Assays mericon Listeria spp 24 mericon Salmonella spp Kit 24 mericon L monocytogenes Kit 24 mericon Campylobacter spp Kit 24 mericon Campylobacter triple Kit 24 mericon VTEC stx1 2 Kit 24 40 For 24 reactions PCR Assay Listeria 290123 spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay Salmonella 290013 spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay L 290023 monocytogenes Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay 290033 Campylobacter spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay 290043 Campylobacter triple Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay VTEC 290053 stx1 2 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Plant and Animal Identification Handbook 04 2013 Product Contents Cat no mericon Cronobacter For 24 r
9. Rotor Gene Q or other real time PCR instrument with fluorescence detection for approximately 520 nm FAM fluorescence and approximately 560 nm for yellow dyes See Table 13 on page 24 for examples M PCR plastics for the thermal cycler to be used For Rotor Gene Q Strip Tubes and Caps 0 1 ml for use with 72 well rotor cat no 981103 or 981106 or PCR Tubes 0 2 ml for use with 36 well rotor cat no 981005 or 981008 E For Rotor Gene Q Loading Block 72 x 0 1 ml Tubes cat no 9018901 or Loading Block 96 x 0 2 ml Tubes cat no 9018905 E Tube rack E Microcentrifuge E Vortexer M Ice bucket with ice or cooled Loading Block Ensure that instruments have been checked and calibrated according to the manufacturer s recommendations Use of yellow dye to detect the internal control of the mericon PCR Assays requires calibration on some instruments See Table 4 page 14 for details ooyyeeeeaeaeaeaeaeaeaeaeaeasaeaeaeSaeaeaeaeaeaeaeaeaeaeaeaeaeaeaeaeaeaeEeEeEeEeEeEeEeEeEeEeEeEeEeESEeEeEeEeEoeeeee Y 22 mericon Plant and Animal Identification Handbook 04 2013 Important Notes General precautions The user should always pay attention to the following M Use gloves as well as sterile pipet tips with filters E All materials and media possibly containing DNA of the tested plant or animal should be autoclaved for 20 min at 120 C M Store and extract positive materials specimens positive controls and amplicons sep
10. of reactions 24 96 Yellow mericon Assay 2x12 1x 96 reactions reactions Red Positive Control DNA 20 20 reactions reactions QuantiTect Nucleic Acid Dilution Buffer 1 5 ml 1 5 ml RNase Free Water 1 9 ml 1 9 ml Blue Multiplex PCR Master Mixt 2x130ul 1040 ul 50x ROX Dye Solution 45 ul 210 ul Quick Start Protocol Contains target specific primers and probes as well as the internal control IC t Contains HotStarTaq Plus DNA Polymerase dedicated multiplex real time PCR buffer and dNTP mix dATP dCTP dGTP dTTP 4 mericon Plant and Animal Identification Handbook 04 2013 Table 1 mericon Plant and Animal Identification Assays Catalog no Product name 24 96 mericon Soy Kit 293013 293045 For specific detection of food components made from soy mericon Corn Kit 293023 293025 For specific detection of food components made from corn mericon Apricot Kernels Kit 293033 293035 For specific detection of food components made from apricot kernels mericon Pig Kit 292013 292015 For specific detection of food components of pig origin mericon Cattle Kit 292023 292025 For specific detection of food components of cattle origin mericon Chicken Kit 292033 292035 For specific detection of food components of chicken origin mericon Turkey Kit 292043 292045 For specific detection of food components of turkey origin mericon Ruminants Kit 292073 292075 For specific detection of food components of ruminant
11. of sheep matter in food and in animal feed Limit of detection The mericon Sheep Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Sheep Kit exhibits high specificity for sheep No cross reactivity was observed with other animal species Table 10 using 50 ng of the tested DNA Table 10 Results from cross reactivity experiment Species Result Species Result Sheep Water buffalo Cattle Goat Deer Salmon Turkey Rabbit Chicken Corn Goose Soy Duck Rice Pig Wheat Horse Potato Cod Cross reactivity experiments are ongoing For up to date information visit www qgiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 19 mericon Goat Kit The mericon Goat Kit is designed for the detection of goat matter in food and in animal feed Limit of detection The mericon Goat Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Goat Kit exhibits high specificity for goat No cross reactivity was observed with other animal species Table 11 using 50 ng of the tested DNA Table 11 Results from cross reactivity experiment Species Result Species Result Goat Water buffalo Cattle Sheep Deer Salmon Turkey Rabbit Chicken Corn Goose Soy Duck Rice Pig Wheat Horse Potato Cod
