CarnoCheck® Instructions For Use
Contents
1. READ BARCODE Damaged barcode Chip was not loaded correctly ERROR MESSAGE MISSING SPOTS PRINTING CONTROL FAILED OR ORIENTATION CONTROL FAILED Dust on the chip Formation of air bubbles during transfer of liquid on the chip HYBRIDISATION CONTROL FAILED Incorrect temperature of washing solution Il Incorrect temperature of water bath Wrong preparation of hybridisation mix PCR CONTROL FAILED No addition of AmpliTaq Gold to the MasterMix Addition of a not proper func tioning AmpliTaq Gold to the MasterMix Insufficient mixing of reaction mix PCR inhibitors are present in the sample Hybridisation was performed without addition of PCR product Insufficient mixing of Hybridisation Mix CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Comments and suggestions Check barcode for damage Enter the barcode manually when the appropriate window appears Check chip orientation and scan the chip in correct orientation Repeat hybridisation of PCR product s on another chip Repeat hybridisation of PCR product s on another chip Pipette carefully to avoid air bubble formation The second washing step must be performed at 50 C Ensure that the washing solution Il is heated to 50 C The second washing step must be performed at 50 C Check the temperature of the water bath Ensure that the water bath is set to a temperature of 50 C If necessary confirm te2. E Geen HO EE EE EE E EE Food DNA Chip CarnoCheck Instructions For Use Parallel qualitative detection of 8 animal species constituents in meat food products REF 462 030 For professional users only Revision BQ 020 01 February 2013 HUBERLAB committed to science g reiner bio one GLOSSARY OF SYMBOLS en de fr es pt ni da SV pl no el tr Store in the dark Im Dunkeln lagern stocker l abri de la lumi re Conser var en un lugar oscuro Conserva re al buio Conservar num local escuro Donker bewaren Opbeva res m rkt F rvaras m rkt Przechow ywa C w ciemnosci Oppbeva res m rkt ATTroQnNkeVeTaI OTA OKOTEIV Karanlik yerde saklayiniz Use by Mindes tens haltbar bis Date limite de conser vation jusqu au Autilizar preferib lemente antes de Da utilizzare entro e non oltre Autilizar prefer velmente antes de Tenminste houdbaar tot Anvendes senest Sista f r bruknings dag Termin zydatnosci holdbar til TO AIVOTEPO diatnpeitai Son kullanma tarihi Consult Instruc tions For Use Vor Gebrauch Anwei sung lesen Lire les in structions avant utilisation Antes de usar lea las instruccio nes Leggere le istruzi oni prima dell uso Antes de usar leia as inst rucdes Gebruik saanwij zing lezen Lees brugsan visningen Las bruk sanvisn
3. while the quality of the hybridisation and the chip hybridisation orientation and printing control is assessed in the green channel excitation wavelength of 532 nm PCR control CarnoCheck monitors the quality of the PCR reaction Amplification of an internal control template present in the CarnoCheck PCR MasterMix generates a signal on the PCR control spots on the CarnoCheck chip The quality of the amplification reaction is also automatically assessed by the CheckReport Software If PCR performance is below a predefined threshold the CheckReport Software will indicate the PCR control as failed and the analysis must be repeated see Chapter 9 If the amount of animal specific DNA in the sample is very high the fluorescence signal of the PCR control spots may be low or even absent due to competition during the PCR reaction In this case the fluorescence signal for at least one animal specific probe must exceed a predefined threshold in order for the test to be considered valid Hybridisation control CarnoCheck monitors the efficiency of the hybridisation through use of a fluorescence labeled probe within the CarnoCheck Hybridisation Buffer which hybridises to specific DNA sequences on the CarnoCheck chip An adequate hybridisation efficiency results in fluorescence signals on each array spot The results of five hybridisation control spots on the CarnoCheck chip are also assessed by the CheckReport Software
4. CarnoCheck Buffer A conc CarnoCheck Buffer B conc Flyer CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Description Slidebox with 5 CarnoCheck chips imprinted with 6 CarnoCheck arrays in wells A1 A6 PCR MasterMix contains all components required for PCR except Taq Polymerase Hybridization Buffer concentrate of Buffer A concentrate of Buffer B Download instructions for Instructions For Use Quantity 5 x 6 arrays 2 x 450 ul 1000 ul 40 ml 15 ml 1 2 CONSUMABLES EQUIPMENT AND HARDWARE REQUIRED Consumables Greiner Bio One Cat No Quantity pieces CarnoCheck test kit 462 030 30 reactions Sterile DNase free micropipette filter tips 0 5 10 ul filter tips 765 288 0 5 20 ul filter tips 774 288 10 100 ul filter tips 772 288 10 200 ul filter tips 739 288 100 1000 ul filter tips 750 288 DNase free reaction tubes Reaction tube 1 5 ml 616 201 Reaction tube 0 2 ml 683 201 8 x 0 2 ml PCR strips 673210 Cap strips for 8 x 0 2 ml PCR strips 373 270 Plastic pipettes for pipettor Pipette 10 ml 607 180 or 607 160 Pipette 50 ml 768 180 or 768 160 50 ml polypropylene tubes optional 210 261 1 Some of the mentioned tip sizes are optional depending on the micropipettes available 2 In principle it is recommended to use 8 tube PCR strips Single reaction tubes 0 2 ml are optional if strips are not available 3 Only required if a slide centrifuge is no
5. Orientation and printing control The orientation control spots of the CarnoCheck chip generate fluorescence signals irrespective of the efficiency of the hybridisation process These spots are used by the CheckReport Software as guidance points for a correct spot finding which is a prerequisite for the correct analysis of the signals In addition the quality of the printing process is monitored by the presence of a green fluorescence signal at each chip spot printing control CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 7 INSTRUCTIONS FOR THE CARNOCHECK WORKFLOW 7 1 General instructions When implementing currently used state of the art techniques in molecular biology into a laboratory the following instructions must be considered to ensure both maximum safety for laboratory staff and high quality results Execution of molecular biology techniques such as DNA extraction amplification and detection of the amplification products require appropriately qualified personnel In addition a clean and well structured workflow is required to prevent erroneous results such as those occurring due to DNA degradation or contamination by amplification products To ensure this it is necessary to separate the areas of extraction amplification and detection as described in Chapter 7 2 Each area should be equipped with separate equipment consumables lab coats and gloves Never transfer lab coats gloves or equipment f
