User Guide
Contents
1. Cat N EXD001 Chemicals n esane Lab Suppliers Tubes 50 ml and 15 ml Lab Suppliers DNAse RNAse Free Water Lab Suppliers Vortexer Lab Suppliers Benchtop Centrifuge for 50 ml Tubes Lab Suppliers Thermal Water Bath or Block Lab Suppliers Pipette sets Lab Suppliers Pipette tips Barrier Lab Suppliers Tube rack for 1 5 ml tubes Lab Suppliers 2 0 and 1 5 ml micro tubes Lab Suppliers Micro centrifuge for 1 5 2 0 ml micro tubes Lab Suppliers DNA Extraction VACUUM BOX Vacuum pump or Venturi meter Lab Suppliers Each step of sample preparation grinding transferring weighing etc must be done according to GLP so that chance of cross contamination between samples is minimized It is recommended to use disposable equipment when possible If the food samples are not in a powdered or granular form they should be processed grinded or blended before DNA extraction The majority of DNA extraction methods supports from 20 to 50 mg of starting material Generon ION Force DNA Extractor FAST Cat N EXD001 allows processing up to 20 grams of starting material in order to maximize sample s lot representation Once the sample has been pulverized homogenized it can be weighed and the appropriate amount extracted according to DNA extraction method selected Refer to manufacturer user manual for extraction procedure details 3 3 Detection via Real Time PCR Material Equipment Source Real Time PCR System 2 Lab Suppliers VERYfinder Equine EU2. Detection Assay Generon Cat N PMA11A GENERase Mastermix Generon Cat N ENGOO1 Optical Adhesive Seal and Optical reaction plate or Lab Suppliers Optical Caps and Strips ae Micropipette sets Lab Suppliers 1 Equipment necessary only when ION Force DNA Extractor FAST Cat N EXDO01 is used 2 The assay can be used with Biorad CFX and MiniOpticon Stratagene MxSeries ABI 7300 7500 7900 Step ONE StepONE Plus Light Cycler 480 Eppendorf realplex Rotor Gene Q etc The assay is not compatible with Roche Light Cycler and Il User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 4 Sennen 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase Mastermix VERYfinder OLIGO MIX Positive Controls and Negative Control Vortex tubes when thawed and spin to collect contents at the bottom of the vial Mix 625 ul of VERYfinder OLIGO Mix with 875 ul of GENERase Mastermix to prepare VERYfinder Working Mastermix WMX Vortex briefly and spin down in order to homogenize the mix Transfer 30 ul of WMX into each well Add 5 ul of Negative Control into wells acting as negative control Add 5 ul of each sample into wells testing the unknown samples Add 5 ul of Positive Control into wells acting as positive control Close wells and ensure no bubbles are present at the bottom of the wells 4 2 Instrument setup With GENERase Mastermix set the following parameters on your thermo
3. GSeneren Advanced Transfer Technologies VERYfinder DETECTION ASSAY EQUINE EU Cat N PMA11A User Guide GSeneren 1 Introduction The recent scandal related to horse meat sold as bovine put under a spotlight the fact that food industry and consumers even though the scope is different share a common interest in the possibility to have a fast and accurate method determining the authenticity of the ingredient used for food preparation The food industry often makes use of products where it is difficult to verify the real content in raw material stated by the manufacturer This problem translates into economic and commercial risks for the company On the other side consumers want to be sure the products they eat deserve the price don t contain risks for health are not infringing religious ethical rules DNA testing allows an efficient and sensitive identification of plant and animal derivatives easily detecting accidental contaminations or potential fraud related to false declaration on the label of the species constituting the food Real Time PCR is the most sensitive method for the detection and quantification of specific DNA sequences of different species The method combined with an appropriate nucleic acid extraction system allows the analysis of raw materials semi finished and finished products as well This assay provides the user with a simple and reliable procedure for detecting the presence of the DNA of a specific organi
4. cycler Total Reaction volume 35 ul Fluorophores Quenchers Target Equine FAM BHQ1 NFQ Thermal profile Duration UNG 2 min Taq Activation 10 min DNA Denaturation 15 sec Annealing Extension Plate Reading 60 sec User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 5 ENER N Advanced Transfer Technologies 5 Data Interpretation Results evaluation must be done according to the analysis software recommended by the Real Time PCR instrument manufacturer After performing PCR each individual sample is analyzed through the instrument software to produce a Cq value quantification cycle for each reporter dye These values are used to determine the presence Qualitative Test of target into the sample See below an example of the graphics obtained for a positive Fig A control for the target amplification blue line Amplification After setting the baseline the analysis outcome should be evaluated following the indications below If the following conditions are met Equine EU FAM Positive Control Negative Control Then the possible results for any sample are Equine EU FAM Positive Sample Negative Sample In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph section 6 for further suggestions User guide VERYfinder Equi