12. of starting materials These solutions were developed to complement the mericon PCR Assays and provide a complete and efficient workflow for food safety testing HotStarTagq Plus DNA Polymerase HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA Polymerase It is provided in an inactive state and has no enzymatic activity at ambient temperature This prevents the formation of misprimed products and primer dimers during reaction setup and the first denaturation step Competition for reactants by PCR artifacts is therefore avoided enabling high PCR specificity and accurate quantification The enzyme is activated first at the start of a reaction by a 5 minute 95 C incubation step which enables reactions to be set up rapidly and conveniently at room temperature In addition the concentration of the polymerase in the master mix is optimized to allow short extension times in the combined annealing extension step of each PCR cycle Multiplex PCR Master Mix The Multiplex PCR Master Mix is specifically developed for fast cycling multiplex real time PCR using sequence specific probes A novel additive in the buffer Q Bond allows short cycling times on standard cyclers and on fast cyclers with rapid ramping rates Q Bond increases the affinity of HotStarTaq Plus DNA Polymerase for short single stranded DNA reducing the time required for primer probe annealing to a few seconds The buffer also contains Factor MP which facilitate
13. of this product signifies the agreement of any purchaser or user of the mericon Plant and Animal Identification Assays to the following terms 1 The mericon Plant and Animal Identification Assays may be used solely in accordance with the mericon Plant and Animal Identification Handbook and for use with components contained in the Kit only QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the mericon Plant and Animal Identification Handbook and additional protocols available at www qiagen com Other than expressly stated licenses QIAGEN makes no warranty that this Kit and or its use s do not infringe the rights of third parties This Kit and its components are licensed for one time use and may not be reused refurbished or resold QIAGEN specifically disclaims any other licenses expressed or implied other than those expressly stated GI Be G0 IND The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court and shall recover all its investigative and Court costs including attorney fees in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and or its components
14. origin mericon Sheep Kit 292063 292065 For specific detection of food components of sheep origin mericon Goat Kit 292053 292055 For specific detection of food components of goat origin mericon Horse Kit 292143 292145 For specific detection of food components of horse origin For more detailed information see Assay specific information starting page 11 pr ee E mericon Plant and Animal Identification Handbook 04 2013 5 Storage mericon Plant and Animal Identification Assays are shipped on dry ice Multiplex PCR Master Mix and ROX dye should be stored immediately at 20 C upon receipt in a constant temperature freezer All remaining not reconstituted kit components should be stored at 2 8 C and protected from light Stored under these conditions and handled correctly assay performance remains unaffected until the date of expiration printed on the quality control label inside the kit box or envelope Reconstituted reagents of mericon Plant and Animal Identification Assays should be dispensed into aliquots to avoid more than 5 freeze thaw cycles and stored at 2 8 C for short term storage 1 month or 20 C for long term storage Product Use Limitations mericon Plant and Animal Identification Assays are intended for molecular biology applications in food animal feed and pharmaceutical product testing These products are not intended for the diagnosis prevention or treatment of a disease P
15. please inquire Larger kit sizes available please inquire mericon Plant and Animal Identification Handbook 04 2013 41 Product Contents Cat no mericon DNA Bacteria 50 Pathogen Lysis Tubes L Fast Lysis 69534 Plus Kit 50 Buffer Instrumentation Rotor Gene Q 5plex Real time PCR cycler with 5 channels Inquire green yellow orange red crimson laptop computer software accessories year warranty on parts and labor For up to date licensing information and product specific disclaimers see the respective QIAGEN kit handbook or user manual QIAGEN kit handbooks and user manuals are available at www giagen com or can be requested from QIAGEN Technical Services or your local distributor SSSSSSS gt gt gt gt E gt gt gt y gt y gt y gt y gt EyEyEyEyEyEyEyEyyo ee 42 mericon Plant and Animal Identification Handbook 04 2013 Trademarks QIAGEN QlAquick DNeasy HotStarTaq mericon Q Bond QuantiTect Rotor Gene QIAGEN Group Mx3005P Mx3000P Stratagene Agilent Technologies SmartCycler Cepheid Roche Hoffman La Roche Inc lowa Black MAX Integrated DNA Technologies Inc Applied Biosystems FAM HEX JOE StepOne StepOnePlus VIC Life Technologies LightCycler Roche Group Developed by ifp Institut fur Produktqualit t Teltowkanalstr 2 12247 Berlin Germany www produktqualitaet com Limited License Agreement Use
16. quality and availability of our technical support Our Technical Service Departments are staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of QIAGEN products If you have any questions or experience any difficulties regarding mericon Plant and Animal Identification Assays or QIAGEN products in general please do not hesitate to contact us QIAGEN customers are a major source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at QIAGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please see our Technical Support Center at www qiagen com Support or call one of the QIAGEN Technical Service Departments or local distributors see back cover or visit www qiagen com Safety Information When working with chemicals always wear a suitable lab coat disposable gloves and protective goggles For more information please consult the appropriate safety data sheets SDSs These are available online in convenient and compact PDF format at www giagen com safety where you can find view and print the SDS for each QIAGEN kit and kit component 24 hour emergency information Emergency medical information in English French and German can be obtain
