Technical Note

Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
SPECIALfinder FISH Assay
[1]
Cat. N. PAV20A-PAV20A-50
Background
Since "Allergen Directive" issuing by the EU (2003/89/EC, 2006/142/CE), followed by Regulation (EU) N° 1169/2014, all
food products on the market must be labeled for the presence (regardless of percentage) of allergenic substances.
Currently in Europe is estimated that 8% of adults and 3% of children suffer from food allergies. For these people the
ingestion of foods containing small quantities of substances to which they are hypersensitive can cause fatal
anaphylactic shock.
Real-Time PCR is the gold-standard method for detection quantification of specific DNA sequences of different
allergens. The method combined with an appropriate nucleic acid extraction system allows both the analysis of raw
materials and of finished products as well.
SPECIALfinder assays
SPECIALfinder is the product line developed by Generon with premium quality molecular biology reagents to provide
the customer a “turn-key“ solution for the detection a wide selection of specific allergens according to EU norms and
DIN EN 15634-1. This real-time PCR assays was developed at Generon for the rapid detection and identification of
FISH, in environmental swabs and in various food/feed matrices after an appropriate DNA extraction.
Preparation of samples
Homogenization of a representative sample and extraction of DNA using ION Force DNA Extractor FAST (Cat.#
EXD001) is recommended. All the validation experiments were performed extracting the DNA of pure allergen, or
spiked test matrices using Generon ION Force DNA Extractor FAST.
Detection using Real-Time PCR
Target detection via Real-Time PCR is performed using a single degradative fluorescent probe FAM-labeled.
Using GENERase PLUS, an additional detection channel (HEX) becomes available to detect the Internal Amplification
Control (IAC), excluding false negative results due to a PCR inhibition.
The detection system is validated on following real-time instruments: Biorad CFX and MiniOpticon, Stratagene
MxSeries, ABI 7300-7500-7900-StepONE-StepONE Plus, Light Cycler 480, Eppendorf realplex, Rotor-Gene Q.
Detection limit
This assay uses the polymerase chain reaction (PCR) to amplify a genetic target typical of the allergenic species.
PCR technique can typically detect down to 1-10 copies of the target sequences. The sensitivity of the assay was
determined using purified genomic DNA from FISH, according to ISO-11843-2:2000 collecting data from at least five
calibration curves to calculate the limit of detection. The lowest detectable copy number was calculated to be at
least ≤ 5 DNA-copies.
Considering many factors like industrial processing, DNA extraction yield, genome size of the ingredients influences
the analysis the real detection/quantification limit is strictly matrix dependant, and needs to be determined through
an in-house validation. Experiments performed at Generon with various food matrices, extracting DNA with Generon
ION Force DNA Extractor Fast (Cat. N. EXD001), shown LOD between 1 and 0.5 ppm and depending on sample
matrix.
See below an example of the curves generated in a Real-time PCR run with SPECIALfinder FISH assay in a 20 µl total
reaction volume with a defined amount of purified DNA template, demonstrating the capacity of the test to detect
down to 500 fg of target DNA (lower than 5 genomic units) as measured by Droplet Digital PCR.
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
[2]
FISH analyzed by means of ddPCR were: Thunnus albacares, Merluccius hubbsi, Anguilla anguilla, Engraulis
encrasicolus, Epinephelus bleekeri, Sardina pilchardus, Brotula barbata, Oncorhynchus keta.
The LOD of the test was challenged on real samples using spiked matrices (beans, corn and wheat flour, chocolate
snacks, spices, olive oil, minced pork meat), prepared as follows: 9 g of the dispersing matrix (FISH free), were spiked
with 1 g of the allergen (lyophilized tuna, Thunnus albacares). The mixture was carefully homogenized and the DNA
of the whole10 g sample was extracted obtaining a 10% (w/w), FISH contaminated sample. This extract was then
scalar diluted using DNA extracted from the FISH-free dispersing matrix down to a concentration 0.01% of FISH. The
LOD showed to be always at least 0.05%.