6. PCR MasterMix already contains all components necessary for performing the PCR reaction PCR buffer MgCl2 dNTPs primers PCR control template The set up of the reaction mix is optimally performed in a protected surrounding e g a PCR hood to avoid reaction contamination mg Prepare the reaction mix consisting of CarnoCheck PCR MasterMix AmpliTaq Gold DNA Polymerase for the required quantity of PCR reactions as outlined in Table 3 Table 3 Set up of the CarnoCheck PCR reaction 1 reaction 7 reactions 1 chip CarnoCheck PCR MasterMix 166 6 yl AmpliTaq Gold DNA Polymerase 5 U ul DNA extract from original sample Total volume per reaction To analyse multiple samples the reaction mix should be prepared in a batch in the quantity required for all analyses To adjust for volume variations during pipetting it is recommended to increase the number of reactions n by 1 for each chip n 1 e g prepare a reaction mix volume for 7 amplification reactions if 6 samples are to be tested see Table 3 Always use one vial of MasterMix for the reactions of one chip Use a precise low volume 0 5 10 ul pipette for pipetting the Taq DNA Polymerase It is recommended to include a negative PCR control for every batch of CarnoCheck PCR reaction mix prepared As negative PCR control the DNA elution buffer of the appropriate DNA extraction kit or PCR grade water may be used i Mix the reaction mix thoroughl
7. Lab coats must be worn throughout the procedure and different sets of lab coats are required for each laboratory room e Gloves must be worn during each step of the analysis and must be frequently changed especially during DNA extraction e The working place must be decontaminated with an appropriate cleaning solution e Never touch the inside of a reaction tube cap To avoid cross contamination open only one tube at a time e Appropriate micropipette filter tips with aerosol barrier must be used free of DNase RNase and human DNA Pipette tips should always be changed between liquid transfers 7 3 2 Instruction for handling DNA chips e DNA chips should be used in a dust free environment The deposition of dust and other particles on the chip surface must be prevented e Do not touch the hybridisation zone on the chip surface e Only the labelled side of the chip is intended for hybridisation e Do not use any marker pens for the identification of DNA chips as they lead to unspecific fluorescence on the chip e DNA arrays are for single use only Hybridised chips cannot be reused e Store unused chips in the original box inside the delivered zipper bag containing the desiccant 7 3 3 General precautions e This kit is exclusively for the qualitative detection of animal constituents in meat products and should only be used by personnel trained in molecular biology laboratory practice e Upon arrival check the kit components fo
8. O O OO OOOO SOCIO I I I I 0000000000 0000000000 0000000000 0000000000 0000000000 0000000000 Figure 2 Design of the CarnoCheck chip a Schematic drawing of the CarnoCheck chip Each CarnoCheck chip contains 6 microarrays designated as well A1 A6 b and c Images displayed by the CheckReport Software for the two different excitation wavelengths used for scanning b red channel 635 nm c green channel 532 nm and schematic drawings of the CarnoCheck microarray layout Species specific probes and on chip controls are indicated CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 horse goat donkey cattle sheep turkey horse goat donkey cattle sheep turkey 6 3 2 On chip controls The design of the CarnoCheck DNA chip incorporates comprehensive on chip controls Several control systems monitor critical steps of both the assay and chip processing including quality of the PCR reaction PCR control the efficiency of the hybridisation hybridisation control as well as spot homogeneity and printing quality orientation control and printing control The CheckReport Software automatically shows both the corresponding values of the controls and the detected animal species in a detailed report For read out of the different controls both excitation wavelengths of the CheckScanner are used For the control of PCR performance PCR control the red channel is used excitation wavelength of 635 nm
9. of the PCR PCR control The PCR products are then hybridised to specific DNA probes and on chip controls attached to the CarnoCheck chip surface Every chip contains 6 DNA microarrays in wells A1 A6 allowing the simultaneous analysis of 6 meat samples Unbound DNA is removed in the subsequent washing steps The hybridisation efficiency is monitored hybridisation control Finally the CarnoCheck chip is automatically scanned analysed and evaluated using the CheckScanner and CheckReport Software respectively see ordering information in Chapter 2 The CheckScanner is a two colour laser scanner excitation wavelengths of 532 nm and 635 nm which enables the detection of the fluorescence signal generated by the presence of cyt b specific amplification products as well as the controls see chapter 6 3 2 The CheckReport Software allows the visualisation analysis and evaluation of the results and automatically shows the corresponding values of both the detected animal species and the controls in a detailed report The report clearly indicates the presence or absence of one or more of the 8 animal species detectable and the comprehensive on chip controls render the analysis highly reliable Table 2 The test is based on species specific differences of cyt b genes from Locus Accession Numbers LOCUS ACCESSION ORGANISM SPECIES Source http www ncbi nim nih gov nuccore CarnoCheck Instructions For Use Revision BQ 020 0
10. of this aliquot without homogenisation Is sufficient Commercial homogenisers for example laboratory blenders such as the stomacher Circulator or bead mills can be used Avoid cross contamination of samples by using disposable homogenisation equipment CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Chapter 8 1 2 DNA extraction MACHERY NACE NucleoSpin Chapter 8 2 PCR Chapter 8 3 2 Hybridisation Chapter 8 3 3 Washing drying Chapter 8 4 Scanning amp evaluation Figure 4 Overview of the different CarnoCheck working steps CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 21 8 1 3 DNA extraction Within the oCheck workflow DNA extraction is the process most sensitive to contamination and so strict adherence to the contamination prevention guidelines outlined in chapter 7 is essential CarnoCheck has been validated using DNA prepared with the following DNA extraction kit and protocol e NucleoSpin Food Macherey Nagel D ren Germany User Manual Protocol for genomic DNA purification from food Please follow the protocol provided by the DNA extraction kit s manufacturer Start the DNA extraction from 100 mg of a homogenous sample Depending on the individual sample complete lysis of the sample can take 1 3 hours or overnight If the sample is not completely lysed after a long incubation time take off the supernatant fo
