User Guide
Contents
1. Coilia mystus Coregonus clupeaformis Acanthurus lineatus Acipenser fulvescens Acipenser nudiventris Acipenser sinensis Alepocephalus bairdii Aluterus scriptus Amphiprion polymnus Anguilla australis schmidti Anguilla dieffenbachii Anguilla marmorata Anguilla reinhardtii Arctogadus glacialis Auxis thazard Barbatula nuda Bodianus mesothorax Botia histrionica Branchiostegus argentatus Callorhinchus milii Caranx melampygus Catla catla Channa argus Chelidonichthys capensis Cirrhinus mrigala Cobitis elongatoides Cobitis takatsuensis Coilia nasus Coregonus lavaretus User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 Achirus lineatus Acipenser gueldenstaedtii Acipenser oxyrhynchus oxyrhynchus Acipenser stellatus Allocyttus niger Ameiurus melas Anabas testudineus Anguilla bengalensis labiata Anguilla interioris Anguilla megastoma Anguilla rostrata Argyrosomus japonicus Bahaba taipingensis synonym Otolithes lini Belonophago hutsebouti Bodianus rufus Botia lohachata Branchiostegus japonicus Campylomormyrus compressirostris Carassius auratus Centrolophus niger Channa maculata Chelon labrosus Cirrhinus molitorella Cobitis granoei Coilia ectenes Coilia reynaldi Coregonus nasus ENERGN Advanced Transfer Technologies Coreoperca whiteheadi Cottus gobio Crossocheilus latius Cynoglossus line2. Epinephelus spilotoceps Epinephelus undulosus Etmopterus baxteri Etmopterus pseudosqualilolus Eugnathichthys macroterolepis Garra mullya Gasterosteus aculeatus Glossogobius olivaceus Gobius niger Harpadon nehereus Hippoglossus stenolepis Huso huso Ictalurus meridionalis Johnius belangerii Cottus aleuticus Cottus poecilopus Cynoglossus abbreviatus Cynoglossus sinicus Cyprinus carpio carpio Cyprinus carpio xingguonensis Diplodus sargus Distichodus fasciolatus Echelus myrus Engraulis japonicus Epinephelus bruneus synonym E moara Epinephelus epistictus Epinephelus lanceolatus Epinephelus polyphekadion Epinephelus stictus Escualosa thoracata Etmopterus fusus Etmopterus princeps Euthynnus alletteratus Garra orientalis Gasterosteus wheatlandi Glyptocephalus zachirus Gobius paganellus Heterotilapia buttifokeri Hoplias malabaricus synonym Macrodon ferox Hyperoglyphe japonica Ictalurus pricei Johnius gripotus User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 Cottus cognatus Cottus reinii Cynoglossus bilinearis Cynoscion arenarius Cyprinus carpio color Cyttus australis Dissostichus eleginoides Distichodus hypostomatus Echelus uropterus Epinephelus amblycephalus Epinephelus clippertonensis Epinephelus fasciatomaculosus Epinephelus latifasciatus Epinephelus quoyanus Epinephelus striatus Esox niger Etmopterus granulosus Et
3. Rev 3 03 12 2014 Labeo cyclorhynchus Labiobarbus lunaris Lagocephalus inermis Lates calcarifer synonym Lates calcifer Lepomis humilis Lethrinus obsoletus Liza ramado Lutjanus argentimaculatus Lutjanus rivulatus Makaira indica synonym Istiompax indica Melanogrammus aeglefinus Mesoborus crocodilus Micropterus floridanus Mogurnda adspersa Mugil curema Mycteroperca acutirostris Mycteroperca olfax Mystacoleucus marginatus Neolebias trilineatus Odontesthes argentinensis Odontobutis sinensis Oreochromis niloticus Padogobius nigricans Pampus argenteus Paralichthys dentatus Paranthias furcifer Patagonothen longipes Phoxinus eos 10 ENERGN Advanced Transfer Technologies Phoxinus oxycephalus jouyi Platichthys stellatus Pollachius pollachius Pristolepis malabarica Puntius semifascicolatus Reinhardtius evermanni synonym Atheresthes evermanni Romanogobio tenuicorpis Salvelinus leucomaenis Sardinops melanostictus Scarus frenatus Schismatorhynchos nutka Schizothorax plagiostomus Scomber colias Scomberomorus munroi Seriola lalandi Siganus puellus Sphyraena barracuda Sprattus sprattus Stolephorus waitei Symphodus tinca Syncrossus reversa Tetrapturus angustirostris Thamnaconus tessellatus Thunnus tonggol Tinca tinca Trisopterus esmarkii Zenopsis nebulosus Phoxinus percnurus mantschuricus Plectropomus leopardus Pomatomus salt