17. April 2013 mericon Plant and Animal Identification Handbook For identification of plant and animal matter in food or animal feed samples using real time PCR QIAGEN Sample amp Assay Technologies QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies enabling the isolation and detection of contents of any biological sample Our advanced high quality products and services ensure success from sample to result QIAGEN sets standards in Purification of DNA RNA and proteins Nucleic acid and protein assays microRNA research and RNAi Automation of sample and assay technologies Our mission is to enable you to achieve outstanding success and breakthroughs For more information visit www qiagen com Contents Kit Contents 4 Storage 6 Quality Control 6 Technical Assistance 7 Safety Information 7 Introduction 8 Equipment and Reagents to Be Supplied by User 22 Important Notes 23 Protocols E Detection of DNA from Specific Plants or Animals by Real Time PCR without ROX 25 E Detection of DNA from Specific Plants or Animals by Real Time PCR with ROX 28 E Analyzing the Results 32 Troubleshooting Guide 34 References 36 Ordering Information 37 SooooOoOo _ U A mericon Plant and Animal Identification Handbook 04 2013 3 Kit Contents mericon Plant and Animal Identification Assays 24 96 See Table 1 for specific kits Number
18. GEN all rights reserved www qiagen com Australia Orders 03 9840 9800 Fax 03 9840 9888 Technical 1 800 243 066 Austria Orders 0800 28 10 10 Fax 0800 28 10 19 Technical 0800 28 10 11 Belgium Orders 0800 79612 Fax 0800 79611 Technical 0800 79556 Brazil Orders 0800 557779 Fax 55 11 5079 4001 Technical 0800 557779 Canada Orders 800 572 9613 Fax 800 713 5951 Technical 800 DNA PREP 800 362 7737 China Orders 021 3865 3865 Fax 021 3865 3965 Technical 800 988 0325 Denmark Orders 80 885945 Fax 80 885944 Technical 80 885942 Finland Orders 0800 914416 Fax 0800 914415 Technical 0800 914413 France Orders 01 60 920 926 Fax 01 60 920 925 Technical 01 60 920 930 Offers 01 60 920 928 Germany Orders 02103 29 12000 Fax 02103 29 22000 Technical 02103 29 12400 Hong Kong Orders 800 933 965 Fax 800 930 439 Technical 800 930 425 Ireland Orders 1800 555 049 Fax 1800 555 048 Technical 1800 555 061 Italy Orders 02 33430 420 Fax 02 33430 426 Technical 800 787980 Japan Telephone 03 6890 7300 Fax 03 5547 0818 Technical 03 6890 7300 Korea South Orders 1544 7145 Fax 1544 7146 Technical 1544 7145 Luxembourg Orders 8002 2076 Fax 8002 2073 Technical 8002 2067 Mexico Orders 01 800 7742 639 Fax 01 800 1122 330 Technical 01 800 7742 639 The Netherlands Orders 0800 0229592 Fax 0800 0229593 Technical 0800 0229602 Norway Orders 800 18859
19. The mericon Soy Kit exhibits high specificity for soy and Roundup Ready soy No cross reactivity was observed with other plant or animal species or genetically modified organisms Table 2 using 50 ng of the tested DNA Table 2 Results from cross reactivity experiment Genetic construct Genetic construct GMO or species Result GMO or species Result Soy Roundup Ready soy Potato Cattle Horse Goat Rye Corn Barley Pig Rice Sheep Canola Wheat Roundup Ready rice CaMV T25 corn CGH351 corn NK603 corn Btl76 corn B 1 1 corn MON 810 corn Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 11 mericon Corn Kit The mericon Corn Kit is designed for the detection of corn in food animal feed and pharmaceutical products Limit of detection The mericon Corn Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Corn Kit exhibits high specificity for corn No cross reactivity was observed with other plant or animal species Table 3 using 50 ng of the tested DNA Table 3 Results from cross reactivity experiment Species Result Species Result Corn Sheep Soy Goat Canola Horse Rice Hazelnut Potato Pecan nut Barley Brazil nut Wheat Almond Rye Musta
20. arately from all other reagents and add them to the reaction mix in a spatially separated facility Thaw all components thoroughly at room temperature 15 25 C before starting an assay E When thawed mix the components by pipetting repeatedly up and down or by pulse vortexing and centrifuge briefly Relevant assay controls Internal control Each vial of mericon Assay contains an internal control to detect possible PCR inhibition Negative PCR control Negative controls should be included in each analysis run to check for possible contamination of the mericon Assay during reaction setup Instead of adding sample DNA to a reaction vial containing Multiplex PCR Master Mix add the same volume of QuantiTect Nucleic Acid Dilution Buffer or RNase free water Positive PCR control Positive controls should be included in each analysis run to check the functionality of the Multiplex PCR Master Mix Instead of adding sample DNA to a reaction vial containing Multiplex PCR Master Mix add the same volume of the positive control included in the kit SoooOo _ gt _ lt _ eee SSS aaa mericon Plant and Animal Identification Handbook 04 2013 23 Internal control calibration for real time cyclers MAX NHS Ester MAX dye is used to detect the internal control of mericon PCR Assays Table 13 lists common thermal cyclers with their calibration requirement and the detection channel or filter