11. reaction tube of a PCR strip with 13 ul of the PCR product Mix thoroughly m Transfer 30 ul of the hybridisation mix into each well A1 A6 of the CarnoCheck chip using a multichannel pipette mg Close the Hybridisation Chamber and incubate the CarnoCheck chip for exactly 20 minutes at room temperature 20 25 C Figure 5 Working steps of the hybridisation procedure CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 29 8 3 3 Washing and drying Special equipment supplied by Greiner Bio One enables the parallel washing of up to four CarnoCheck chips see Chapter 2 The additional equipment required for processing the CarnoCheck chips is comprised of two oCheck WashBoxes and a handle for the magnetic slide holder of the Hybridisation Chamber The different working steps are shown in Figure 6 II II II IT DI II Carefully remove the magnetic slideholder containing the hybridised slides from the Hybridisation Chamber Drop the slideholder containing the slides directly into the oCheck WashBox containing washing solution Ensure that the magnetic side is facing up Attach the oCheck Handle to the slideholder and begin the first of three washing steps Wash the chip at room temperature 20 25 C in washing solution by moving it quickly up and down for 20 seconds The arrays must stay covered with washing solution at all times Wash the chip for 20 seconds in washing s
12. the needed volumes for one chip by the number of processed chips see Table 4 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Preparation of washing solutions I and Il for one CarnoCheck chip mg Washing solution I Mix 60 ml double distilled water with 6 ml Buffer A and 750 ul Buffer B and fill it into the WashBox mg Washing solution II Mix 73 5 ml double distilled water with 900 ul Buffer A and 600 ul Buffer B and fill it into the other WashBox washing solution Number of CarnoCheck chips Distilled deionised water 60 ml 120 ml 180 ml 240 ml 1 2 3 4 CarnoCheck Buffer A 6 ml 12 ml 18 ml 24 ml CarnoCheck Buffer B 0 75 ml 1 5 ml 2 25 ml 3 ml total volume 66 75 ml 133 5 ml 200 25 ml 267 ml washing solution Il Number of CarnoCheck 1 2 3 Distilled deionised water 73 5 ml 147 ml 220 5 ml 294 ml CarnoCheck Buffer A 0 9 ml 1 8 ml 2 ml 3 6 ml CarnoCheck Buffer B 0 6 ml 1 2 ml 1 8 ml 2 4 ml total volume 75 ml 150 ml 225 ml 300 ml Label them as washing solution and Il Each oCheck WashBox contains an engraved scale indicating the correct amount of washing solution needed for up to 4 chips Please use this scale to check the buffer quantity mg Preheat washing solution II to 50 C in a temperature controlled water bath for at least 20 minutes prior to use Ensure that the fill level of the water bath equals the fill level of the washing solution ll Never reuse the washing so
13. water for several minutes Remove contact lenses if present and easy to do Continue rinsing Supplemental Hazard Statements None according to European Directive 67 548 EEC Hazard symbols Harmful Risk phrases R21 22 Harmful in contact with skin and if swallowed R36 37 38 Irritating to eyes respiratory system and skin Safety phrases 526 In case of contact with eyes rinse immediately with plenty of water and seek medical advice S36 37 Wear suitable protective clothing and gloves The current version of the Safety Data Sheet for this product can be downloaded from the Greiner Bio One website www gbo com bioscience biochips_ download Regulatory Information According to regulation EC No 1272 2008 CLP inner packages must be only labelled with symbol s and product identificator According to EU directives 67 548 EEC or 1999 45 EC Special permission has been granted in accordance with EU Directive 88 379 Article 9 which permits small packages of up to 125 ml or g containing materials that are harmful to health or irritant to be unlabelled with the hazard identification information given in the safety data sheet CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 9 WASTE DISPOSAL After washing and drying of the CarnoCheck chip the washing solutions I and II can be discarded without any special precautions Dispose the used CarnoCheck chip unused kit components as well as unused hybridisation mix
14. 1 February 2013 1 PCR reaction TTT MAA 2 Hybridisation 3 Washing and drying ges DIEU hal l t Figure 1 CarnoCheck assay procedure 1 PCR reaction After DNA extraction a 389 bp fragment of the cyt B gene and a control target are amplified by PCR and fluorescently labeled The amplification products are then hybridised to complementary DNA probes on the chip 2 Hybridisation Each species is detected by a specific DNA probe present in five replicates 3 Washing amp drying Unbound DNA is removed in the subsequent washing steps 4 Scanning amp analysis The CarnoCheck chip is scanned analysed and evaluated using the CheckScanner and CheckReport Software A report is created that clearly indicates the presence or absence of one or more of the animal species detectable 14 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 6 3 6 3 1 Design of the CarnoCheck DNA chip CarnoCheck chip layout Each CarnoCheck chip contains 6 microarrays designated as well A1 A6 Each CarnoCheck microarray comprises 12 different probes and is bordered by an elevated rim Each probe is spotted in five replicates The CarnoCheck microarray layout is illustrated in Figure 2 and the on chip controls are further explained in Chapter 6 3 2 E LO e ak o E te O VG D ho green channel 532 nm QOOOO OOOO OOOO COCO COCA I I I I OOOO OOOO
15. NA Polymerase from Applied Biosystems see ordering information in Chapter 2 It is mandatory to use this enzyme in order to achieve the established performance 8 2 1 Thermal cycler set up The CarnoCheck test kit has been validated with the following thermal cyclers e GeneAmp PCR system 9700 Applied Biosystems e Veriti 96 Well Thermal Cycler Applied Biosystems It is absolutely necessary to use one of the thermal cyclers mentioned above in order to achieve the established performance The thermal cycler program of the CarnoCheck PCR is summarised in Table 2 Table 2 Thermal cycler program of the CarnoCheck PCR No of cycles In addition the following run parameters must be set for each thermal cycler For a description on how to set these parameters see the Instructions For Use of the respective thermal cycler GeneAmp PCR system 9700 Applied Biosystems Set the reaction volume to 25 ul the ramp speed to 9600 and use lid temperature of 103 C Veriti 96 Well Thermal Cycler Applied Biosystems Use the Convert Method tool of the Veriti 96 Well Thermal Cycler to enter the CarnoCheck PCR program and choose the 9600 Emulation Mode Set the reaction volume to 25 ul and the temperature of the lid to 103 C 23 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 8 2 2 PCR reaction set up With the exception of the AmpliTaq Gold DNA Polymerase the CarnoCheck