4. auriga Pampus echinogaster Paraneetroplus synsipilus Paraplagusia blochii Peprilus triacanthus Labeo calbasu Labeo rohita Laemonema barbatulum Larimichthys polyactis Lepidopsetta bilineata Leptobotia rubrilabris Liza affinis Lophius piscatorius Lutjanus kasmira Luvarus imperialis Mastacembelus favus Merluccius australis Microphysogobio tafangensis Microstomus pacificus Morone chrysops Muraena helena Mycteroperca jordani Mycteroperca tigris Nemacheilus rueppelli Nibea albiflora Odontesthes incisa Oncorhynchus tshawytscha Oxyeleotris marmorata Pagrus major Pampus punctatissimus Paramisgurnus dabryanus Paraplagusia japonica Perca flavescens Labeo chrysophekadion Labeo senegalensis Laemonema goodebeanorum Lateolabrax japonicus Lepomis cyanellus Lethrinus lentjan Liza aurata Lota lota Lutjanus malabaricus Macquaria australasica Mastacembelus mastacembelus Merluccius senegalensis Micropterus dolomieu Misgurnus anguillicaudatus Morone saxatilis Muraena lentiginosa Mycteroperca microlepis Mycteroperca venenosa Neocyttus rhomboidalis Notopterus notopterus Odontesthes smitti Oreochromis mossambicus synonym Tilapia mossambica Oxyeleotris urophthalmoides Pagrus pagrus Paracheirodon axelrodi Paranthias colonus Parastromateus niger Phago boulengeri User guide SPECIALfinder Fish Detection Assay
5. 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 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 Fish Detection Assay Rev 3 03 12 2014 13 ENER N Advanced Transfer Technologies Quick Reference Guide Page 1 Product Line SPECIALfinder Part Number PAV20A Type Qualitative Storage Frozen Execution time about 120 minutes Expiry date see date on the packaging p
6. 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 extrac
7. s evene Advanced Transfer Technologies SPECIALfinder DETECTION ASSAY FISH Cat N PAV20A 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 Fish Detection Assay provide the user with a simple and reliable procedure for the detection of DNA related to species potentiall
8. acunda Atherina hepsetus Balistes capriscus Beryx decadactylus Boleophthalmus boddarti Botia kubotai Branchiostegus albus Carangoides armatus Carassius carassius Cephalopholis argus Channa marulius Chitala blanci Citharinus latus Cobitis lutheri Coilia grayii Colisa lalia Species expected positive on the basis of in silico analysis Acanthocybium solandri Acipenser brevirostrum Acipenser mikadoi Acipenser ruthenus Aetobatus flagellum Alosa pseudoharengus Amphiprion clarkii Anarhichas lupus Anguilla bicolor pacifica Anguilla luzonensis Anguilla nebulosa nebulosa Anoplopoma fimbria Atherina presbyter Balistes polylepis Beryx mollis Boops boops Botia rostrata Callorhinchus callorynchus Carangoides malabaricus Carassius cuvieri Cephalopholis sexmaculata Chanos chanos Chitala ornata Clupea pallasii Cobitis sinensis Coilia lindmani Colistium nudipinnis Acanthopagrus latus Acipenser dabryanus Acipenser naccarii Acipenser schrenckii Alburnus alburnus Alosa sapidissima Amphiprion percula Anguilla australis Anguilla celebensis Anguilla malgumora Anguilla obscura Aphia minuta Auxis rochei Balistes vetula Beryx splendens Botia almorhae Botia striata Callorhinchus capensis Caranx ignobilis Carassius gibelio Cephalopholis sonnerati Cheimerius nufar Chondrostoma lemmingii Cobitis choii Cobitis striata