Additionally, different food preparations labelled to contain low FISH (lower than 0.1%) percentages were analyzed
(see statistical data paragraphs).
Repeatability and Reproducibility
To test the repeatability of the SPECIALfinder FISH Detection Assay, 20 copies of FISH DNA extracts were amplified by
Real-Time PCR in triplicate, 3 times at weekly intervals with three different operators. The assay exhibited a high
degree of both repeatability and reproducibility. The relative standard deviation within a test did not exceed 5%.
Specificity - Exclusivity
The method presents 100% specificity as none of the following species DNA extracts shows a positive result in a 20 µl
total reaction volume (40 ng DNA).
Vegetable Allergens:
Almond (Prunus dulcis), Barley (Hordeum vulgare), Brasilian Walnut (Bertholletia excelsa), Cashew (Anacardium occidentalis),
Celery (Apium graveolens), Durum Wheat (Tritucum durum), Hazelnut (Corylus avellana), Kamut (Triticum turgidum), Lupine (Lupinus
albus), Macadamia Nut (Macadamia integrifolia), Mustard (Brassica nigra), Oat (Avena sativa), Peanut (Arachis hypogaea), Pecan
Nut (Carya illinoiensis), Pistachio (Pistacia vera), Rye (Secale cereale), Sesame (Sesamum indicum), Soft Wheat (Triticum aestivum),
Soybean (Glycine max), Spelt (Triticum monococcum), Walnut (Junglas regia).
Other vegetables:
Ananas (Ananas comossus), Apple (Malus domestica), Apricot (Prunus armeniaca), Arugula (Eruca vesicaria) Banana (Musa
acuminata), Basil (Ocimum basilicum), Broccoli (Brassica oleracea), Buckwheat (Fagopyrum esculentum), Carrot (Daucus carota),
Chard (Beta vulgaris var cicla), Cherry (Prunus avium), Chestnut (Castanea vulgaris), Clementine (Citrus x clementina), Cocoa
(Theobroma cacao), Coconut (Cocos nucifera), Corn (Zea mays), Cucumber (Cucumis sativus), Eggplant (Solanum melongena),
Fennel (Foeniculum vulgare), Garlic (Allium sativum), Grape (Vitis vinifera), Grapefruit (Citrus × paradisi), Green Beans (Phaseolus
vulgaris), Lemon (Citrus limon), Mandarine (Citrus reticulata), Mango (Mangifera indica), Mushroom (Agaricus campestris), Olive
(Olea europaea), Onion (Allium cepa), Orange (Citrus aurantium), Parsley (Petroselinum crispum), Peach (Prunus persica), Pepper
(Capsicum annuum), Pear (Pyrus communis), Peas (Pisum sativum), Pine Nuts (Pinus pinea), Plum (Prunus domestica), Pomelo (Citrus
maxima), Poplar (Populus spp.), Potato (Solanum tuberosum), Radish (Raphanus sativus), Rapeseed (Brassica napus), Rice (Oryza
sativa), Spinach (Spinacia oleracea), Strawberry (Fragaria x ananassa), Sunflower (Helianthus annuus), Tomato (Solanum
lycopersicon), Zucchini (Cucurbita pepo).
Other Allergens (ShellFISH and Crustaceans)
American Lobster (Homarus americanus), Argentine Shortfin Squid (Illex argentinus), Big Blue Octopus (Octopus cyanea), Coastal
Mud Shrimp (Solenocera crassicornis), CuttleFISH (Sepia officinalis), Ensis Siliqua (Ensis minor), Kiddi Shrimp (Parapenaeopsis stylifera),
King Scallop (Pecten maximus), Lobster (Palinurus spp.), Manila Clam (Tapes semidecussatus), Mantis Shrimp (Squilla mantis), Mitre
Squid (Loligo chinensis, synonym: Uroteuthis chinensis), Mussel (Mytilus edulis), Northern Prawn (Pandalus borealis), Norway lobster
(Nephrops norvegicus), Pacific Shrimp (Heterocarpus affinis), Prawn (Penaeus vannamei), Red Squat Lobster (Pleuroncodes
monodon), Snow Crab (Chinoecetes opilio), Squid (Loligo edulis).
