PAV02A SPECIALFinder Peanut User_Manual
Contents
1. After setting the baseline the analysis outcome should be evaluated following the indications below If the following conditions are met TEST Peanut FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are TEST Peanut FAM Internal Amplification Control HEX Positive Sample Negative Sample M 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 6 Inclusivity Panel Species tested for inclusivity Peanut Arachis hypogaea User guide SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 6 ENER N Advanced Transfer Technologies 7 Exclusivity Panel The following DNA extracts showed no amplification curve in a 20 ul total reaction volume Beef Bos taurus Buffalo Bubalus bubalis Donkey Equus asinus Duck Anas spp Clam Venus gallina Norway lobster Nephrops norvegicus Hake Merluccius merluccius Almond Prunus dulcis Barley Hordeum vulgare Celery Apium graveolens Cashew Anacardium occidentalis Brasilian Walnut Bertholletia excelsa Ananas Ananas comossus Apple Malus domestica Apricot Prunus armeniaca Banana Musa acuminata Basil Ocimum basilicum2. Broccoli Brassica oleracea var italica Buckwheat Fagopyrum esculentum Carrot Daucus carota Chard Beta vulgaris var cicla Cherry Prunus avium Chestnut Castanea vulgaris Goat Capra hircus Horse Equus caballus Poultry Gallus gallus domesticus Quail Coturnix coturnix Rabbit Oryctolagus cuniculus Sheep Ovis aries Swine Sus scrofa domesticus Turkey Meleagris gallopavo Crustaceans and Molluscs Cuttlefish Sepia officinalis Prawn Penaeus vannamei Salmon Oncorhynchus kisutch Mussel Mytilus edulis Sea bream Sparus aurata Vegetables Allergens Durum Wheat Triticum durum Hazelnut Corylus avellana Kamut Triticum turgidum Lupine Lupinus albus Macadamia Nut Macadamia integrifolia Cocoa Theobroma cacao Coconut Cocos nucifera Corn Zea mays Cucumber Cucumis sativus Eggplant Solanum melongena Fennel Foeniculum vulgare Garlic Allium sativum Grapefruit Citrus x paradisi Grapes Vitis vinifera Green Beans Phaseolus vulgaris Lemon Citrus limonia Mustard Brassica nigra Sesame Sesamum indicum Oat Avena sativa Pecan Nut Carya illinoinensis Pistachio Pistacia vera Mandarine Citrus reticulata Mushroom Agaricus campestris Olive Olea europaea Onion Allium cepa Orange Citrus aurantium Parsley Petroselinum crispum Peach Prunus persica Pear Pyrus communis Peas Pisum sativum Pepper Capsicum a
3. When used along with GENERase PLUS Mastermix Cat N ENGOO2 this Real Time PCR assay detects a specific DNA sequence in the DNA of Peanut 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 SPECIALfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 250 2 250 Positive Control 1 85 2 85 Negative Control 1 200 1 reagents are supplied with a 596 of extra volume We suggest to use SPECIALfinder Peanut Detection Assay along with the following Polymerase Enzyme Ready to use mastermix GENERase PLUS Mastermix Cat N ENGOO2 When using this GENERase 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 SPECIALfinder Peanut Detection Assay Rev 1 26 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
4. s evene Advanced Transfer Technologies SPECIALfinder DETECTION ASSAY PEANUT Cat N PAVO2A User Guide ENER N Advanced Transfer Technologies 1 Introduction Food allergies are an adverse immune response to a food protein that are the most common allergic compound Food allergies are an important concern for human health in fact the presence of specific proteins in any food matrix can cause an allergic reaction IgE mediated Allergic reactions may have a broad spectrum which varies on the basis of the individual sensitivity thus generating in some cases severe anaphylactic reactions Indeed food correct labeling is of great importance to inform consumers about presence of any allergic substance other than achieve a high level of health protection Unfortunately although known allergens can be included in the product and in the product label by the food producer potentially hazardous allergenic residues contaminants can be present as result of common industrial practices Cross contamination between raw materials production lines or equipment is a common cause of unwanted allergen transfer between products intended for different scopes For all these reasons developing a detection method for allergic substances ensures customers protection in accordance with food labeling regulations The SPECIALfinder Peanut Detection Assay provide the user with a simple and reliable procedure for the detection of DNA related to species potent
5. 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 SPECIALfinder OLIGO Mix OLIGOS and Probe pre blended mix 1 250 2 250 Positive Control 1 85 2 85 Negative Control 1 1 All reagents are supplied with a 5 of extra volume Not Provided Article GENERase PLUS Mastermix Cat N ENG002 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 PLUS Mastermix before opening it Prepare SPECIALfinder WORKING Mastermix by adding 250 ul of SPECIALfinder OLIGO Mix into each tube prefilled with 500 ul of GENERase PLUS Mastermix Cat N ENGOO2 in order to obtain a single volume of 750 ul of SPECIALfinder WORKING Mastermix Vortex briefly SPECIALfinder 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 PLUS vials with target name after OLIGO Mix addition Vortex briefly Positive Control and samples before proceeding furthe
