User Guide
Contents
1. Vortex briefly OLIGO mix afterwards spin to collect contents at the bottom of the vials Spin GENERase ULTRA PLUS Mastermix Cat N ENGOO9 before opening it Prepare VERYfinder WORKING Mastermix by adding 150 ul of VERYfinder OLIGO Mix into each tube prefilled with 750 ul of GENERase ULTRA PLUS Mastermix Cat N ENGOO9 in order to obtain a single volume of 900 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 12 ul Negative Control VERYfinder WORKING Mastermix 18 ul Final Volume 30 ul Detector Setup Target Reporter Dye Quencher Dye EQUINE Target FAM BHQ1 NFQ IAC Internal Amplification Control HEX BHQ1 NFQ According to your thermocycler you can replace HEX detector in the plate setting with VIC or JOE in case your own Real Time Platform does not possess the HEX reading channel User guide VERYfinder Equine Semi Quantitative Detection Assay Rev 1 27 11 2014 9 ENER N Advanced Transfer Technologies Quick2. 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 then used to determine the presence and afterwards semi quantify the amount of equine DNA material in each sample Set the Baseline to Auto The analysis outcome should be evaluated following this table If the following conditions are met Equine FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are Internal Amplification Entine capt Control HEX Cq Unknown sample Cq Semi QT Control Target species Semi QT Control Cq Unknown sample gt Cq Semi QT Control Target species lt Semi QT Control Invalid Sample inhibited RED LINE Semi QT Control for EQUINE target GREEN LINE Unknown 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 8 for further suggestions User guide VERYfinder Equine Semi Quantitative Detection Assay Rev 1 27 11 2014 ENER N Advanced Transfer Technologies 6 Inclusivity Panel Horse Equus caballus Donkey Equus asinus 7 Exclusivity Panel The following DNA extracts showed no amplification curve whe
3. GSeneren Advanced Transfer Technologies VERYfinder DETECTION ASSAY EQUINE SEMI QUANTITATIVE Cat N PMAO8S User Guide G Sennen Advanced Transfer Technologies 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 detectin
4. Reference Guide Thermal cycling Step Duration Taq Activation 95 3 min DNA Denaturation 95 10 sec Annealing Extension Plate Reading 60 45 sec The thermal profile presented above was optimized for GENERase ULTRA PLUS Mastermix Cat N ENGOO9 Results analysis If the following conditions are met Internal Amplification Control HEX Positive Control TEST Equine FAM Negative Control Then the possible results for any sample are TEST Equine FAM ine ye Cq Unknown sample lt Cq Semi QT Control Target species gt Semi QT Control Cq Unknown sample gt Cq Semi QT Control Target species Semi QT Control Invalid Sample inhibited 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 8 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
5. as 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 Real Time PCR System VERYfinder EQUINE Semi QT Detection Assay GENERase ULTRA PLUS Mastermix Optical Adhesive Seal and Optical reaction plate or Optical Caps and Strips Micropipette sets DNA Quantification System Source Generon or other Lab Suppliers Generon Cat N PMAO8S Generon Cat N ENGOO9 Generon or other Lab Suppliers Generon or other Lab Suppliers Generon or other Lab Suppliers 1 Equipment necessary only when ION Force DNA Extractor FAST Cat N EXDOO1 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 Semi Quantitative Detection Assay Rev 1 27 11 2014 s evene 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase ULTRA PLUS 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 150 ul of VERYfinder OLIGO Mix with 750 ul of GENERase ULTRA PLUS Mastermix to prepare VERYfinder Wo
6. ective 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 Semi Quantitative Detection Assay Rev 1 27 11 2014 8 ENER N Advanced Transfer Technologies Quick Reference Guide Page 1 Product Line VERYfinder Type Semi Quantitative 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 ul N vials Volume ul VERYfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 150 2 150 Positive Control R 0 196 1 300 1 Positive Control HT 196 1 300 1 Negative Control 1 1000 T All reagents are supplied with a 596 of extra volume Not Provided Article GENERase ULTRA PLUS Mastermix Cat N ENGOO9 or equivalent Reaction Set Up Protect reagents 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
7. esponsibility 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 without fail if the package is still sealed the product can be returned and the user can be refunded User guide VERYfinder Equine Semi Quantitative Detection Assay Rev 1 27 11 2014 10
8. g the presence of the DNA of a specific organisms in food matrices The assay utilizes the Polymerase Chain Reaction PCR to amplify a genetic target typical of the equine species of interest The validation performed at Generon exploited ION Force DNA Extractor FAST Cat N EXDOO1 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 controls provided in the assay Cut off is strictly dependent from the positive control provided User guide VERYfinder Equine Semi Quantitative Detection Assay Rev 1 27 11 2014 2 s evene 2 VERYfinder Equ