Meat:
Beef (Bos taurus), Buffalo (Bubalus bubalis), Donkey (Equus asinus), Duck (Anas crecca, Cairina muschata, Anas spp.), Goat (Capra
hircus), Goose (Anser spp.), Horse (Equus caballus), Poultry (Gallus gallus domesticus), Quail (Coturnix coturnix), Rabbit (Oryctolagus
cuniculus), Sheep (Ovis aries), Swine (Sus scrofa domesticus), Wild boar (Sus scrofa), Turkey (Meleagris gallopavo).
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
[3]
Specificity - Inclusivity
The inclusivity of the FISH assay was verified either in vitro (82 FISH species) or in silico (546 FISH species).
FISH species tested in vitro
Atlantic Cod (Gadus morhua), Pacific Cod (Gadus macrocephalus), Alaska Pollock (Theragra chalcogramma, synonym: Gadus
chalcogrammus), Atlantic Mackerel (Scomber scombrus), Yellowfin tuna (Thunnus albacares), Bigeye Tuna (Thunnus obesus),
Atlantic Bluefin Tuna (Thunnus thynnus), Pink Salmon (Oncorhynchus gorbuscha), Keta Salmon (Oncorhynchus keta), Coho Salmon
Oncorhynchus kisutch), Rainbow Trout (Oncorhynchus mykiss), Anchovy (Engraulis encrasicolus), Peruvian Anchovy (Engraulis
ringens), European Pilchard (Sardina pilchardus), SwordFISH (Xiphias gladius), Argentine Seabass (Acanthistius brasilianus), Atlantic
Sturgeon (Acipenser sturio), Eel (Anguilla anguilla), Big-Scale Sand-Smelt (Atherina boyeri), Atlantic Pomfret (Brama brama), Tusk
(Brosme brosme), Bearded Brotula (Brotula barbata), Herring (Clupea harengus), Rock Grenadier (Coryphaenoides rupestris),
LumpFISH (Cyclopterus lumpus), Senegalese Tonguesole (Cynoglossus senegalensis), Seabass (Dicentrarchus labrax), Duskytail
Grouper (Epinephelus bleekeri), Northern Pike (Esox lucius), Pink Cusk-Eel (Genypterus blacodes), Witch Flounder (Glyptocephalus
cynoglossus), Halibut (Hippoglossus hippoglossus), Shortfin Mako (Isurus oxyrinchus), Skipjack Tuna (Katsuwonus pelamis), Nile Perch
(Lates niloticus), Sand Steenbras (Lithognathus mormyrus), Megrim (Lepidorhombus whiffiagonis), Winter Skate (Leucoraja ocellata),
Yellowfin Sole (Limanda aspera), Yellow GooseFISH (Lophius litulon), Devil AnglerFISH (Lophius vomerinus), Patagonian Whiphake
(Macruronus novaezelandiae), Capelin (Mallotus villosus), Whiting (Merlangius merlangus), Shallow-Water Cape Hake (Merluccius
capensis), South Pacific Hake (Merluccius gayi), Argentine Hake (Merluccius hubbsi), European Hake (Merluccius merluccius),
Deep-Water Cape Hake (Merluccius paradoxus), North Pacific Hake (Merluccius productus), Withemouth Croaker (Micropogonias
fournieri), Grey Mullet (Mugil cephalus), Red Mullet (Mullus barbatus), Smooth-Hound (Mustelus mustelus), Narrownose SmoothHound (Mustelus schmitti), IceFISH (Neosalanx taihuensis), Pandora (Pagellus erythrinus), Pink Snapper (Pagrus auratus, synonym:
Chrysophrys auratus), GoatFISH (Parupenaeus spp.), European Perch (Perca fluviatilis), European Plaice (Pleuronectes platessa),
Saithe (Pollachius virens), Blue Shark (Prionace glauca), Blackspotted Croaker (Protonibea diacanthus), Turbot (Psetta maxima,
synonym: Scophthalmus maximus), Smooth Oreo (Pseudocyttus maculatus), Thornback Ray (Raja clavata), Atlantic Salmon (Salmo