21. col Analyzing the Results on page 32 26 mericon Plant and Animal Identification Handbook 04 2013 Table 15 Cycling protocol for Rotor Gene Q or Rotor Gene 6000 Step Time Temperature Comments ens oO OO Eee 3 step cycling Denaturation 15s JDC S ee a Data collection at 60 C Extension 10s 72 C Number of cycles 45 Detection Reporter Excitation emission Channel Target FAM 495 520 nm Green Internal control MAX 524 557 nm Yellow Table 16 Cycling protocol for real time cyclers other than Rotor Gene Q or Rotor Gene 6000 Step Time Temperature Comments ge ee e 3 step cycling Denaturation 15s JSC hae ae TE oe Data collection at 60 C Extension 10s 722C Number of cycles 45 Detection Reporter Excitation emission Channel Target FAM 495 520 nm Green FAM Internal control MAX 524 557 nm Yellow VIC t For some instruments the shortest annealing time possible is longer than 23 s in the range of 32 s Use the shortest annealing time permitted by the instrument t See Table 13 page 24 for information on instrument specific detection channel or filter set mericon Plant and Animal Identification Handbook 04 2013 27 Protocol Detection of DNA from Specific Plants or Animals by Real Time PCR with ROX For certain real time thermal cyclers the use of a ROX passive reference dye during the PCR is necessary to compensate for variations in the fluorescence signal that are not relat
22. d store the aliquots at 2 8 C for short term storage 1 month or 20 C for long term storage E Dissolve the dried Positive Control DNA tube with red lid Add 200 ul of QuantiTect Nucleic Acid Dilution Buffer to the vial and mix by pipetting up and down 5 times or vortexing Note If the dissolved positive control will not be used entirely in one assay run make appropriate aliquots to avoid more than 5 freeze thaw cycles and store the aliquots at 2 8 C for short term storage 1 month or 20 C for long term storage Before each use all reagents need to be thawed completely mixed by repeated up and down pipetting or by quick vortexing and centrifuged briefly Procedure 1 Set up the sample and control reactions according to Table 18 page 29 depending on the thermal cycler to be used Keep all samples and reaction tubes on ice during setup Table 18 Setup of sample and control reactions for PCR instruments requiring ROX Dye at a low concentration Positive PCR Negative Component Sample control PCR control Reconstituted PEN 10 4 ul 10 4 ul 10 4 ul Sample DNA 9 6 ul Dissolved Positive Control DNA 7 vor j QuantiTect Nucleic Acid Dilution Buffer or 7 z 9 6 pl RNase free water Total volume 20 pl 20 pl 20 pl f e ss mericon Plant and Animal Identification Handbook 04 2013 29 2 Close the PCR tubes or strips and place them in the reaction chamber of the thermal cycler securing them according
23. did not new kit if necessary comply with the instructions given in Storage page 6 e The mericon PCR Assay Check the storage conditions and the expiration has expired date see the kit label of the reagents and use a new kit if necessary 34 mericon Plant and Animal Identification Handbook 04 2013 Comments and suggestions Weak or no signal in the internal amplification control a The PCR conditions do Check that PCR conditions match the cycling not comply with the protocols in Table 15 or Table 16 0n page 27 protocol or Table 19 on page 32 Repeat the PCR with corrected settings if necessary 2 The PCR was inhibited Use the recommended DNA isolation method and closely follow the manufacturer s instructions QIAGEN offers dedicated sample preparation kits developed to complement mericon PCR Assays and provides a complete and efficient workflow for food safety testing c The storage conditions Check the storage conditions and the expiration for one or more kit date see the kit label of the reagents and use a components did not new kit if necessary comply with the instructions given in Storage page 6 The mericon PCR Assay Check the storage conditions and the expiration has expired date see the kit label of the reagents and use a new kit if necessary Signals present for the negative controls a Contamination Repeat the PCR with new reagents in replicates occurred during PCR If possib
24. eactions PCR Assay 290063 spp Kit 24 Cronobacter spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon S aureus Kit For 24 reactions PCR Assay S aureus 290073 24 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Quant For 24 reactions PCR Assay Legionella 290083 Legionella spp Kit 96 spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Quant L For 24 reactions PCR Assay L 290093 pneumophila Kit 96 pneumophila Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Shigella spp Kit For 24 reactions PCR Assay Shigella 290103 24 spp Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Y For 24 reactions PCR Assay Y 290113 enterocolitica Kit 24 enterocolitica Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon sample preparation kits DNeasy mericon Food 50 QlAquick Spin Columns 69514 Kit 50 Proteinase K buffers mericon DNA Bacteria Fast Lysis Buffer 69525 Kit 100 Larger kit sizes available