16. ave any questions or experience any difficulties concerning oCheck products please do not hesitate to contact your local Greiner Bio One distributor 34 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 11 PERFORMANCE CHARACTERISTICS OF CARNOCHECK The CarnoCheck was tested for the specificity and sensitivity of the analysis of meat products As reference material sausages with known composition see table 1 were obtained from the Federal Research Centre for Nutrition and Food in Germany BfEL Kulmbach Two different grades of preservation were tested cooked preserves F lt 0 9 and full preserves F 3 4 Table 4 Composition of reference material sausages Sample Cattle Pig Chicken Turkey Sheep Duck Horse product g product meat product g product meat product g product meat product g product meat product g product meat product g product meat product g product meat product percentage of meat fractions in total product g product weight proportion of meat fractions in total product meat percentage of meat fractions in total meat Detection of duck species is not the intended use of CarnoCheck there is no duck specific probe included the chicken probe might show cross hybridisation to some but not all duck species Composition of the tested saussages 50 meat 25 plant oil 23 ice 1 5 nitrit pickling salt 0 25 sp
17. cken or beef CarnoCheck determined the presence of lamb and pork and absence of beef and chicken or other contaminants So CarnoCheck participated successfully at the FAPAS Proficiency Test 2911 and 2918 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 12 CARNOCHECK SHORT PROTOCOL 12 1 Room 2 PCR Set up of reaction mix Sample collection homogenisation and DNA extraction have to be performed in a separate room and are not part of this short protocol Prepare the reaction mix for the required quantity of PCR reactions m Mix the PCR MasterMix carefully before pipetting 1 reaction 7 reactions 1 chip CarnoCheck PCR MasterMix 166 6 yl AmpliTaq Gold DNA Polymerase 5 U ul Total volume m Mix the reaction mix carefully m Aliquot the reaction mix add 24 ul of the reaction mix for each PCR reaction into a 0 2 ml PCR reaction tube of a PCR strip CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 le ffe fl Bn O ff ffe 12 2 Room 2 PCR DNA template addition PCR reaction mg Add 1 ul of DNA template for each PCR reaction m Mix thoroughly Start the PCR reaction with the prepared thermal cycler program No of cycles 1 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 sj ef A le ffe ffe Ba le fk y Begin preparation at least 30 minutes prior
18. d DNA extraction rnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnunnn 20 o O NE EE 20 A o Ne EE 20 Sid DNA ai cocaina 22 8 2 Polymerase chain reaction PAR electos 23 RENEE o NTE 23 ALA A 24 0 3 Elebpddisattetratrtwte bugs 26 A NNN NNN 26 Eek 28 ESN AN NN 30 8 4 Scanning and evaluation of the CarnoCheck chip arnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnvnnennnne 32 9 TROUVUBEESNO OHNNG se 33 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 10 TESTINICAL ASISTAN Ea 34 11 PERFORMANCE CHARACTERISTICS OF CARNOCHECK i mennnnnnnnnnnnennnnnnnnnnnnnenen 35 Fer 36 ME Fe 36 11 4 3 Successful participation at FAPAS Proficiency Test ccocoooccccccccnccoccnnconnnnconcnnncnnnnnnnnons 38 12 CARNOCHECK SHORT PROTOCOL eunnnnernnnvnnnnnnnnnnnennnnennnnnnnnnnnennnnennnnnennnnvnnnnnennnn 39 121 Room 2 PCR Set up Of reaction MIX aria 39 12 2 Room 2 PCR DNA template addition PCR reaction arrrnnnnnnnnnnnnnnnennnnnnnnnnnnennnnnnnnennnnnnnnennne 40 12 3 Room 3 Hybridisation Preparation Hybridisation reactiOn sxxrrnnnavnnnnnvnnnnnnnnnnnavnnnnnennnnnunr 41 12 4 Room 3 Hybridisation Preparation Hybridisation reaction asxarrnnnnnnnnnnnvnnnnnvnnnnnnnnnnnnnnnnnn 42 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 1 KIT CONTENTS Components CarnoCheck 5x6 Arrays CarnoCheck PCR MasterMix CarnoCheck Hyb Buffer
19. es in total product Cattle Sheep Turkey Horse Chicken Figure B Validation of CarnoCheck using full preserves F 3 4 Signal to noise ratios SNR depending on of meat fractions from different species in total product 37 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Table 5 Limit of detection LOD as percentage of meat fractions in total product Belg LOD Species cooked preserves full preserves F lt 0 9 F 3 4 openen or C vomer Ja Only tested in mixtures of raw meet 11 4 3 Successful participation at FAPAS Proficiency Test The FAPAS Food Analysis Performance Assessment Scheme UK proficiency testing is an essential element of laboratory quality assurance and is an important requirement of the EU Additional Measures Directive 93 99 EEC applying to laboratories entrusted with the official control of food The analysis of an external quality check sample provides objective standards for quality of the analysis of an individual laboratory In FAPAS test 2911 freeze dried raw chicken test material was dispatched to the participants and was analysed for contaminating meat fractions These contaminations beef and pork were distinctively identified by using the CarnoCheck test system The FAPAS test material for FAPAS test 2918 was freeze dried raw pork contaminated with lamb The test partcipants were told that it contained at least one contaminant from lamb chi
20. following components of the CarnoCheck test kit contain harmful or hazardous contents Kit Component Hazardous Content Hybridisation Buffer 25 60 Guanidin thiocyanate Labelling according Regulation EC No 1272 2008 CLP d Warning Hazard statements H302 Harmful if swallowed H332 Harmful if inhaled H412 Harmful to aquatic life with long lasting effects Precautionary statement P273 Avoid release to the environment Supplemental Hazard information EU EUHO32 Contact with acids liberates very toxic gas according to European Directive 67 548 EEC Hazard symbol s Harmful Risk phrases R 20 21 22 Harmful by inhalation in contact with skin and if swallowed R 32 Contact with acids liberates very toxic gas R 52 53 Harmful to aquatic organisms may cause long term adverse effects in the aquatic environment Safety phrases S 13 Keep away from food drink and animal feeding stuffs S 36 37 Wear suitable protective clothing and gloves S 61 Avoid release to the environment Refer to special instructions safety data sheet CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Buffer B lt 20 of Sodium dodecylsulfate according Regulation EC No 1272 2008 CLP Danger Hazard statements H315 Causes skin irritation H318 Causes serious eye damage Precautionary statements P280 Wear protective gloves eye protection face protection P305 P351 P338 IF IN EYES Rinse cautiously with
21. heckScanner and the CheckReport Software CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 31 8 4 Scanning and evaluation of the CarnoCheck chip Place the CarnoCheck chip s into the CheckScanner and proceed with scanning as described in detail in the User Guide of the CheckReport Software For more detailed information about the installation of the CheckScanner and the CheckReport Software as well as computer system requirements please consult the corresponding Instructions For Use of the CheckScanner and the CheckReport Software Whenever analysing data using the CheckReport Software ensure that the version of the CheckReport Software installed on your computer matches the version indicated on the currently used CarnoCheck kit If the versions do not match update the CheckReport Software The latest Software version can be downloaded from the Greiner Bio One website www gbo com bioscience biochips download CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 32 9 TROUBLESHOOTING If one of the following error messages occurs during chip scanning or if the CarnoCheck analysis fails due to specific on chip controls proceed as described below Please do not hesitate to contact your local Greiner Bio One distributer if you have any questions or experience any difficulties while using CarnoCheck PROBLEM and cause ERROR MESSAGE COULD NOT