salar), Spanish Sardine (Sardinella aurita), Goldstripe Sardinella (Sardinella gibbosa), Brown Meagre (Sciaena umbra), Brill
(Scophthalmus rhombus), RedFISH (Sebastes marinus, synonym: Sebastes norvegicus), Greater Amberjack (Seriola dumerili), French
Sole (Solea lascaris, synonym: Pegusa lascaris), Common Sole (Solea vulgaris), Gilthead Seabream (Sparus aurata), Spiny DogFISH
(Squalus acanthias), Oceanic Sole (Synaptura spp.), Mediterranean Horse Mackerel (Trachurus mediterraneus), Derbio (Trachinotus
ovatus), Sapphirine Gurnard (Trigla lucerna, synonym: Chelidonichthys lucerna), Canary Drum (Umbrina canariensis), Dory (Zeus
faber).
FISH species verified in silico
Abramis brama, Acanthocybium solandri, Acanthopagrus latus, Acanthurus lineatus, Achirus lineatus, Acipenser baerii, Acipenser
brevirostrum, Acipenser dabryanus, Acipenser fulvescens, Acipenser gueldenstaedtii, Acipenser medirostris, Acipenser mikadoi,
Acipenser naccarii, Acipenser nudiventris, Acipenser oxyrhynchus oxyrhynchus, Acipenser persicus, Acipenser ruthenus, Acipenser
schrenckii, Acipenser sinensis, Acipenser stellatus, Acipenser transmontanus, Aetobatus flagellum, Alburnus alburnus, Alepocephalus
bairdii, Allocyttus niger, Alosa alosa, Alosa pseudoharengus, Alosa sapidissima, Aluterus scriptus, Ameiurus melas, Amphilophus
citrinellus, Amphiprion clarkii, Amphiprion percula, Amphiprion polymnus, Anabas testudineus, Anarhichas lupus, Anarhichas minor,
Anguilla australis, Anguilla australis schmidti, Anguilla bengalensis labiata, Anguilla bicolor bicolor, Anguilla bicolor pacifica, Anguilla
celebensis, Anguilla dieffenbachii, Anguilla interioris, Anguilla japonica, Anguilla luzonensis, Anguilla malgumora, Anguilla
marmorata, Anguilla megastoma, Anguilla mossambica, Anguilla nebulosa nebulosa, Anguilla obscura, Anguilla reinhardtii,
Anguilla rostrata, Anodontostoma chacunda, Anoplopoma fimbria, Aphia minuta, Arctogadus glacialis, Argyrosomus japonicus,
Atherina hepsetus, Atherina presbyter, Auxis rochei, Auxis thazard, Bahaba taipingensis (synonym: Otolithes lini), Balistes capriscus,
Balistes polylepis, Balistes vetula, Barbatula nuda, Belonophago hutsebouti, Beryx decadactylus, Beryx mollis, Beryx splendens,
Bodianus mesothorax, Bodianus rufus, Boleophthalmus boddarti, Boops boops, Botia almorhae, Botia histrionica, Botia kubotai, Botia
lohachata, Botia rostrata, Botia striata, Branchiostegus albus, Branchiostegus argentatus, Branchiostegus japonicus, Callorhinchus
callorynchus, Callorhinchus capensis, Callorhinchus milii, Campylomormyrus compressirostris, Carangoides armatus, Carangoides
malabaricus, Caranx ignobilis, Caranx melampygus, Carassius auratus, Carassius carassius, Carassius cuvieri, Carassius gibelio, Catla
catla, Centrolophus niger, Cephalopholis argus, Cephalopholis sexmaculata, Cephalopholis sonnerati, Channa argus, Channa
maculata, Channa marulius, Chanos chanos, Cheimerius nufar, Chelidonichthys capensis, Chelon labrosus, Chitala blanci, Chitala
ornata, Chondrostoma lemmingii, Cirrhinus molitorella, Cirrhinus mrigala, Citharinus latus, Clupea pallasii, Cobitis choii, Cobitis