25. ed 24 hours a day from Poison Information Center Mainz Germany Tel 49 6131 19240 mericon Plant and Animal Identification Handbook 04 2013 7 Introduction mericon Plant and Animal Identification Assays are a ready to use system for the detection of DNA sequences from specific animal or plant material found in food animal feed and pharmaceutical products using real time polymerase chain reaction PCR These assays perform optimally on the Rotor Gene Q but have been validated for block thermal cyclers as well The Multiplex PCR Master Mix included in each kit contains QIAGEN proprietary technology including HotStarTag Plus DNA Polymerase patented multiplex PCR technology such as Factor MP and fast cycling technology including Q Bond Multiplex PCR Master Mix is also highly tolerant to PCR inhibitors Each mericon Assay is an optimized mixture of PCR primer sets for a plant or animal specific target sequence and an internal control IC plus probes labeled with 2 distinct fluorescent dyes the test sample is detected with FAM reporter 495 520 nm and the internal control is detected with MAX NHS Ester reporter MAX 524 557 nm In addition each kit includes positive control DNA and all of the reagents necessary to perform the analysis Principle Detection of plant or animal material by the polymerase chain reaction PCR is based on the amplification of a genetic construct used in GMOs In real time PCR the amplifi
26. ed product is detected via target specitic fluorescent probes that bind to the amplified product Accumulation of PCR product results in an increased fluorescent signal from the bound probes Monitoring the fluorescence intensities during the PCR run i e in real time allows the detection of the accumulating PCR product without having to re open the reaction tubes after the PCR run The probes of mericon PCR Assays are sequence specific oligonucleotides with a fluorophore and a quencher moiety attached The fluorophore is at the 5 end of the probe and the quencher moiety is located at the 3 end If the target DNA sequence is present the probe is cleaved by the 5 gt 3 exonuclease activity of HotStarTagq Plus DNA Polymerase during the extension phase of PCR This separates the fluorophore and the quencher moiety resulting in a detectable fluorescence that is proportional to the amount of accumulated PCR product The PCR primer set for each assay is highly specific and targets a unique and conserved DNA region of the tested plant or animal that is verified bioinformatically and experimentally Cross reactivity has been bioinformatically investigated and thoroughly tested with a panel of selected targets for each mericon PCR Assay Each assay can detect down to 10 target copies in a reaction 8 mericon Plant and Animal Identification Handbook 04 2013 Dedicated mericon sample preparation solutions are available from QIAGEN for a broad range
27. ed to the PCR Important points before starting E Before beginning the procedure read Important Notes page 23 E Take time to familiarize yourself with the real time PCR instrument to be used before starting the protocol See the instrument user manual E Make sure that at least one positive and one negative control are included per PCR run Things to do before starting E Prepare the mericon Assay tube with yellow lid by adding Multiplex PCR Master Mix tube with blue lid and ROX dye to the tube s of mericon Assay according to Table 17 Mix by pipetting up and down 5 times or vortexing and centrifuge briefly Note The 50x ROX Dye solution provided is suitable for PCR instruments requiring a low ROX Dye concentration Table 20 For PCR instruments requiring a high ROX Dye concentration please contact QIAGEN Technical Service Table 17 Components to add to the mericon Assay Kit Multiplex PCR Thermal cycler size ROX dye Master Mix Applied Biosystems 24 5 4 ul per vial 130 ul per vial model 7500 and 7500 Rotor Gene models Stratagene Mx models LightCycler 480 ROX reference dye not necessary SmartCycler models Bio Rad instruments EEE a SSSSSSS gt yEyy gt EEE gt yeyxyeEEy oy 28 mericon Plant and Animal Identification Handbook 04 2013 Note If the reconstituted mericon Assay will not be used entirely in one assay run make appropriate aliquots to avoid more than 5 freeze thaw cycles an
28. es and reaction tubes on ice during setup If using the Rotor Gene Q place the desired number of PCR tubes or strips into the adapters of the cooling block for the Rotor Gene Q mericon Plant and Animal Identification Handbook 04 2013 25 Table 14 Setup of sample and control reactions Positive PCR Negative PCR Component Sample control contre Reconstituted mericon Assay 10 pl 10 ul 10 ul Sample DNA 10 ul z 5 Dissolved Positive Control DNA 10 ul z QuantiTect Nucleic Acid Dilution Buffer 10 ul or RNase free water Total volume 20 pl 20 pl 20 pl 2 Close the PCR tubes or strips and place them in the reaction chamber of the thermal cycler securing them according to the instrument manual If using the Rotor Gene Q make sure that the locking ring is placed on top of the rotor to prevent accidental opening of the tubes during the run 3 Program the thermal cycler If using the Rotor Gene Q or Rotor Gene 6000 use the cycling protocol in Table 15 page 27 For all other real time cyclers use the cycling protocol in Table 16 page 27 Note For information on instrument detection settings for the MAX NHS Ester dye MAX used to detect the internal control of mericon Assays see Table 17 on page 28 4 For the Rotor Gene Q or Rotor Gene 6000 make sure that Perform Optimisation Before 1st Acquisition in the Gain Optimisation menu is activated 5 Start the PCR run 6 Proceed to the proto