9. along with GENERase ULTRA PLUS Mastermix Cat N ENGOO9 this Real Time PCR assay detects a specific DNA sequence in the DNA of Fish 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 Fish Detection Assay along with the following Polymerase Enzyme Ready to use mastermix GENERase ULTRA PLUS Mastermix Cat N ENGOOS 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 SPECIALfinder Fish Detection Assay Rev 3 03 12 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
10. atrix Protosalanx chinensis Puntius snyderi Reinhardtius hippoglossoides Ruvettus pretiosus Sander canadensis Saurida microlepis Scarus ghobban Schizopyge niger Schizothorax prenanti Scomber japonicus Scomberomorus niphonius Seriola quinqueradiata Siganus unimaculatus Sphyraena japonica Squalidus argentatus Symphodus cinereus Syncrossus beauforti Takifugu oblongus Tetrapturus audax synonym Kajikia audax Theragra finmarchica Thymallus arcticus Trachinotus blochii Trisopterus minutus Phoxinus percnrus sachalinensis Plesiomyzon baotingensis Pomatoschistus knerii Psettodes erumei Rachycentron canadum Rexea solandri Salmo trutta trutta Sarda sarda Saurida undosquamis Scarus rubroviolaceus Schizothorax dolichonema Schizothorax progastus Scomberomorus cavalla Scomberomorus semifasciatus Sicyopterus lagocephalus Sillago sihama Spicara maena Squalus megalops Symphodus melanocercus Syncrossus berdmorei Tenualosa ilisha Tetrapturus belone Thunnus alalunga Thymallus grubii Trachurus japonicus Upeneus moluccensis Phycis blennoides Pleurogrammus azonus Pomatoschistus minutus Pseudobrama simoni Rasbora borapetensis Rhabdosargus globiceps Salvelinus alpinus Sardinella albella Scardinius erythrophtalmus Scarus schlegeli Schizothorax esocinus Sciaenops ocellatus Scomberomorus commerson Scophthalmus aquosus Siganus fuscescens Sillago vincenti Sprattus ant
11. ection 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 authorisation for PCR use are not inc
12. icus Pandora Pagellus erythrinus Pink Snapper Pagrus auratus synonym Chrysophrys auratus Saithe Pollachius virens Shallow Water Cape Hake Merluccius capensis South Pacific Hake Merluccius gayi Turbot Psetta maxima synonym Scophthalmus maximus Withmouth Croaker Micropogonias furnieri French Sole Solea lascaris synonym Pegusa lascaris Grey Mullet Mugil cephalus Big Scale Sand Smelt Atherina boyeri Bigeye Tuna Thunnus obesus Canary Drum Umbrina canariensis Brown Meagre Sciaena umbra Deep Water Cape Hake Derbio Trachinotus ovatus Merluccius paradoxus European Hake Merluccius Eel Anguilla Anguilla maluas Gilthead Seabream Sparus aurata Halibut Hippoglossus hippoglossus Mediterranean Horse Mackerel Trachurus mediterraneus Lumpfish Cyclopterus lumpus North Pacific Hake Merluccius productus Patagonian Whiphake Macruronus novaezelandiae Northern Pike Esox lucius Peruvian Anchovy Engraulis ringens Rainbow Trout Onein PES Red Mullet Mullus barbatus Sapphirine Gurnard Trigla lucerna synonym Chelidonichthys lucerna Skipjack Tuna Katsuwonus pelamis Sand Steenbras Lithognathus mormyrus Shortfin Mako Isurus oxyrhincus Spanish Sardine Sardinella aurita Spiny Dogfish Squalus acanthias Whiting Merlangius Tusk Brosme brosme ESTA Yellowfin Sole Limanda aspera Yellow Goosefish Lophiu