elongatoides, Cobitis granoei, Cobitis lutheri, Cobitis sinensis, Cobitis striata, Cobitis takatsuensis, Coilia ectenes, Coilia grayii, Coilia
lindmani, Coilia mystus, Coilia nasus, Coilia reynaldi, Colisa lalia, Colistium nudipinnis, Coregonus clupeaformis, Coregonus lavaretus,
Coregonus nasus, Coreoperca whiteheadi, Coryphaena equiselis, Coryphaena hippurus, Cottus aleuticus, Cottus cognatus, Cottus
gobio, Cottus hangiongensis, Cottus nozawae, Cottus poecilopus, Cottus reinii, Crossocheilus latius, Ctenopharyngodon idella,
Cyclocheilichthys enoplos, Cynoglossus abbreviatus, Cynoglossus bilinearis, Cynoglossus lineolatus, Cynoglossus puncticeps,
Cynoglossus semilaevis, Cynoglossus sinicus, Cynoscion arenarius, Cynoscion nebulosus, Cynoscion regalis, Cyprinus carpio,
Cyprinus carpio carpio, Cyprinus carpio color, Cyprinus carpio haematopterus, Cyprinus carpio wananensis, Cyprinus carpio
wuyanensis, Cyprinus carpio xingguonensis, Cyttus australis, Dasyatis akajei, Dasyatis bennetti, Diagramma pictum, Diplodus sargus,
Dissostichus eleginoides, Dissostichus mawsoni, Distichodus affinis, Distichodus decemmaculatus, Distichodus fasciolatus, Distichodus
hypostomatus, Distichodus noboli, Distichodus sexfasciatus, Drepane africanus, Echelus myrus, Echelus uropterus, Echeneis
naucrates, Echiichthys vipera, Eleginops maclovinus, Engraulis japonicus, Epinephelus akaara, Epinephelus amblycephalus,
Epinephelus areolatus, Epinephelus awoara, Epinephelus bruneus, (synonym: E. moara), Epinephelus clippertonensis, Epinephelus
coeruleus, Epinephelus coioides, Epinephelus daemelii, Epinephelus epistictus, Epinephelus fasciatomaculosus, Epinephelus
fuscoguttatus, Epinephelus goreensis, Epinephelus howlandi, Epinephelus lanceolatus, Epinephelus latifasciatus, Epinephelus
macrospilos, Epinephelus merra, Epinephelus miliaris, Epinephelus polyphekadion, Epinephelus quoyanus, Epinephelus
septemfasciatus , Epinephelus sexfasciatus, Epinephelus spilotoceps, Epinephelus stictus, Epinephelus striatus, Epinephelus
trimaculatus, Epinephelus tukula, Epinephelus undulosus, Escualosa thoracata, Esox niger, Esox reichertii, Ethmalosa fimbriata,
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
[4]
Etmopterus baxteri, Etmopterus fusus, Etmopterus granulosus, Etmopterus lucifer, Etmopterus polli, Etmopterus princeps, Etmopterus
pseudosqualilolus, Etmopterus unicolor, Etmopterus virens, Etroplus maculatus, Eugnathichthys macroterolepis, Euthynnus
alletteratus, Gadus ogac, Gambusia affinis, Garra flavatra, Garra mullya, Garra orientalis, Garra rufa, Garra spilota, Gasterochisma
melampus, Gasterosteus aculeatus, Gasterosteus wheatlandi, Genypterus capensis, Glossogobius celebius, Glossogobius
circumspectus, Glossogobius olivaceus, Glyptocephalus zachirus,, Gobius bucchichi, Gobius cobitis, Gobius cruentatus, Gobius
niger, Gobius paganellus, Gudusia chapra, Gymnodiptychus pachycheilus, Harpadon mirochir, Harpadon nehereus,
Heteropneustes fossilis, Heterotilapia buttifokeri, Hilsa kelee, Hippoglossoides platessoides, Hippoglossus stenolepis, Hoplias
malabaricus (synonym: Macrodon ferox), Hoplostethus japonicus, Hucho bleekeri, Huso dauricus, Huso huso, Hyperoglyphe