29. final point on the fluorescence curve that lies clearly above the threshold Figures 1 3 are examples of possible outcomes which are summarized in Table 20 page 21 10 oa Green channel tn Yellow channel o gt o D N o O O t B o o rz oS nN Normalized fluorescence Normalized fluorescence t 2 Ezd D 5 10 15 25 30 35 40 5 10 15 25 30 35 40 20 Cycle Cycle Figure 1 The sample is negative for the tested plant or animal The 3 sample curves in the green channel left are at the baseline and below a preset threshold The corresponding curves of the internal control in the yellow channel right are above the threshold indicating that the PCR was successful a 6 O tn Green channel Yellow channel Y 7 3 a D S ny a w Y 3 4 W g Normalized fluorescence Normalized fluorescence E 25 30 35 40 20 Cycle Cycle Figure 2 The sample is positive for the tested plant or animal The 3 sample curves in the green channel left and the corresponding curves of the internal control in the yellow channel right are above a preset threshold indicating the presence of target DNA in the sample and a successful PCR SSS 32 mericon Plant and Animal Identification Handbook 04 2013 ro o th 3 33 Green channel 241 Yellow channel scence tn w ormalized fluore
30. gative control are included per PCR run Things to do before starting Prepare the mericon Assay tube with yellow lid 24 sample kit Add 130 ul Multiplex PCR Master Mix tube with blue lid to each vial of mericon Assay yellow lid Mix by pipetting up and down 5 times or vortexing and centrifuge briefly 96 sample kit Add 1040 ul Multiplex PCR Master Mix tube with blue lid to the vial of mericon Assay yellow lid Mix by pipetting up and down 5 times or vortexing and centrifuge briefly Note If the reconstituted mericon Assay will not be used entirely in one assay run make appropriate aliquots to avoid more than 5 freeze thaw cycles and store the aliquots at 2 8 C for short term storage 1 month or 20 C for long term storage Dissolve the dried Positive Control DNA tube with red lid Add 200 ul of QuantiTect Nucleic Acid Dilution Buffer to the vial and mix by pipetting up and down 5 times or vortexing Centrifuge briefly Note If the dissolved positive control will not be used entirely in one assay run make appropriate aliquots to avoid more than 5 freeze thaw cycles and store the aliquots at 2 8 C for short term storage 1 month or 20 C for long term storage Before each use all reagents need to be thawed completely mixed by repeated up and down pipetting or by quick vortexing and centrifuged briefly Procedure l Set up the sample and control reactions according toTable 14 page 26 Keep all sampl
31. iTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Goat Kit 24 For 24 reactions PCR Assay Turkey 292053 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Horse Kit 24 For 24 reactions PCR Assay Turkey 292143 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution Related products mericon GMO Detection Assays mericon Screen 35S Kit For 24 reactions PCR Assay Screen 291013 24 35S Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Screen Nos Kit For 24 reactions PCR Assay Screen 291043 24 Nos Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution Larger kit sizes available please inquire 38 mericon Plant and Animal Identification Handbook 04 2013 Product mericon RR Soy 24 mericon GMO Screen 35S pat 24 mericon GMO Screen CTP2 CP4EPSPS 24 mericon GMO Screen bar 24 mericon Mon 810 Corn 24 Contents For 24 reactions PCR Assay RR Soy Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution For 24 reactions PCR Assay GMO Screen 35S pat Positive Control Multiplex PCR Master
32. le close the PCR tubes directly after SeIUP addition of the sample to be tested Make sure to pipet the positive controls last Make sure that the work space and instruments are decontaminated at regular intervals b Contamination Repeat the extraction and PCR of the sample to occurred during be tested using new reagents extraction Make sure that the work space and instruments are decontaminated at regular intervals mericon Plant and Animal Identification Handbook 04 2013 35 References QIAGEN maintains a large up to date online database of scientific publications utilizing QIAGEN products Comprehensive search options allow you to find the articles you need either by a simple keyword search or by specifying the application research area title etc For a complete list of references visit the QIAGEN Reference Database online at www qgiagen com RefDB search asp or contact QIAGEN Technical Services or your local distributor p E e r T 36 mericon Plant and Animal Identification Handbook 04 2013 Ordering Information Product Contents mericon Soy Kit 24 For 24 reactions PCR Assay Soy Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Corn Kit 24 For 24 reactions PCR Assay Corn Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Apricot Kernels F
33. nd in animal feed Limit of detection The mericon Pig Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Pig Kit exhibits high specificity for domesticated pig and wild boar No cross reactivity was observed with other animal species Table 5 using 50 ng of the tested DNA Table 5 Results from cross reactivity experiment Species Result Species Result Domesticated pig Chicken Wild boar Sheep Turkey Goat Duck Cattle Goose Horse Deer Ostrich Kangaroo Reindeer Moose Cod Salmon Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens 14 mericon Plant and Animal Identification Handbook 04 2013 mericon Cattle Kit The mericon Cattle Kit is designed for the detection of cattle matter excluding water buffalo in food and in animal feed Limit of detection The mericon Cattle Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Cattle Kit exhibits high specificity for cattle No cross reactivity was observed with other plant or animal species Table 6 using 50 ng of the tested DNA Table 6 Results from cross reactivity experiment Species Result Species Result Cattle Deer Water buffalo Cod Turkey Salmon Chicken Rabbit Goose Corn Duck S Soy Pig Rice Sheep Wheat Goat Potato Horse
