PMA99A VERYFinder Total Meat QUALITATIVE User_Manual
Contents
1. 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 TOTAL MEAT 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 Total Meat Detection Assay Rev 1 27 11 2014 ENER N Advanced Transfer Technologies Quick Reference Guide Thermal cycling Step Duration Taq Activation 95 3 min DNA Denaturation 95 10 sec Annealing Extension Plate Reading 60 42. 30 Veryfinder Total meat Assay Quantiscale Bovine Std Points 0 196 1 10 100 100 24 ng Ox DNA Effpcr 94 R 1 00 i Unknown sample 12 30 Example of a quantification of contamination in a complex matrix tomato meat sauce using the double standard curve approach The product was claimed to contain 1596 w w bovine meat 24 ng reaction of the DNA extracted from the sample underwent analysis Tests were performed using the VERYfinder equine and VERYfinder total meat assays referencing results to a standard curve prepared using VERYfinder Quantiscale horse and bovine respectively Graphs show the calculated amount of equine and total meat DNA in the sample and through an easy proportioning Equinepca Realeguine Otal Meatpce 100 it becomes evident all of the meat DNA contained in the sample is equine User guide VERYfinder Total Meat Detection Assay Rev 1 27 11 2014 6 ENER N Advanced Transfer Technologies 6 Inclusivity Panel Meat Raw and Heat treated matrices Beef Bos taurus Buffalo Bubalus bubalis Donkey Equus asinus Duck Anas spp Anchovy Engraulis encrasicolus Cod Merluccius merluccius Sardine Sardina pilchardus 7 Exclusivity Panel Goat Capra hircus Horse Equus caballus Poultry Gallus gallus domesticus Quail Coturnix coturnix Fish Raw matrices Mackerel Scombrus scombru Salmon Onchorthynchus kisutch R
3. 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 Total Meat Detection Assay Rev 1 27 11 2014 10
4. 5 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 Total Meat FAM Negative Control Then the possible results for any sample are TEST Total Meat FAM Internal Amplification Control HEX Positive Sample Negative Sample 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 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 res
5. GSeneren Advanced Transfer Technologies VERYfinder DETECTION ASSAY TOTAL MEAT Cat N PMA99A User Guide ENER N Advanced Transfer Technologies 1 Introduction The horse meat scandal dating 2012 demonstrated the need for food industry and consumers to have reliable and sensitive analytical tools facilitating routine control tests of meat species in different foods and feedstuffs These analysis would avoid unfair market competition and protect consumers from false labeled meat products for economical religious and health reasons Although Real time PCR is widely accepted as a robust method for the identification of undesired contaminating ingredient species due to its high sensitivity and specificity there are few published reports on its application for quantitation of species in complex DNA samples Some of the drawbacks found are inherent in any procedure based on DNA because DNA yields may depend on the source material method of extraction or fragmentation of DNA that takes place in highly processed food When several species are present in the sample ascertaining the relative amounts of each species in the mix requires a unique reference target sequence identical in all of the possible species User guide VERYfinder Total Meat Detection Assay Rev 1 27 11 2014 2 s evene Advanced Transfer Technologies 2 VERYfinder Total Meat Detection Assay This assay can serve three different purposes it provides a positi
6. 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 Working 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 If VERYfinder Total Meat Assay is used along with specific animal target Quanticast or Quantiscale products proceed adding 12 ul of each standard points in order to carry a regression curve for a specific animal target quantification 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 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 vegan FAM BHQ1 NFQ Target IAC HEX BHQ1 NFQ Thermal profile Step Duration Taq Activation DNA Denaturation Annealing Extension Plate Reading User guid
7. abbit Oryctolagus cuniculus Swine Sus scrofa domesticus Sheep Ovis aries Wild boar Sus crofa Trout Salmo trutta Tuna Thunnus albacares The following DNA extracts showed no amplification curve when tested according to the general assay instruction Vegetables Barley Hordeum vulgare Basil Ocinum Basilicum Beans Phaseolus vulgaris Carrot Daucus carota Corn Zea mays Cucumber Cucumis sativus Eggplant Solanum melongena Garlic Allium sativum Lupine Lupinus albus Mushroom Agaricus campestris Mustard Brassica nigra Oat Avena sativa Olive Olea europaea Onion Allium cepa Parsley Petroselinum crispum Pepper Capsicum annuum Rice Oryza sativa Rye Secale cereale Sesame Sesamum indicum Soybean Glycine max Spelt Triticum monococcum Garlic Allium sativum Spinach Spinacia oleracea Tomato Solanum lycopersicon Wheat Triticum aestivum Zucchini Cucurbita pepo User guide VERYfinder Total Meat Detection Assay Rev 1 27 11 2014 7 s evene Advanced Transfer Technologies 8 Troubleshooting Concomitant no target or IAC amplification or amplification plots grossly abnormal Possible causes and corrective actions 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 dil
8. e VERYfinder Total Meat 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 then used to determine the presence and afterwards semi quantify the amount of animal 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 TEST Total Meat FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are TEST Total Meat FAM Internal Amplification Control HEX Positive Sample Negative Sample 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 for further suggestions In case of VERYfinder animal target Quanticast or Quantiscale maching see below an example of quantification example Veryfinder Equine Assay Quantiscale Horse Std Points 0 1 1 5 10 100 100 24 ng Horse DNA Effpca 108 R2 0 988 H Unknown sample 17
9. 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 EXDOO1 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 Lab Suppliers VERYfinder Total Meat Detection Assay Generon Cat N PMA99A GENERase ULTRA PLUS Mastermix Generon Cat N ENGOOS VERYfinder Quantiscale or Quanticust Generon Cat N PMAxxW or X Optical Adhesive Seal and Optical reaction plate or L li Optical Caps and Strips eb oupplicts Micropipette sets 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 Total Meat Detection Assay Rev 1 27 11 2014 4 s evene 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase ULTRA PLUS Mastermix VERYfinder
10. le 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 Total Meat Detection Assay Rev 1 27 11 2014 8 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 ul N vials Volume ul VERYfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 150 2 150 Positive Control 1 300 1 300 Negative Control 1000 1000 All reagents are supplied with a 5 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 Vortex briefly OLIGO mix afterwards spin to collect contents at the bottom of the vials Spin GENERase ULTRA PLUS Mastermix Cat N
11. o 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 Total Meat 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 Cat N EXDOO1 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
12. ponsible 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 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
13. utions 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 if analysis aimed for a animal target quantification and semi quantification In case of a qualitative analysis 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 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 defective Generon will provide a replacement product Generon shall not be liab
14. ve control on the total PCR amplifiable animal DNA present in the sample it validates the quantitation of different species DNA allowing the calculation of the percentages in the mixed sample third it traces the presence of DNA that has not been detected by the species specific detectors The second and third applications require the use of quantitative standards dispersed in water Quantiscale or in a customizable dispersive matrix Quanticust provided separately by Generon e g according to the animal target users would like to quantify Quantiscale and Quanticust products are Cat N PMBxxW or PMBxxX 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 animals meat presence 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 1 300 1 300 Negative Control 1 1 reagents are supplied with a 5 of extra volume We suggest to use VERYfinder Total Meat Detection Assay along with the following Polymerase Enzyme Ready to use mastermix GENERase ULTRA PLUS Mastermix Cat N ENGOO9 When using this GENERase ULTRA PLUS an additional detection channel HEX becomes available t
Download Pdf Manuals
Related Search