9. 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 PCR run should be repeated 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 9 Disclaimers 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 def
10. ine Semi Quantitative Detection Assay When used along with GENERase ULTRA PLUS Mastermix Cat N ENGOO9 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 ul N vials Volume ul VERYfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 150 2 150 Positive Control R 0 1 96 1 300 1 300 Positive Control HT 1 96 1 300 1 300 Negative Control 1 reagents are supplied with a 5 of extra volume We suggest to use VERYfinder Equine Semi Quantitative Detection Assay VERYfinder Equine Semi QT along with the following Polymerase Enzyme Ready to use mastermix GENERase ULTRA PLUS Mastermix Cat N ENG009 When using this GENERase ULTRA PLUS an additional detection channel HEX becomes available to detect the Internal Amplification Control IAC to excluding false negative results due to a PCR inhibition 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 Semi Quantitative Detection Assay Rev 1 27 11 2014 3 ENER N Advanced Transfer Technologies 3 Materia
11. ls and equipments needed 3 1 Extraction Material Equipment Source Extraction Kit Generon ION Force DNA extractor FAST Cat N EXDOO1 Chemicals n esane Lab Suppliers Tubes 50 ml and 15 ml Generon or other Lab Suppliers DNAse RNAse Free Water Generon or other Lab Suppliers Vortexer Generon or other Lab Suppliers Benchtop Centrifuge for 50 ml Tubes Generon or other Lab Suppliers Thermal Water Bath or Block Generon or other Lab Suppliers Pipette sets Generon or other Lab Suppliers Pipette tips Barrier Generon or other Lab Suppliers Tube rack for 1 5 ml tubes Generon or other Lab Suppliers 2 0 and 1 5 ml micro tubes Generon or other Lab Suppliers Micro centrifuge for 1 5 2 0 ml micro tubes Generon or other Lab Suppliers DNA Extraction VACUUM BOX Vacuum pump or Venturi meter Generon or other 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 h
12. n tested according to the general assay instruction Meat Raw and Heat Treated matrices Beef Bos taurus Poultry Gallus gallus domesticus Swine Sus scrofa domesticus Buffalo Bubalus bubalis Quail Coturnix coturnix Turkey Meleagris gallopavo Duck Anas spp Rabbit Oryctolagus cuniculus Wild boar Sus crofa Goat Capra hircus Sheep Ovis aries Vegetables Barley Hordeum vulgare Mushroom Agaricus campestris Sesame Sesamum indicum Basil Ocinum Basilicum Mustard Brassica nigra Soybean Glycine max Beans Phaseolus vulgaris Oat Avena sativa Spelt Triticum monococcum Carrot Daucus carota Olive Olea europaea Garlic Allium sativum Corn Zea mays Onion Allium cepa Spinach Spinacia oleracea Cucumber Cucumis sativus Parsley Petroselinum crispum Tomato Solanum lycopersicon Eggplant Solanum melongena Pepper Capsicum annuum Wheat Triticum aestivum Garlic Allium sativum Rice Oryza sativa Zucchini Cucurbita pepo Lupine Lupinus albus Rye Secale cereale Fish Raw matrices Anchovy Engraulis encrasicolus Mackerel Scombrus scombru Trout Salmo trutta Cod Merluccius merluccius Salmon Onchorthynchus kisutch Tuna Thunnus albacares Sardine Sardina pilchardus User guide VERYfinder Equine Semi Quantitative Detection Assay Rev 1 27 11 2014 7 s evene Advanced Transfer Technologies 8 Troubleshooting Concomitant no target or IAC amplification or amplification plots
13. rking Mastermix WMX Vortex briefly and spin down in order to homogenize the mix Transfer 18 ul of WMX into each well Add 12 ul of Negative Control into wells acting as negative control Add 12 ul of each sample into wells testing the unknown samples in order to perform a proper semi quantification all the unknown samples should be normalized at the concentration of 2 ng ul as the positives controls supplied within the assay Quantification should be executed using a suitable DNA quantification system we suggest Quantus Fluorometer Promega Cat N E6150 Add 12 ul of Positive Controls into wells acting as Semi QT controls we strongly recommend to use the Positive Control R Raw when testing raw matrices and Positive Control HT Heat Treated when testing cooked high processed food matrices Close wells and ensure no bubbles are present at the bottom of the wells 4 2 Instrument setup With GENERase ULTRA PLUS Mastermix set the following parameters on your thermocycler Total Reaction volume 30 ul Fluorophores Quenchers Target Equine FAM BHQ1 NFQ Target IAC HEX BHQ1 NFQ Thermal profile Step Duration Taq Activation DNA Denaturation Annealing Extension Plate Reading User guide VERYfinder Equine Semi Quantitative 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
14. 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 for 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 r
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