22. ices pepper muscat cardamon cinnamon CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 11 4 1 Procedure DNA extraction was done using the CTAB method Binke et al 2003 e Lysis buffer pH 8 20 g l Cetyltrimethylammonium bromide CTAB 1 4 mol l Sodium chloride 0 1 mol l Tris hydroxymethyl aminomethane Tris 20 mmol l Ethylenediaminetetraacetate EDTA e Proteinase K solution 10 mg ml e RNAse A solution 20 mg ml e Extraction solution Chloroform isoamylalcohol V V 24 1 e TE buffer 1 mmol l Tris HCI pH 8 1 mmol l EDTA pH 8 For lysis 25 to 100 mg of meat or 50 mg of meat product is weighed out in a 1 5 ml reaction tube and to this 400 ul lysis buffer and 20 ul Proteinase K solution are added and mixed The mixture is allowed to lyse overnight at 56 C with intermittent shaking for 10 s every 10 min For RNA digestion the sample solution is cooled down to room temperature and 20 ul RNAse A solution is added for incubation for 1 h at 37 C For nucleic acid extraction 300 ul of the extraction solution is pipetted into the sample mixed and centrifuged gt 10000 rpm for 10 min For nucleic acid precipitation 300 ul isopropanol is added to a new 1 5 ml reaction tube and 300 ul of the supernatant is pipetted in and mixed well The solution is incubated at room temperature for 10 min and then centrifuged 10 min at gt 10000 rpm The supernatant is carefully decanted and discarded and the pelle
23. ignal on the chicken probe detection of duck species is not the intended use of CarnoCheck 3 Composition of the tested saussages 50 meat 25 plant oil 23 ice 1 5 nitrit pickling salt 0 25 spices pepper muscat cardamon cinnamon Since the limit of detection of CarnoCheck depends on the species compostition processing and preservation grade the testing of complex food products should be validated first by verification of the analysis procedure by reference samples with known species content CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 6 2 Assay principle CarnoCheck is a microarray based test kit for the detection of species specific differences in the sequence of the cytochrome b cyt b gene see Table 2 The assay procedure is summarised in Figure 1 Prior to the CarnoCheck analysis DNA must be extracted from a meat sample Sample collection homogenisation and DNA extraction are not part of the CarnoCheck test kit After the extraction of genomic DNA from a meat sample a 389 bp fragment of cyt b gene of all the animal species present in the sample is amplified and fluorescently labeled with the aid of the polymerase chain reaction This is achieved using a primer pair matching two highly conserved sequences such that only a single reaction is necessary In the same reaction an internal control template present in the CarnoCheck PCR MasterMix is amplified to monitor the performance
24. in gen fore anvand ning Przed uzyciem przeczytac instrukcje Les bruksan visning for bruk TIpIv TNV xenon 1aB OTE TIC odnyieg Kullanma dan nce talimati okuyun CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Catalog Number Katalog nummer Num ro de r f rence Numero de catalo go Numero catalogo Numero de catalo go Catalo gusnum mer Katalog nummer Katalog nummer Numer katalo gowy katalog nummer Api udc KataAdyou Katalog numaras Manufac turer Hersteller Fabricant Fabri cante Produt tore Fabri cante Fabrikant Producent Tillverkare Producent produsent Mapaywy s Uretici firma Tempe rature limitation Tempera turbegren zung Limite de temp ra ture Limitaci n de tempera tura Limitazio ne tempe ratura Limitac o de tempera tura Tempera tuurbeper king Tempera turbegra ens r Tempe ratur be gr nsning Ograni czenie tempera tury tempe raturbe grensning TIEPIOPIOM G Bepuokpao iac Sicaklik sinirlamasi harmful gesund heits schadlich nocif nocivo nocivo nocivo schadelijk sundhe dsfarlig halsofarlig szkodliwy helsefarlig ENIBAABH2 Zararli Warning Achtung Attention Atenci n Attenzi one Atencao Waar schuwing Advarsel Varning Uwaga Ob
25. ini Centrifuge e PCR thermal cycler e GeneAmp PCR system 9700 Applied Biosystems or Veriti 96 Well Thermal Cycler Applied Biosystems e Water bath or heating block 65 C and 70 C e Micropipettes different ranges from 1 1000 ul e 8 Channel multipipette range 5 50 ul e g Brand Transferpette 8 Brand e Pipettor for glass and plastic pipettes e Vortex shaker e Racks for different reaction tubes e Waste container e Timer e Photometer for determination of DNA concentration Additional hardware required e Computer for system requirements see Instructions For Use of the CheckSanner and the CheckReport Software CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 3 SHIPMENT AND STORAGE The shipment of the CarnoCheck test kit takes place at room temperature Nevertheless the kit has to be stored immediately upon receipt at 4 8 C and should be protected from light Stored correctly the CarnoCheck test kit and its components can be used until the indicated expiration date Furthermore under these conditions the shelf life does not deviate from the expiration date after the first opening of the kit and its components 4 SAFETY INSTRUCTIONS The CarnoCheck test kit is for laboratory use only not for drug household or other purposes Always wear a suitable lab coat disposable gloves and protective goggles and follow the safety instructions given in this section The
26. lutions as this could lead to an accumulation of washed off PCR product that possibly interferes with CarnoCheck results Use fresh washing solutions for each assay The prepared washing solution mix can be stored up to one week at room temperature Check if precipitation of SDS has occured If so warm up the washing solution mix until the precipitate is dissolved and equilibrate to room temperature again Then prepare for the next hybridisation experiment CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 8 3 2 Hybridisation Hybridisation must be performed at room temperature 20 25 C The principle working steps for hybridising the PCR products of the CarnoCheck PCR reaction onto the CarnoCheck chip are shown in Figure 5 mg Mix the PCR products before use Briefly spin down If PCR products were stored at 20 C until hybridisation the PCR products must be heated for 3 minutes at 95 C mixed spin briefly down and then proceed as described mw Vortex the Hybridisation Buffer before use Briefly spin down mg Mix 24 pl of the CarnoCheck Hybridisation Buffer in a fresh reaction tube of an 8x PCR strip with 13 ul of the PCR product by either vortexing or by pipetting up and down several times m Briefly spin down mg Transfer 30 ul of the hybridisation mix into each chip well by using six channels of a multichannel pipette Avoid air bubble formation It is recommended to process six samples in