japonica, Hyporhamphus sajori, Ictalurus balsanus, Ictalurus furcatus, Ictalurus meridionalis, Ictalurus pricei, Ictalurus punctatus,
Istiophorus albicans, Istiophorus platypterus, Johnius belangerii, Johnius gripotus, Kuhlia mugil, Labeo angra, Labeo bata, Labeo
calbasu, Labeo chrysophekadion, Labeo cyclorhynchus, Labeo lineatus, Labeo pierrei, Labeo rohita, Labeo senegalensis,
Labiobarbus lunaris, Labrus merula, Labrus viridis, Laemonema barbatulum, Laemonema goodebeanorum, Lagocephalus inermis,
Lagocephalus spadiceus, Larimichthys crocea, Larimichthys polyactis, Lateolabrax japonicus, Lates calcarifer (synonym: Lates
calcifer), Lemonema longipes, Lepidocybium flavobrunneum, Lepidopsetta bilineata, Lepomis cyanellus, Lepomis humilis, Lepomis
macrochirus, Leptobotia microphthalma, Leptobotia rubrilabris, Lethrinus lentjan, Lethrinus obsoletus, Leuciscus rutilus, Leuciscus
waleckii, Liza affinis, Liza aurata, Liza ramado, Lophius americanus, Lophius budegassa, Lophius piscatorius, Lota lota, Lutjanus
argentimaculatus, Lutjanus bengalensis, Lutjanus johnii, Lutjanus kasmira, Lutjanus malabaricus, Lutjanus rivulatus, Lutjanus russellii,
Lutjanus sebae, Luvarus imperialis, Macquaria australasica, Makaira indica (synonym: Istiompax indica), Makaira mazara (synonym:
M. nigricans), Mastacembelus armatus, Mastacembelus favus, Mastacembelus mastacembelus, Melanogrammus aeglefinus,
Merluccius albidus, Merluccius angustimanus, Merluccius australis, Merluccius senegalensis, Mesoborus crocodilus, Metzia
mesembrinus, Micromesistius poutassou, Microphysogobio tafangensis, Micropterus dolomieu, Micropterus floridanus, Micropterus
salmoides salmoides, Microstomatichthyoborus bashforddeani, Microstomus pacificus, Misgurnus anguillicaudatus, Mogurnda
adspersa, Molva molva, Monotrete leiurus, Morone chrysops, Morone saxatilis, Mugil curema, Mullus surmuletus, Muraena augusti,
Muraena helena, Muraena lentiginosa, Mycteroperca acutirostris, Mycteroperca bonaci, Mycteroperca interstitialis, Mycteroperca
jordani, Mycteroperca microlepis, Mycteroperca olfax, Mycteroperca prionura, Mycteroperca rosacea, Mycteroperca tigris,
Mycteroperca venenosa, Mystacoleucus marginatus, Nannaethiops bleheri, Nannaethiops unitaeniatus, Nemacheilus rueppelli,
Nematalosa nasus, Neocyttus rhomboidalis, Neolebias trilineatus, Neolebias unifasciatus, Nibea albiflora, Notopterus notopterus,
Odontesthes argentinensis, Odontesthes bonariensis, Odontesthes hatcheri, Odontesthes incisa, Odontesthes smitti, Odontobutis
sinensis, Oncorhynchus clarkii henshawi (synonym: Salmo clarkii henshawi), Oncorhynchus masou masou, Oncorhynchus
tshawytscha, Oreochromis mossambicus, (synonym: Tilapia mossambica), Oreochromis niloticus, Osmerus eperlanus, Osmerus
mordax, Oxyeleotris marmorata, Oxyeleotris urophthalmoides, Padogobius nigricans, Pagellus bogaraveo, Pagrus auriga, Pagrus
major, Pagrus pagrus, Pampus argenteus, Pampus chinensis, Pampus echinogaster, Pampus punctatissimus, Paracheirodon
axelrodi, Paralichthys dentatus, Paralichthys olivaceus, Paramisgurnus dabryanus, Paraneetroplus synsipilus, Paranthias colonus,