5. enaturation 95 15 sec Annealing Extension Plate Reading 50 60 sec The thermal profile presented above was optimized for GENERase Mastermix Cat N ENGOO1 Results analysis If the following conditions are met TEST Equine EU FAM Positive Control Negative Control Then the possible results for any sample are Equine EU FAM Positive Sample Negative Sample In case of inhibition DNA isolation and purification for the sample need to be improved or you may need to dilute your sample before performing a new test Refer to the Troubleshooting paragraph section 6 in the User Guide for further suggestions Warning and Precaution Please do not interchange components of assays with different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions GLP Disclaimer Generon s r l guarantees the buyer exclusively concerning the quality of reagents and of the components used to produce the Assay Generon S r l is not responsible and cannot anyway be considered responsible or jointly responsible for possible damages resulting from the utilization of the product by the user The user consciously and under his own responsibilities decides for the utilization purposes of the product and uses it the way he considers most suitable in order to reach his goals and or objectives Generon S r l is not responsible for the data resulting from the use of the products f
6. isclaimers The product is intended for research use only Generon makes no warranty of any kind either expressed or implied except that the materials from which its products are made of standard quality If any materials are defective Generon will provide a replacement product Generon shall not be liable for any damages including special or consequential damage or expense arising directly or indirectly from the use of this product Please do not interchange components between assays of different lot numbers This assay is designed to be used by laboratory personnel following the common molecular biology precautions User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 7 ENER N Advanced Transfer Technologies Quick Reference Guide Page 1 Product Line VERYfinder Type Qualitative Storage Frozen Execution time about 120 minutes Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging and constantly under suitable temperature conditions as mentioned above Assay Box Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume yl VERYfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 625 2 625 Positive Control 1 125 1 125 Negative Control al 1000 al 1000 All reagents are supplied with a 5 of extra volume Not Provided Article GENERase Mastermix Cat N ENG001 or equivalent Reaction Set Up Protect reagent
7. ls provided in the assay Cut off is strictly dependent from the positive control provided User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 2 GSeneren 2 VERYfinder Equine EU Semi Quantitative Detection Assay When used along with GENERase Mastermix Cat N ENGOO1 this Real Time PCR assay detects a specific DNA sequence in the DNA of equine in less than 1 5 hours The amplification of the target sequence is measured by the use of a specific fluorescence labeled probe FAM 2 1 Assay Content Box 50 reactions Box 100 reactions N vials Volume pl N vials Volume pl VERYfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 625 2 625 Positive Control 125 125 Negative Control reagents are supplied with a 5 of extra volume We suggest to use VERYfinder Equine EU Detection Assay along with the following Polymerase Enzyme Ready to use mastermix GENERase Mastermix Cat N ENGOO1 2 2 Storage amp Expiry information Expiry date see date on the packaging product validity refers to the product kept intact in its original packaging Protect reagents from light exposure as far as OLIGO Mix reagents are photosensitive Store frozen User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 3 ENER N Advanced Transfer Technologies 3 Materials and equipments needed 3 1 Extraction Material Equipment Source Extraction Kit Generon ION Force DNA extractor FAST
8. ne EU Detection Assay Rev 1 27 11 2014 6 GSeneren Advanced Transfer Technologies 6 Troubleshooting Concomitant no target or IAC amplification or amplification plots grossly abnormal Possible causes and corrective actions e An excess of DNA in the target might inhibit the reaction may be affected due to an excess of DNA and or PCR inhibitors Test samples diluted 1 10 and 1 100 Please use DNase RNase Free Water to prepare dilutions Inadequate sealing of optical caps film caused sample evaporation Redo the analysis using proper tools and proper optical caps film to secure perfect sealing Did not use the proper consumables Redo the analysis and use only optical grade 96 well plates and optical adhesive seal or optical 8 well strips and caps Samples were not properly prepared Remake the sample DNA preps Ensure that the DNA extraction method is properly performed Positive Control reactions failed to amplify but other reactions appear correct e g the IAC is amplified Positive Controls DNA were not added to the reaction wells If other reactions look normal there may be no need to repeat the run Ill Negative Control reactions are positive e Contamination of the negative control vial or the VERYfinder PCR mix with VERYfinder positive DNA Use more care to prevent contamination while handling assay reagents and setting up assays In case support is needed contact Generon at support generon it 7 D