27. mperature using a thermometer Repeat preparation of hybridisation mix with the correct volumes and hybridise PCR products on another chip Repeat CarnoCheck analysis starting with the preparation of the PCR reaction Repeat CarnoCheck analysis starting with the preparation of the PCR reaction Repeat CarnoCheck analysis starting with the PCR reaction Take care to mix the reaction mix thoroughly Repeat DNA extraction and CarnoCheck analysis Repeat hybridisation Repeat hybridisation 33 PROBLEM and cause Comments and suggestions Problems with the thermal Check both the performance of the thermal cycler and the correct program cycler ming of the thermal cycler PCR steps heat ramp volume Attention Use either the thermal cycler GeneAmp PCR System 9700 Ap plied Biosystems or the Veriti 96 Well Thermal Cycler Applied Biosystems in combination with CarnoCheck PCR AND OR SAMPLE This result is rated as valid if the CheckReport Software detects at least one CONTROLS HAVE NOT FAILED animal species in the sample with a signal above a defined threshold The BUT DISPLAY A SNR VALUE OF 0 PCR control may then show low or even absent fluorescence signals due to competition during PCR 10 TECHNICAL ASSISTANCE Greiner Bio One employs a technical service department staffed with experienced scientists with extensive practical and theoretical expertise in molecular biology and oCheck products If you h
28. ndle Wash the CarnoCheck chip s in washing solution at room temperature for 20 seconds Wash the CarnoCheck chip s in preheated washing solution Il in a water bath at 50 C for 20 seconds Remove any liquid from the CarnoCheck chip surface by centrifugation Scan the CarnoCheck chip s with the CheckScanner Perform scanning and analysis as described in the User Guide of the CheckReport Software Create reports 3 minutes or 500 g 1 minute max speed T P d
29. olution II at 50 C by vigorously moving the slide holder up and down Immediately remove any liquid from the chip surface by centrifugation Ifa special microcentrifuge for microarrays is used centrifuge for 1 minute If a centrifuge applicable for 50 ml tubes is used place every washed CarnoCheck chip into a 50 ml tube and centrifuge at room temperature for 3 minutes at 500 g The CarnoCheck chip is now ready for scanning and should be scanned immediately For cleaning of the oCheck WashBoxes rinse several times with water after each completed washing and drying procedure CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 30 First washing step II II IT II Carefully remove the magnetic slideholder from the Hybridisation Chamber Quickly drop the slideholder into the oCheck Washbox with washing solution I Attach the oCheck Handle Wash the CarnoCheck chip s in washing solution at room temperature for 20 seconds by moving the slideholder up and down Second washing step II Wash the CarnoCheck chip s in washing solution II in a water bath 50 C for 20 seconds by moving the slideholder up and down Drying II Immediately remove any liquid from the surface of the CarnoCheck chips by centrifugation 3 minutes 1 minute 500 g max speed Figure 6 Working steps of the washing and drying procedure prior to the analysis of the CarnoCheck chip with the C
30. parallel using an 8 channel multipipette and 8x PCR Strips see Figure 5 This increases handling efficiency speed and thereby reduces the risk of evaporation If more than one slide is to be processed at once the usage of a multipipette is mandatory in order to achieve the correct hybridisation time If possible hybridise all 6 wells of a chip In case of processing fewer than 6 samples leave the unused wells empty Unused wells on a processed chip cannot be used for future samples p Handle the chip carefully to avoid spilling of the hybridisation mix Spilling can lead to cross contamination of samples and to false positive results Incubate the chip for exactly 20 minutes at room temperature 20 25 C within the prepared Hybridisation Chamber in a dark humidity saturated atmosphere Be careful not to move the Hybridisation Chamber during the hybridisation Never change the incubation time or temperature of the hybridisation reaction as this may cause a loss of fluorescence signal intensity or an increase in unspecific fluorescence Do not expose the hybridised chips to direct sunlight CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Prepare a humidity saturated atmosphere in the Hybridisation Chamber Incubate the required amount of CarnoCheck chips in the Hybridisation Chamber at room temperature 20 25 C see Chapter 8 3 1 Mix 24 ul of the CarnoCheck Hybridisation Buffer in a 0 2 ml
31. r DNA extraction and discard the remaining unlysed sample After DNA extraction we recommend a determination of the concentration of the DNA The concentration of DNA template for the PCR should be between 5 15 ng ul The detection limit of all species is below 0 35 in a mixed sample However the detection limit of CarnoCheck depends on the extent to which the sample has been processed and the purity of the DNA e the presence of possible PCR inhibitors It is recommended to include a negative extraction control for every time a DNA extraction is performed As negative extraction control sample the DNA elution buffer of the appropriate DNA extraction kit or PCR grade water may be used If a homogenisation of the tested samples is performed perform also the homogenisation procedure with this negative extraction control sample in order to monitor carry over contamination in the homogenisation and DNA extraction procedure CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 8 2 Polymerase chain reaction PCR PCRis avery sensitive method which can detect extremely small amounts of DNA Special precautions must be observed in order to avoid reaction contamination see Chapter 7 AmpliTaq Gold DNA Polymerase Applied Biosystems 5U ul is required but not provided with the CarnoCheck test kit and must be purchased separately see Chapter 2 The CarnoCheck test kit has been validated using AmpliTaq Gold D
32. r damage If one of the components is damaged e g buffer bottles contact your local Greiner Bio One distributor Do not use damaged kit components as their use may lead to poor kit performance e Do not use the CarnoCheck test kit after the expiry date e Do not use expired reagents e Do not mix reagents from different batches e Use only reagents equipment provided with the kit and those recommended by the manufacturer e Regular calibration maintenance should be performed for micropipettes waterbath and heating block e Pipetting of small amounts of liquid in the microliter range is a challenge Therefore take care to pipette as accurately as possible e To avoid microbial contamination of the reagents take care when removing aliquots from reagent tubes e All centrifugation steps should be carried out at room temperature 18 25 C CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 7 3 4 Working safely e Never pipette solutions by mouth e Do not eat drink smoke or apply cosmetic products in the work areas e Avoid direct contact with the biological samples as well as splashing or spraying of the samples e Always wear a lab coat gloves and goggles while working with human samples e Wash hands carefully after handling of samples and reagents CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 8 CARNOCHECK PROCEDURE The following chapter describes the different