Paranthias furcifer, Paraplagusia bilineata, Paraplagusia blochii, Paraplagusia japonica, Parastromateus niger, Patagonothen
longipes, Pelthorhamphus novaezeelandiae, Peprilus triacanthus, Perca flavescens, Phago boulengeri, Phoxinus eos, Phoxinus
oxycephalus jouyi, Phoxinus percnrus sachalinensis, Phoxinus percnurus mantschuricus, Phycis blennoides, Pinjalo pinjalo, Platichthys
stellatus, Plectropomus leopardus, Plesiomyzon baotingensis, Pleurogrammus azonus, Pleurogrammus monopterygius, Pollachius
pollachius, Pomatomus saltatrix, Pomatoschistus knerii, Pomatoschistus minutus, Pristipomoides multidens, Pristolepis malabarica,
Protosalanx chinensis, Psettodes erumei, Pseudobrama simoni, Pseudorasbora parva, Puntius semifascicolatus, Puntius snyderi,
Rachycentron canadum, Rasbora borapetensis, Rastrelliger kanagurta, Reinhardtius evermanni (synonym: Atheresthes evermanni),
Reinhardtius hippoglossoides, Rexea solandri, Rhabdosargus globiceps, Rhodeus pseudosericeus, Romanogobio tenuicorpis,
Ruvettus pretiosus, Salmo trutta trutta, Salvelinus alpinus, Salvelinus fontinalis, Salvelinus leucomaenis, Sander canadensis, Sarda
sarda, Sardinella albella, Sardinella maderensis, Sardinops melanostictus, Saurida microlepis, Saurida undosquamis, Scardinius
erythrophtalmus, Scarus forsteni, Scarus frenatus, Scarus ghobban, Scarus rubroviolaceus, Scarus schlegeli, Schedophilus velaini,
Schismatorhynchos nutka, Schizopyge niger, Schizothorax dolichonema, Schizothorax esocinus, Schizothorax labiatus, Schizothorax
plagiostomus, Schizothorax prenanti, Schizothorax progastus, Sciaenops ocellatus, Scomber australasicus, Scomber colias, Scomber
japonicus, Scomberomorus munroi,, Scomberomorus cavalla, Scomberomorus commerson, Scomberomorus maculatus,
Scomberomorus niphonius, Scomberomorus semifasciatus, Scophthalmus aquosus,, Selar crumenophthalmus, Seriola lalandi, Seriola
quinqueradiata, Sicyopterus lagocephalus, Siganus fuscescens, Siganus guttatus, Siganus puellus, Siganus unimaculatus, Sillago
sihama, Sillago vincenti, Solea senegalensis, Sphyraena barracuda, Sphyraena japonica, Spicara maena, Sprattus antipodum,
Sprattus muelleri, Sprattus sprattus, Squalidus argentatus, Squalus megalops, Stizostedion lucioperca (synonym: Sander lucioperca),
Stolephorus chinensis, Stolephorus waitei, Symphodus cinereus, Symphodus melanocercus, Symphodus ocellatus, Symphodus
roissali, Symphodus tinca, Syncrossus beauforti, Syncrossus berdmorei, Syncrossus helodes, Syncrossus hymenophysa, Syncrossus
reversa, Takifugu oblongus, Tenualosa ilisha, Tetraodon nigroviridis, Tetrapturus albidus (synonym: Kajikia albida), Tetrapturus
angustirostris, Tetrapturus audax (synonym: Kajikia audax), Tetrapturus belone, Tetrapturus georgii, Tetrapturus pfluegeri,
Thamnaconus tessellatus, Theragra finmarchica, Thunnus alalunga, Thunnus maccoyii, Thunnus orientalis, Thunnus tonggol, Thymallus
arcticus, Thymallus grubii, Thymallus thymallus, Thynnichthys polylepis, Tinca tinca, Trachinotus blochii, Trachurus japonicus, Trachurus
trachurus, Trinectes maculata, Trisopterus esmarkii, Trisopterus minutus, Upeneus moluccensis, Variola albimarginata, Variola louti,