9. or the utilization that the user independently decides to make of them or for the damages possibly resulting from the disclosure or transmission of the data themselves to third parties under any form or circumstance This clause is automatically accepted by the user when purchasing the products The patent for performing PCR is held by Hoffmann La Roche Authorization to use PCR can be obtained on licence from Hoffmann LaRoche The product equipment and information included in the assay consists of assembled and reagents The licence and licence and authorisation for PCR use are not included in the assay The user is responsible for setting prefixed goals choosing whether or not to perform the PCR reaction and to apply for register his own licence The use assay is designed for the services supply quality control or any other application that is not exclusively an internal company s research and requires a specific licence for PCR use This PCR use licence to supply a service on food analysis field has to be requested directly from Applied Biosystems This assay requires the use of Taq Polymerise enzyme The product was internally tested by our quality control Any responsibility is waivered if the warranty of quality control does not refer to the specific product The user is personally responsible for data that he will obtained and or he will supply to third parties using this assay Once the sealed package is open the user accepts all the conditions wi
10. s from light exposure as far as OLIGO Mix reagents are photosensitive Before setting the analysis we strongly advise to leave the reagents to warm up at room temperature Vortex briefly OLIGO mix afterwards spin to collect contents at the bottom of the vials Spin GENERase Mastermix Cat N ENG001 before opening it Prepare VERYfinder WORKING Mastermix by adding 625 ul of VERYfinder OLIGO Mix into each tube prefilled with 875 ul of GENERase Mastermix Cat N ENG001 in order to obtain a single volume of 1500 ul of VERYfinder WORKING Mastermix Vortex briefly VERYfinder WORKING Mastermix with the aim of homogenizing the mix and excluding MgCl gradient that could impair the results Spin to collect contents at the bottom of the vial Note label GENERase vials with target name after OLIGO Mix addition Vortex briefly Positive Control and samples before proceeding further spin to collect contents at the bottom of the vial Transfer VERYfinder WORKING Mastermix and samples into the plate as follows Reagents per well Volume Unknown Sample Positive Control 5 ul Negative Control VERYfinder WORKING Mastermix 30 ul Final Volume 35 ul Detector Setup Target Reporter Dye Quencher Dye EQUINE EU Target BHQ1 NFQ User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 ENER N Advanced Transfer Technologies Quick Reference Guide Thermal cycling Step Duration UNG 50 2 min Taq Activation 95 10 min DNA D
11. sms in food matrices The assay utilizes the Polymerase Chain Reaction PCR to amplify a genetic target typical of the equine species of interest according with the official note by the European Union Reference Laboratory for Animal Proteins in feeding stuffs EURL AP recommended protocol version 1 0 application and publication day 18 02 2013 http eurl craw eu en 187 method of reference and sops The validation performed at Generon exploited ION Force DNA Extractor FAST Cat N EXD001 as DNA extraction method from raw and heat treated matrices 20 at 121 C The Limit of detection LOD 0 01 and the Limit of Quantification LOQ 0 05 have been calculated based upon a solution concentrated at 2 ng ul DNA DNA containing equine DNA on a background of DNA from a simulating matrix These representing the minimum detectable amount of sought species after spiking a simulating matrix This approach has been used in both raw matrices and heat treated matrices due to the impossibility of having a real standard as each matrix undergoes a different industrial process Species can moreover be unevenly distributed in the matrix or separated in the homogenization process Due to the high sensibility of the test some matrices might cause a background signal we therefore suggest to operate a DNA quantification after the extraction and to normalize its concentration accordingly to our validation The plot should then be evaluated using the positive contro
12. thout fail if the package is still sealed the product can be returned and the user can be refunded User guide VERYfinder Equine EU Detection Assay Rev 1 27 11 2014 9
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