6. ction procedure details 3 3 Detection via Real Time PCR Material Equipment Source Real Time PCR System Generon or other Lab Suppliers Specialfinder Peanut Detection Assay Generon Cat N PAVO2A GENERase PLUS Mastermix Generon Cat N ENGOO2 Optical Adhesive Seal and Optical reaction plate or Gres C ael oe Generon or other Lab Suppliers Micropipette sets Generon or other 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 StepONE StepONE Plus Light Cycler 480 Eppendorf realplex Rotor Gene Q etc The assay is not compatible with Roche Light Cycler and Il User guide SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 4 s evene 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase PLUS Mastermix SPECIALfinder OLIGO MIX Positive Control and Negative Control Vortex tubes when thawed and spin to collect contents at the bottom of the vial Mix 250 yl of SPECIALfinder OLIGO Mix with 500 ul of GENERase PLUS Mastermix to prepare SPECIALfinder Working Mastermix WMX Vortex briefly and spin down in order to homogenize the mix Transfer 15 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 int
7. horisation 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 Polymerase 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 without fail if the package is still sealed the product can be returned and the user can be refunded User guide SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 10
8. ially allergenic in food and feed matrices as well as swabs Such a detection is an ndirect Proof of the potential presence of the Peanut proteins into the matrix being proteins the real allergens This assay utilizes the polymerase chain reaction PCR to amplify a genetic target typical of the allergenic species PCR technique can typically detect up to 1 10 copies of the target sequence but the real detection quantification limit depends on industrial processing degree sample matrix DNA extraction and lastly on the DNA content of the sample Genome size of the complex samples under investigation can deeply impact the Limit Of Detection LOD also in addition it does exist a theoretical LOD you cannot go below given an advised maximum load between 2 and 4 ng DNA ul reaction mix Generon in house validation the LOD has been calculated as copy number by means of ddPCR Droplet Digital PCR a novel technique capable to count physically the copy number of a selected amplifiable target With SPECIALfinder Peanut an average count of 5 10 copies was obtained DNA was extracted using Generon ION Force DNA Extractor FAST Cat N EXDOO1 The LOD for this assay was experimentally determined between 1 and 0 5 ppm and depends on sample matrix processing grade DNA preparation and DNA content User guide SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 2 GSeneren Advanced Transfer Technologies 2 SPECIALfinder Peanut Detection Assay
9. ive Control reactions failed to amplify but other reactions appear correct e g the IAC is amplified Positive Control DNA was 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 SPECIALfinder PCR mix with SPECIALfinder 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 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 SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 8 ENER N Advanced Transfer Technologies Quick Reference Guide Page 1 Product Line SPECIALfinder Part Number PAVO2A Type Qualitative Storage Frozen Execution time about 120 minutes
10. 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 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 extra
11. nnuum Pine Nuts Pinus pinea User guide SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 Wild boar Sus scrofa Squid Loligo edulis Tuna Thunnus albacares Rye Secale cereale Soft Wheat Triticum aestivum Soybean Glycine max Spelt Triticum monococcum Walnut Juglans regia Plum Prunus domestica Pomelo Citrus maxima Potato Solanum tuberosum Radish Raphanus sativus Rapessed Brassica napus Rice Oryza sativa Strawberry Fragaria x ananassa Sunflower Helianthus annuus Tomato Solanum lycopersicon Zucchini Cucurbita pepo s evene Advanced Transfer Technologies 8 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 and IAC 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 Posit
12. o 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 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 aut
13. o wells acting as positive control Close wells and ensure no bubbles are present at the bottom of the wells Spin briefly optical PCR tubes or plates 4 2 Instrument setup With GENERase PLUS Mastermix set the following parameters on your thermocycler Total Reaction volume 20 ul Fluorophores Quenchers Target Peanut FAM BHQ1 NFQ Internal Amplification Control HEX BHQ1 NFQ Depending on 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 Thermal profile Step Duration UNG 2 min Taq Activation 10 min DNA Denaturation 15 sec Annealing Extension Plate Reading 60 sec User guide SPECIALfinder Peanut Detection Assay Rev 1 26 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 allergen into the sample See below an example of the graphics obtained for a positive Fig A and a negative Fig B control respectively for the allergen target amplification blue line and for the IAC amplification green line Amplification Amplification
14. r spin to collect contents at the bottom of the vial Transfer SPECIALfinder WORKING Mastermix and samples into the plate as follows Reagents per well Volume Unknown Sample Positive Control Sul Negative Control SPECIALfinder WORKING Mastermix 15 ul Final Volume 20 ul Detector Setup Target Reporter Dye Quencher Dye Peanut 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 SPECIALfinder Peanut Detection Assay Rev 1 26 11 2014 9 ENER N Advanced Transfer Technologies Quick Reference Guide Thermal cycling Step Duration UNG 50 2 min Taq Activation 95 10 min DNA Denaturation 95 15 sec Annealing Extension Plate Reading 60 60 sec The thermal profile presented above was optimized for GENERase PLUS Mastermix Cat N ENGOO2 Results analysis If the following conditions are met TEST Peanut FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are TEST Peanut 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 t
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