33. reserved foods Raw cooked and full preserves can be analysed with CarnoCheck but tropical preserves cannot The extreme heating in the making of tropical preserves breaks the DNA into very small fragments As a result amplification by PCR for CarnoCheck analysis is not possible CarnoCheck is validated for meat mixtures of meat and saussages with a sterilisation value of F lt 3 4 The low limit of detection 0 05 0 35 depending on species processing of the food product and purification permits the detection of even small traces of animal constituents 1 For complete definition of sterilisation symbols D Z F refer to http www fda gov ICECI Inspections InspectionGuides ucm070835 htm CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 6 1 Intended use CarnoCheck is an analysis kit for the detection of pig cattle sheep turkey chicken duck horse donkey goat in raw cooker preserved boiled or fully preserved meat mixtures of meat and saussages with a sterilization value of F lt 3 4 Table 1 Matrices preservation grades and animal species analysable with CarnoCheck matrices preservation grade species meat raw pig mixtures of meat cooker preserved boiled cattle saussages fully preserved sheep FS34 turkey chicken horse donkey goat 2 In case of detection of a chicken species signal the presence of duck species cannot be excluded Not all duck species give a positive s
34. rom one distinct area to another 7 2 Room separation Figure 3 shows an example of how a laboratory may be separated into three distinct sections One is used only for DNA extraction another is for the set up and running of PCR reactions and the last is for hybridisation and analysis Each room is used exclusively for the application or technique indicated to prevent sample contamination The use of colour coding could be advantageous to avoid the accidental exchange of equipment and consumables between areas Figure 3 Room separation Room 1 The entire DNA extraction procedure must be performed in this room Room 2 Within this room the reaction mix for the PCR is set up and aliquoted optimally under a PCR hood The addition of the DNA samples extracted in room 1 must be carried out in a separate space within room 2 Rooms Within the third laboratory room the hybridisation reaction washing steps and chip drying take place Additionally the CheckScanner in conjunction with the CheckReport Software is used for the final analysis of the CarnoCheck assay Neither equipment nor consumables should be interchanged between the different laboratory rooms and spaces Hence duplications in equipment and consumables are a necessity and should be taken into account when equipping the laboratory CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 7 3 Warnings and precautions 7 3 1 Contamination prevention e
35. s NPOZOXH Dikkat Danger Gefahr Danger Peligro Pericolo Perigo Gevaar Fare Fara NIEBEZPIECZ ENSTWO Fare KINAYNOZ Tehlikeli Batch code Chargen bezeich nung Code du lot C digo de lote Codice del lotto C digo do lote Lot nummer Lotnum mer Lot nummer Kod partii batch nr KWOIKOG Traptidas Partikodu Important Note Wichtiger Hinweis Note im portante Nota im portante Nota im portante Aviso im portante Belangri jke op merking Vigtig henvis ning Viktigt medde lande Wazne Viktig merknad 2NMAVTIK UTT DEIEN nemli Not TABLE OF CONTENTS EE ss 5 2 CONSUMABLES EQUIPMENT AND HARDWARE REQUIRED occocccocccnccconcnccnnnooo 6 3 SHIPMENT AND STORAGE suelos adela 8 4 SAFETY INSTRUCT IONS ee 8 2 NASLE DDPO E 10 A o IN S E EN otc ee RE NA 11 A e O EE 12 62 FASSI ne E 13 6 3 Design of the CarnoCheck DNA chip ae LE 15 Eer EEE CHIP Ia CUM tea a o E ARE 15 AA E o AP ER 16 7 INSTRUCTIONS FOR THE CARNOCHECK WORKFLOW ennnnernnnnnennnnnnvnnnnnvennnnn 17 TA General IAS TUCASA e e MA e e 17 T2 ROOM lt eparatloN main ela ia alado a rd 17 TS WWarnimgs and precautions NS oras 18 La DE eeh El Ee Ta ei OM tt 18 Rorz Anstucitontor handing DNA nl al 18 ars Generali DEC AUNO A a a An 18 TAr NOKT Sae NS e a de e 19 8 CARNOCHECK PROCEDURE cion 20 8 1 Sample collection homogenisation an
36. shing buffers expose them to room temperature 20 25 C for 30 minutes and mix well before use Storage of the CarnoCheck test kit at 4 8 C may result in precipitates in the Hybridisation Buffer and Buffer B Allow the solutions to equilibrate to room temperature and then vortex the tube or agitate the bottle until any precipitate is dissolved mg Prepare the oCheck Hybridisation Chamber Put a fresh wet paper towel into the Hybridisation Chamber and close the lid to create a humidity saturated atmosphere To avoid evaporation of the small volume of used hybridisation mix on the chip it is necessary to perform the hybridisation in a humidity saturated atmosphere A dedicated Hybridisation Chamber for CarnoCheck analysis is available from Greiner Bio One see Chapter 2 mg Incubate the required amount of CarnoCheck chips in the prepared Hybridisation Chamber at room temperature 20 25 C for at least 10 minutes The magnetic slide holder of the Hybridisation Chamber contains a magnet only at one of two ends If less than four CarnoCheck chips are to be hybridised in parallel take care to fill the slideholder with CarnoCheck chips from the opposite side of the magnet Otherwise the CarnoCheck chips will not be covered with liquid during the washing procedure mg Prepare the washing solutions I and II appropriate for the number of CarnoCheck chips being analysed If more than one CarnoCheck chip is processed multiply
37. t available Equipment Greiner Bio One Cat No Quantity CheckScanner 862 070 1 CheckReport Software Basic 862 080 1 CheckReport Software CarnoCheck plugin 862 084 oCheck Hybridisation Chamber with slideholder 447 070 Handle for slideholder 447 001 oCheck Washbox 447 020 Enzymes required e AmpliTaq Gold DNA Polymerase Applied Biosystems Cat No N808 0240 5 U ul 250 U DNA Extraction Kits e NucleoSpin Food Macherey Nagel Cat No 740945 50 for 50 purifications e Gen Elute Sigma Aldrich Cat No G1N 50 for 50 purifications Additional consumables required e PCR grade water e ethanol puriss p a 2 99 8 e distilled or deionised water e single use gloves cleaning solution for DNA decontamination e scalpel blades e disposables for homogenization depending on the homogenization procedure CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Additional equipment required e Precision scales e Scalpel with disposable blades e Equipment for homogenisation e Clean bench for PCR set up e Cooling block for Taq e Microcentrifuge for 1 5 and 2 ml reaction tubes e Centrifuge for 50 ml polypropylene tubes e g BeckmanCoulter Allegra X 22 Centrifuge C0650 Fixed Angle Rotor or slide centrifuge e g Labnet Slide Spinner VWR International Galaxy MiniArray Centrifuge e Microcentrifuge for single 0 2 ml reaction tubes or 8 tube PCR strips e g Labnet Spectrafuge M