13. ipodum Stizostedion lucioperca synonym Sander lucioperca Symphodus ocellatus Syncrossus helodes Tetraodon nigroviridis Tetrapturus georgii Thunnus maccoyii Thymallus thymallus Trachurus trachurus Variola albimarginata User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 Pinjalo pinjalo Pleurogrammus monopterygius Pristipomoides multidens Pseudorasbora parva Rastrelliger kanagurta Rhodeus pseudosericeus Salvelinus fontinalis Sardinella maderensis Scarus forsteni Schedophilus velaini Schizothorax labiatus Scomber australasicus Scomberomorus maculatus Selar crumenophthalmus Siganus guttatus Solea senegalensis Sprattus muelleri Stolephorus chinensis Symphodus roissali Syncrossus hymenophysa Tetrapturus albidus synonym Kajikia albida Tetrapturus pfluegeri Thunnus orientalis Thynnichthys polylepis Trinectes maculata Variola louti 11 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 Goose Anser spp Sheep Ovis aries Clam Tapes semidecussatus American Lobster Homarus americanus Northern Prawn Pandalus borealis Arugula Eruca vesicaria Eggplant Solanum melongena Maize Zea mays Parsley Petroselinum crispum Rapeseed Brassica napus Buffalo Bubalus bubalis Horse Equus caballus S
14. itive Control into 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 ULTRA PLUS Mastermix set the following parameters on your thermocycler Total Reaction volume 20 ul Fluorophores Quenchers Target Fish 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 Taq Activation 3 min DNA Denaturation 10 sec Annealing Extension Plate Reading 60 sec User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 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 Amplificati
15. luded 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 Fish Detection Assay Rev 3 03 12 2014 15
16. mopterus unicolor Gadus ogac Garra rufa Genypterus capensis Gobius bucchichi Gudusia chapra Heteropneustes fossilis Hoplostethus japonicus Hyporhamphus sajori Ictalurus punctatus Kuhlia mugil ENERGN Advanced Transfer Technologies Labeo angra Labeo lineatus Labrus merula Lagocephalus spadiceus Lemonema longipes Lepomis macrochirus Leuciscus rutilus Lophius americanus Lutjanus bengalensis Lutjanus russellii Makaira mazara synonym M nigricans Merluccius albidus Metzia mesembrinus Micropterus salmoides salmoides Molva molva Mullus surmuletus Mycteroperca bonaci Mycteroperca prionura Nannaethiops bleheri Neolebias unifasciatus Odontesthes bonariensis Oncorhynchus clarkii henshawi synonym Salmo clarkii henshawi Osmerus eperlanus Pagellus bogaraveo Pampus chinensis Paralichthys olivaceus Paraplagusia bilineata Pelthorhamphus novaezeelandiae Labeo bata Labeo pierrei Labrus viridis Larimichthys crocea Lepidocybium flavobrunneum Leptobotia microphthalma Leuciscus waleckii Lophius budegassa Lutjanus johnii Lutjanus sebae Mastacembelus armatus Merluccius angustimanus Micromesistius poutassou Microstomatichthyoborus bashforddeani Monotrete leiurus Muraena augusti Mycteroperca interstitialis Mycteroperca rosacea Nannaethiops unitaeniatus Nematalosa nasus Odontesthes hatcheri Oncorhynchus masou masou Osmerus mordax Pagrus
17. olatus Cynoscion regalis Cyprinus carpio haematopterus Dasyatis akajei Dissostichus mawsoni Distichodus noboli Echeneis naucrates Epinephelus akaara Epinephelus coeruleus Epinephelus fuscoguttatus Epinephelus macrospilos Epinephelus septemfasciatus Epinephelus trimaculatus Esox reichertii Etmopterus lucifer Etmopterus virens Gambusia affinis Garra spilota Glossogobius celebius Gobius cobitis Gymnodiptychus pachycheilus Hilsa kelee Hucho bleekeri Ictalurus balsanus Istiophorus albicans Coryphaena equiselis Cottus hangiongensis Ctenopharyngodon idella Cynoglossus puncticeps Cynoscion nebulosus