34. or 24 reactions PCR Assay Apricot Kit 24 Kernels Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Pig Kit 24 For 24 reactions PCR Assay Pig Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Cattle Kit 24 For 24 reactions PCR Assay Cattle Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Chicken Kit For 24 reactions PCR Assay Chicken 24 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Turkey Kit 24 For 24 reactions PCR Assay Turkey Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution Larger kit sizes available please inquire mericon Plant and Animal Identification Handbook 04 2013 Cat no 293013 293023 293033 292013 292023 292033 292043 37 Product Contents Cat no mericon Ruminant Kit For 24 reactions PCR Assay Turkey 292073 24 Positive Control Multiplex PCR Master Mix QuantiTect Nucleic Acid Dilution Buffer RNase Free Water 50x ROX Dye Solution mericon Sheep Kit 24 For 24 reactions PCR Assay Turkey 292063 Positive Control Multiplex PCR Master Mix Quant
35. rd Cattle Lupine Pig Cross reactivity experiments are ongoing For up to date information visit www qiagen com mericonPathogens 12 mericon Plant and Animal Identification Handbook 04 2013 mericon Apricot Kernels Kit The mericon Apricot Kernels Kit is designed for the detection of apricot kernels in food animal feed and pharmaceutical products Limit of detection The mericon Apricot Kernels Kit can detect down to 10 copies of target DNA in a reaction Specificity The mericon Apricot Kernels Kit exhibits high specificity for apricot No cross reactivity was observed with other plant or animal species Table 4 using 50 ng of the tested DNA Table 4 Results from cross reactivity experiment Species Result Species Result Apricot South Africa Apricot China sweet Apricot Turkey Apricot Iran F Apricot Greece Apricot Syria Almond Potato Peach Barley Plum Wheat Rice Rye Cherry Cattle Hazelnut Pig Peanut Sheep Walnut Goat Pecan nut Horse Corn Celery Canola Mustard Soy Lupine Cross reactivity experiments are ongoing For up to date information visit www qgiagen com mericonPathogens mericon Plant and Animal Identification Handbook 04 2013 13 mericon Pig Kit The mericon Pig Kit is designed for the detection of domesticated pig Sus scrofa domestica and wild boar Sus scrofa matter in food a
36. roduct Warranty and Satisfaction Guarantee QIAGEN guarantees the performance of all products in the manner described in our product literature The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse QIAGEN will replace it free of charge or refund the purchase price We reserve the right to change alter or modify any product to enhance its performance and design If a QIAGEN product does not meet your expectations simply call your local Technical Service Department or distributor We will credit your account or exchange the product as you wish Separate conditions apply to QIAGEN scientific instruments service products and to products shipped on dry ice Please inquire for more information A copy of QIAGEN terms and conditions can be obtained on request and is also provided on the back of our invoices If you have questions about product specifications or performance please call QIAGEN Technical Services or your local distributor see back cover or visit www qiagen com Quality Control In accordance with QIAGEN s ISO certified Quality Management System each lot of mericon Plant and Animal Identification Assays is tested against predetermined specifications to ensure consistent product quality 6 mericon Plant and Animal Identification Handbook 04 2013 Technical Assistance At QIAGEN we pride ourselves on the
37. s multiplex PCR This synthetic factor increases the local concentration of primers and probes at the DNA template and stabilizes specifically bound primers and probes allowing efficient annealing and extension In addition the Multiplex PCR Buffer is carefully formulated to be highly tolerant to inhibitors commonly present in food QuantiTect Nucleic Acid Dilution Buffer QuantiTect Nucleic Acid Dilution Buffer is an optimized solution to dilute the nucleic acids used as positive controls for mericon PCR Assays The buffer stabilizes DNA controls during dilution and reaction setup and prevents loss of nucleic acids on plastic surfaces such as tubes or pipet tips The buffer is ready to use and is free of DNases Proper use of the buffer enables safe and accurate dilution of the small amounts of nucleic acids typically used as controls for analysis of nucleic acids Aliquots of diluted positive control can be stored in QuantiTect Nucleic Acid Dilution Buffer at 15 to 30 C for up to 6 months Repeated freezing and thawing should be avoided mericon Plant and Animal Identification Handbook 04 2013 9 ROX Dye Solution 50x For certain real time cyclers the presence of ROX passive reference dye in real time PCR compensates for non PCR related variations in fluorescence detection The use of ROX dye is necessary for most instruments from Applied Biosystems and is optional for Stratagene cyclers from Agilent ROX dye is not necessary for