Zenopsis nebulosus,
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
[5]
Robustness
Robustness of the present method was determined introducing experimental and technical variations in the
parameters (user, annealing temperature, RT equipment, type of plate) using five positive and five negative samples
and performed in triplicate.
Result:
Congruent results were obtained for all samples in all parameters variants tested. All samples also presented the
expected test results.
Trueness
Trueness of the method was evaluated using 10 positive and 10 negative food samples All the samples were tested in
triplicate.
Result:
All samples presented the expected test results for all the replicates, corresponding to a trueness of 100%
Performance Characteristics for validation
The determination of false positive and false negative rate was performed in 20 food samples (10 positive and 10
negative samples) in 10 extraction replicates and 3 detection replicates.
False positive rate corresponds to the probability that a known negative sample has been classified as positive by
the method. The false positive rate (that can be expressed as a percentage) is calculated by the expression:
Pfpos=nfpos/nrneg+nfpos x 100%
In which:
Pfpos: False positive rate
nrneg: Number of real negative test results
nfpos: Number of misclassified known negative samples
Result:
For the present method, false positive rate was calculated based on 20 samples. The final result was 0%
False negative rate corresponds to the probability that a known positive sample has been classified as negative by
the method. The false negative rate (that can be expressed as a percentage) is calculated by the expression:
Pfneg=nfneg/nrpos+nfneg x 100%
In which:
Pfneg: False negative rate
nrpos: Number of real positive test results
nfneg: Number of misclassified known positive samples
Result:
For the present method, false positive rate was calculated based on 20 samples. The final result was 0%
Positive predictive value (PPV) is calculated by the expression:
PPV=nrpos/nrpos+nfpos x 100%
In which:
nrpos: Number of real positive test results
nfneg: Number of misclassified known positive samples
Result:
The positive predictive value determined was 100%.
Negative predictive value (NPV) is calculated by the expression:
NPV=nrneg/nrneg+nfneg x 100%
In which:
nrneg: Number of real negative test results
nfneg: Number of misclassified known negative samples
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
Technical Note SPECIALfinder FISH Assay Rev. 1 - 04/01/16
Result:
The negative predictive value determined was 100%.
Criteria for acceptance and declaration of performance
Generon validation requirements are the following:
Inclusivity: 100%
Exclusivity: 99%
Selectivity: 99%
Trueness: 99%
False positive rate: 1%
False negative rate: 1%
The tests performed and the here reported results are in line with Generon minimum validation requirements. The
product must be considered valid and fitting the analytical purposes.
Via San Geminiano, 4
41030 San Prospero (MO) – Italy
: +39 059 8637161
: [email protected]
[6]