Cyprinus carpio wananensis Dasyatis bennetti Distichodus affinis Distichodus sexfasciatus Echiichthys vipera Epinephelus areolatus Epinephelus coioides Epinephelus goreensis Epinephelus merra Epinephelus sexfasciatus Epinephelus tukula Ethmalosa fimbriata Etmopterus polli Etroplus maculatus Garra flavatra Gasterochisma melampus Glossogobius circumspectus Gobius cruentatus Harpadon mirochir Hippoglossoides platessoides Huso dauricus Ictalurus furcatus Istiophorus platypterus Coryphaena hippurus Cottus nozawae Cyclocheilichthys enoplos Cynoglossus semilaevis Cyprinus carpio Cyprinus carpio wuyanensis Diagramma pictum Distichodus decemmaculatus Drepane africanus Eleginops maclovinus Epinephelus awoara Epinephelus daemelii Epinephelus howlandi Epinephelus miliaris
18. on After setting the baseline the analysis outcome should be evaluated following the indications below If the following conditions are met TEST Fish FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are TEST Fish FAM Internal Amplification Control HEX Positive Sample Negative Sample e 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 User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 6 ENER N Advanced Transfer Technologies Inclusivity Panel Aee Palesk Gate Argentine Hake Merluccius Argentine Seabass Atlantic Cod Gadus morhua chalcogrammus synonym hubbsi Wes aeg lcu Theragra chalcogramma Atlantic Mackerel Scomber scombrus Anchovy Engraulis encrasicolus Atlantic Sturgeon Acipenser sturio Atlantic Pomfret Brama brama Atlantic Bluefin Tuna Ginn Atlantic Salmon Salmo salar Bearded Brotula Brotula barbata Brill Scophthalmus rhombus Common Sole Solea vulgaris Duskytail Grouper Epinephelus bleekeri European Plaice Pleuronectes platessa Greater Amberjack Seriola dumerili Keta Salmon Oncorhynchus keta Nile Perch Lates nilot
19. roduct 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 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 before opening it Prepare SPECIALfinder WORKING Mastermix by adding 250 ul of SPECIALfinder OLIGO Mix into each tube prefilled with 500 ul of GENERase ULTRA PLUS Mastermix Cat N ENGOO9 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 ULTRA PLUS vials with target name after OLIGO Mix addition Vortex briefly Positive Control and samples before proceeding further spin to collec
20. s litulon Blackspotted Protonibea diacanthus Capelin Mallotus villosus Devil Anglerfish Lophius vomerinus European Perch Perca fluviatilis Goatfish Parupeneus spp Herring Clupea harengus Megrim Lepidorhombus whiffiagonis Oceanic Sole Synaptura spp Pink Cusk Eel Genypterus blacodes Redfish Sebastes marinus synonym Sebastes norvegicus Seabass Dicentrarchus labrax Smooth Oreo Pseudocyttus maculatus Blue Shark Prionace glauca Coho Salmon Oncorhynchus kisutch Dory Zeus faber European Pilchard Sardina pilchardus Goldstripe Sardinella Sardinella gibbosa Icefish Neosalanx taihuensis Narrownose Smooth Hound Mustelus schmitti Pacific Cod Gadus macrocephalus Pink Salmon Oncorhynchus gorbuscha Rock Grenadier Coryphaenoides rupestris Senegalese Tonguesole Cynoglossus senegalensis Smooth Hound Mustelus mustelus Swordfish Xiphias gladius Thornback Ray Raja clavata Winter Skate Leucoraja ocellata Yellowfin Tuna Thunnus albacares User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 Witch Flounder Glyptocephalus cynoglossus ENER N Advanced Transfer Technologies Abramis brama Acipenser baerii Acipenser medirostris Acipenser persicus Acipenser transmontanus Alosa alosa Amphilophus citrinellus Anarhichas minor Anguilla bicolor bicolor Anguilla japonica Anguilla mossambica Anodontostoma ch