38. set for this dye Refer to the manufacturer s manual of the thermal cycler to be used for detailed calibration instructions Table 13 Calibration requirements and detection channel for MAX NHS Ester MAX dye Filter suitable for MAX NHS Ester Thermal cycler Dye calibration detection Rotor Gene Q Not required Yellow Rotor Gene 6000 Applied Biosystems models 7000 7300 7500 7500 Required for new VIC Fast 7700 7900HT instrumentst StepOne StepOnePlus Filter set 535 550 nm HEX JOE VIC Stratagene Agilent models Mx3005P Mx3000P Not required For information on detection channel settings for instruments not listed in Table 13 contact QIAGEN Technical Services t If the instrument is new a dye calibration for the individual channels e g VIC of the real time cycler must be performed See the manufacturer s manual for details on calibration EEEEEEEE gt E gt yxy gt eeeye lll 24 mericon Plant and Animal Identification Handbook 04 2013 Protocol Detection of DNA from Specific Plants or Animals by Real Time PCR without ROX Important points before starting Before beginning the procedure read Important Notes page 23 Take time to familiarize yourself with the Rotor Gene Q or other real time PCR instrument to be used before starting the protocol See the instrument user manual Make sure that at least one positive and one ne
39. so the Frequently Asked Questions page at our Technical Support Center www qiagen com FAQ FAQList aspx The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or sample and assay technologies for contact information see back cover or visit www giagen com Comments and suggestions No signal with positive control a The selected For data analysis select the green channel FAM fluorescence channel for the samples and the yellow channel VIC or for PCR data analysis corresponding filter set for the internal control does not comply with See Table 15 or Table 16 on page 27 or Table the protocol 19 on page 31 For Applied Biosystems software the channel for the internal control is specified during calibration with MAX NHS Ester dye Refer to the manufacturer s manual of the cycler to be used 2 Incorrect programming Compare the temperature profile with the of the real time PCR protocol See Table 15 or Table 16 0n page 27 instrument or Table 19 on page 31 Refer to the manufacturer s manual of the cycler to be used c Incorrect configuration Check that reactions were set up according to of the PCR Table 14 page 26 or Table 18 page 29 Repeat the PCR if necessary d The storage conditions Check the storage conditions and the expiration for one or more kit date see the kit label of the reagents and use a components
40. the Rotor Gene Q LightCycler systems from Roche SmartCycler instruments from Cepheid and Bio Rad instruments ROX dye is provided in a 50x solution suitable for PCR instruments requiring a low ROX Dye concentration such as the Applied Biosystems models 7500 and 7500Fast Instructions for using the dye are provided in the protocol Detection of GMO DNA by real time PCR with ROX on page 28 Primer probe mix with internal control Each mericon PCR Assay includes rigorously designed primers and probes in a carefully balanced mix that amplify a target sequence and an internal control IC with high specificity This internal control provides information regarding the presence of inhibitors in the tested samples and the overall success of the PCR MAX NHS Ester is employed as the reporter dye for the internal control With excitation emission maxima of 524 557 nm and a non fluorescent quencher lowa Black the MAX dye has a spectral profile comparable to HEX JOE or VIC and can be used with most real time cyclers EE EEESE gt gt y gt y gt y gt y gt E gt yEyEyEeEEyEeyeyE ee 10 mericon Plant and Animal Identification Handbook 04 2013 Assay specific information mericon Soy Kit The mericon Soy Kit is designed for the detection of soy Glycine max in food animal feed and pharmaceutical products Limit of detection The mericon Soy Kit can detect down to 10 copies of target DNA in a reaction Specificity
41. to the instrument manual 3 Program the real time cycler according to Table 19 page 31 Note For information on instrument detection settings for the MAX NHS Ester dye MAX used to detect the internal control of mericon Assays see Table 13 on page 24 4 Start the PCR run 5 Proceed to the protocol Analyzing the Results on page 32 30 mericon Plant and Animal Identification Handbook 04 2013 Table 19 Cycling protocol for real time cyclers using ROX reference dye Step Time Temperature Comments ens oO OO A a 3 step cycling Denaturation 15s JDC reai Ana a Data collection at 60 C Extension 10s 722C Number of cycles 45 Detection Reporter Emission maximum Channel Target FAM 520 nm Green FAM Internal control MAX 560 nm Yellow VIC For some instruments the shortest annealing time possible is longer than 23 s in the range of 32 s Use the shortest annealing time permitted by the instrument t See Table 13 page 24 for information on instrument specific detection channel or filter set mericon Plant and Animal Identification Handbook 04 2013 31 Protocol Analyzing the Results Determining the presence or absence of DNA from genetically moditied organisms GMO is carried out based on the amplification of the target sequence and is visualized in real time on the amplification plot generated by the application software of the real time PCR instrument used A positive result is visible as a
Download Pdf Manuals
Related Search