38. t is washed with 500 ul ethanol 70 and centrifuged for 5 min at gt 10000 rpm The supernatant is discarded and the washing step is repeated with ethanol 100 The pellet is dried at 70 C for 10 min and taken up in 100 ul TE buffer prewarmed to 70 C 15ng DNA was used as template for the PCR reaction PCR hybridisation and washing was done as described in the CarnoCheck Instructions for Use 11 4 2 Results In cooked preserves F lt 0 9 all tested animal species could be detected down to a percentage of meat fractions of 0 25 in total product and for pig cattle turkey and horse even down to 0 05 see figure A In full preserves F 3 4 all tested animal species could be detected down to a percentage of meat fractions of 0 25 in total product and for pig cattle and turkey even down to 0 05 see figure B In a further analysis the limit of detection LOD was explored more precisely For a detailed overview of the determined LODs for each animal species see table 4 For animal species goat and donkey the limit of detection was only determined in mixtures of raw meet Here LODs of 0 1 or 0 35 respectively were obtained CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 36 0 05 PI 0 25 mi 0 5 P H 25 Wm 45 7 Cattle Sheep Turkey Horse Chicken Figure A Validation of CarnoCheck using cooked preserves F lt 0 9 Signal to noise ratios SNR depending on of meat fractions from different speci
39. to hybridisation Prepare the washing solution I and II for the number of CarnoCheck chips to be analysed Distilled deionised water CarnoCheck Buffer A CarnoCheck Buffer B total volume Distilled deionised water CarnoCheck Buffer A CarnoCheck Buffer B total volume 60 ml 6 ml 0 75 ml 66 75 ml 73 5 ml 0 9 ml 0 6 ml 75 ml 120 ml 12 ml 1 5 ml 133 5 ml 147 ml 1 8 ml 1 2 ml 150 ml 180 ml 18 ml 2 25 ml 200 25 ml 220 5 ml 2 7 ml 1 8 ml 225 ml Fill washing solution and II in separate oCheck WashBoxes Preheat washing solution Il in a water bath at 50 C 240 ml 24 ml 3 ml 267 ml 294 ml 3 6 ml 2 4 ml 300 ml Incubate the number of CarnoCheck chips to be analysed in the prepared oCheck Hybridisation Chamber at room temperature Mix PCR products and briefly spin down Mix Hybridisation Buffer and briefly spin down Mix 24 pl CarnoCheck Hybridisation Buffer with 13 ul PCR product Mix thoroughly and briefly spin down Transfer 30 ul of the hybridisation mix into each well of the CarnoCheck chip using a multichannel pipette Avoid air bubble formation Incubate the CarnoCheck chip for exactly 20 minutes at room temperature 20 25 C Remove the magnetic slideholder from the Hybridsation Chamber Drop the slideholder into the oCheck Washbox with washing solution Attach the oCheck Ha
40. with the laboratory chemical waste Observe all national state and local regulations regarding disposal CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 6 INTRODUCTION CarnoCheck is a DNA Chip for the parallel qualitative detection of eight different animal species constituents in meat containing food products With CarnoCheck the animal species can be rapidly and simultaneously identified in raw and preserved food matrices derived from meat Preserved foods differ in the extent to which they have been heated and thereby in their shelf life The extent of preservation is defined by the sterilization value F The F value for a sterilisation process is the number of minutes required to kill a known population of microorganisms in a given food under specified conditions When F is used without a subscript indicating temperature 121 1 C is assumed so an F value of 1 corresponds to 1 minute at 121 C e Cooker preserves boiled food Heating is at temperatures up to a maximum of 100 C The shelf life when kept cooled at temperatures lower than 10 C is limited to 1 year F 2 0 4 e Fully preserved food Heating is at temperatures over 100 C The shelf life is limited to 4 years at 25 C without cooling F 2 3 e Tropical preserves Heating is at temperatures over 100 C The shelf life is limited to 1 year at 40 C without cooling F 2 12 Cooked and full preserves comprise approximately 90 of all p
41. working steps which finally lead to the production of a detailed report indicating the presence or absence of one or more of the 8 animal species detectable in the analysed meat sample Figure 4 shows an overview of the necessary different working steps It also indicates the corresponding subchapter describing the specific assay step The working steps must be performed in the order outlined in this chapter Each specific hands on step is indicated by a blue arrow mm Sample collection homogenisation DNA extraction and analysis with the CheckReport Software are not part of the CarnoCheck test kit Therefore the description of these working steps is abbreviated within this chapter For more detailed information please refer to the corresponding Instructions For Use e g from the DNA Extraction Kits and the CheckReport Software 8 1 Sample collection homogenisation and DNA extraction 8 1 1 Sample collection For sample collection knifes or scalpels with sterile disposable blades are recommended In order to avoid cross contamination of samples use for each sample a new disposable blade 8 1 2 Homogenisation If a homogenisation step is necessary depends on the homogeneity of the starting material For inhomogeneous samples representative random samples should be taken at several sites and combined in a single homogenate In a homogenous sample e g raw meat the excision of one representative aliquot of the sample and direct lysis
42. y by either vortexing for 2 seconds and then spinning down or by pipetting up and down several times mg Aliquot the reaction mix by pipetting 24 ul of the reaction mix for each PCR reaction into a 0 2 ml thin walled PCR reaction tube 24 CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 Carry out addition of the template DNA in a separate work space than the set up of the reaction mix see Chapter 7 2 mg Add 1 ul of DNA extract 5 15 ng ul to each PCR reaction and mix either by vortexing for 2 seconds and then spinning down or by pipetting up and down several times The total volume of one PCR reaction is 25 ul mg Place the reaction tubes in the thermal cycler and start the PCR reaction using the thermal cycler program described in Chapter 8 2 1 Table 2 After the PCR has been completed the amplification products should be used immediately for hybridisation or stored in the dark at 20 C for one week If so the PCR products must be heated for 3 minutes at 95 C before hybridisation CarnoCheck Instructions For Use Revision BQ 020 01 February 2013 25 8 3 Hybridisation and washing 8 3 1 Preparation and set up Hybridisation must be performed at room temperature 20 25 C Begin with the necessary preparations for the hybridisation and washing steps at least 30 minutes in advance of starting the hybridisation procedure mg To dissolve potential precipitates in the hybridisation and wa
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