21. t 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 Fish FAM BHO1 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 Fish Detection Assay Rev 3 03 12 2014 14 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 57 60 sec The thermal profile presented above was optimized for GENERase ULTRA PLUS Mastermix Cat N ENGOO9 Results analysis If the following conditions are met TEST Fish FAM Internal Amplification Control HEX Positive Control Negative Control Then the possible results for any sample are TEST Fish 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 s
22. tion procedure details 3 3 Detection via Real Time PCR Material Equipment Source Real Time PCR System Generon or other Lab Suppliers Specialfinder Fish Detection Assay Generon Cat N PAV20A GENERase ULTRA PLUS Mastermix Generon Cat N ENGOOS Optical Adhesive Seal and Optical reaction plate or Gies Ce ael ein 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 Fish Detection Assay Rev 3 03 12 2014 4 s evene 4 Real Time PCR detection 4 1 Reaction setup Allow the reagents to thaw GENERase ULTRA 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 ul of SPECIALfinder OLIGO Mix with 500 ul of GENERase ULTRA 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 Pos
23. wine Sus scrofa domesticus Cuttlefish Sepia officinalis Coastal Mud Shrimp Solenocera crassicornis Norway lobster Nephrops norvegicus Basil Ocimum basilicum Fennel Foeniculum vulgare Mango Mangifrea indica Pepper Capsicum annum Rice Oryza sativa Donkey Equus asinus Poultry Gallus gallus domesticus Wild boar Sus scrofa scrofa Crustaceans and Molluscs Poulpe Octopus cyaena Kiddi Shrimp Parapenaeopsis stylifera Pacific Shrimp Heterocarpus affinis Vegetabile Bean Phaseolus vulgaris Garlic Allium sativum Mushrooms Agaricus campestris Pine Nuts Pinus pinea Spinach Spinacia oleracea Duck Anas crecca Cairina muschata Quail Coturnix coturnix Turkey Meleagris gallopavo Squid Loligo edulis Lobster Palinurus spp Prawn Penaeus vannamei Buckwheat Fagopyrum esculentum Grape Vitis vinifera Olive Olea europaea Plum Prunus domestica Sunflower Helianthus annuus Goat Capra hircys Rabbit Oryctolagus cuniculus Lobster Palinurus spp Mantis Shrimp Squill mantis Red Squat Lobster Pleuroncodes monodon Carrot Daucus carota Lemon Citrus limon Onion Allium cepa Potato Solanum tuberosum Tomato Solanum lycopersicon En AN extus poem pu Almond Prunus dulcis Durum Wheat Triticum durum Mustard Brassica nigra Rye Secale cereale Walnut Juglans regia Barle
24. y Hordeum vulgare Hazelnut Corylus avellana Oat Avena sativa Sesame Sesamum indicum Brasilian Walnut Bertholletia excelsa Kamut Triticum turgidum Peanut Arachis hypogaea Soft Wheat Triticum aestivum Cashew Anacardium occidentalis Lupine Lupinus albus Pecan Nut Carya illinoinensis Soybean Glycine max User guide SPECIALfinder Fish Detection Assay Rev 3 03 12 2014 Celery Apium graveolens Macadamia Nut Macadamia integrifolia Pistachio Pistacia vera Spelt Triticum monococcum 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 Il Positive Control reactions failed to amplify
25. y allergenic in food and feed matrices as well as swabs Such a detection is an ndirect Proof of the potential presence of the Fish 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 Fish 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 Fish Detection Assay Rev 3 03 12 2014 2 s evene Advanced Transfer Technologies 2 SPECIALfinder Fish Detection Assay When used
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