User Manual

SPECIALfinder
Fish
DNA Detection Assay
Cat. N. PAV20A / PAV20A-50
User Manual
Via San Geminiano, 4
41030 San Prospero (MO)- Italy
: +39 059 8637161
: +39 059 7353024
: [email protected]
: www.generon.it
[1]
User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
1 - Introduction
The SPECIALfinder Detection Assay provide the user with a simple and reliable procedure for the
detection of DNA related to species potentially allergenic, in food and feed matrices as well as swabs.
Such a detection is an Indirect Proof of the potential presence of the allergenic proteins into the matrix,
being proteins the real allergens.
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.
The LOD of all SPECIALfinder assays in genomic copies number was validated using ddPCR (Droplet
Digital PCR), a novel technique capable to count physically the copy number of a selected amplifiable
target.
Many factors like industrial processing, DNA extraction yield, genome size of the ingredients, influence the
analysis. The real detection/quantification limit is thus 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), have shown LOD
between 1 and 0.5 ppm depending on sample matrix.
Generon SPECIALfinder catalogue offers some ancillary products for each allergen target to support inhouse method validations:
SPECIALfinder DigiCount (Cat. N. PAV20R): DNA buffered solution at specific genomic units
concentration.
SPECIALfinder SpyX (Cat. N. PAV20X): a tube with 5 g at specific contamination level to be used
as DNA extraction and qPCR process control.
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
2 - SPECIALfinder Fish Detection Assay
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 Box Content
Box 50 reactions
Box 100 reactions
Volume
N.vials
(µl)
2
250
SPECIALfinder OLIGO Mix *
1
Volume
(µl)
250
Positive Control
1
85
2
85
Negative Control
GENERase ULTRA PLUS
Mastermix
1
300
1
300
1
500
2
500
N.vials
* reagents are supplied with a 5% of extra volume.
When 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.
2.2 – Storage & Expiry information
Refer to the date on the packaging. Product validity refers to the product kept intact in its original
packaging. Protect reagents from light exposure as OLIGO Mix reagents are photosensitive. Store frozen.
2.3 – Disclaimers
Generon s.r.l. guarantees the buyer exclusively concerning the quality of reagents and of the
components used to manufacture the product. Generon S.r.l. is not responsible and cannot anyway be
considered responsible or jointly responsible for any possible damages resulting from the utilization of the
product by the user and from the data obtained.
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
3 – Materials and equipments needed
3.1 – Extraction(1)
Material/Equipment
Source
Extraction Kit
Generon ION Force DNA Extractor FAST (Cat. N. EXD001)
Chemicals: n-Hexane
Lab Suppliers
Tubes, 50 ml and 15 ml
DNAse/RNAse Free Water
Vortexer
Benchtop Centrifuge for 50 ml Tubes
Thermal Water Bath or Block
Pipette sets
Pipette tips (Barrier)
Generon or other Lab Suppliers
Tube rack for 1.5 ml tubes
2.0 and 1.5 ml micro-tubes
Micro centrifuge for 1.5-2.0 ml micro-tubes
DNA Extraction VACUUM BOX
Vacuum pump or Venturi meter
(1) Equipment necessary only when ION Force DNA Extractor FAST (Cat. N. EXD001) is used.
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
extraction procedure details.
3.2 – Detection via Real-Time PCR
Material/Equipment
Source
Real-Time PCR System (2)
Generon or other Lab Suppliers
Specialfinder Fish Detection Assay
Generon (Cat. N. PAV20A PAV20A-50)
Optical Adhesive Seal or Caps
Optical Reaction Plate or Strips
Generon or other Lab Suppliers
Micropipette sets
(2) The assay can be used with Biorad CFX and MiniOpticon, Stratagene MxSeries, ABI 7300-7500-7900-StepONEStepONE Plus, Light Cycler 480, Eppendorf realplex, Rotor-Gene Q etc. The assay is not compatible with Roche Light
Cycler I and II.
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
4 – Real-Time PCR detection
4.1 – Detection via Real-Time PCR
I.
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.
II.
Mix 250 µl of SPECIALfinder OLIGO Mix with 500 µl of GENERase PLUS Mastermix to prepare
SPECIALfinder Working Mastermix (WMX).
III.
Vortex briefly and spin down in order to homogenize the mix.
IV.
Transfer 15 µl of WMX into each well.
V.
Add 5 µl of Negative Control into wells acting as negative control.
VI.
Add 5 µl of each sample into wells testing the unknown samples.
VII.
Add 5 µl of Positive Control into wells acting as positive control.
VIII.
Close wells and ensure no bubbles are present at the bottom of the wells.
IX.
Spin briefly optical PCR tubes or plates.
4.2 – Instrument setup
Set the following parameters on your thermocycler:
I.
Total Reaction volume: 20 µl
II.
Fluorophores/Quenchers: Target Fish (FAM/BHQ1-NFQ); Internal Amplification Control (HEX/BHQ1NFQ). If HEX dye is not included in the list of calibrated dyes in the qPCR instrument select
alternatively VIC or JOE.
III.
Thermal profile:
[5]
Step
T (°C)
Duration
Loops
Taq Activation
95
3 min
1
DNA Denaturation
95
10 sec
Annealing/Extension + Plate Reading
57
60 sec
User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
45
5 – Data Interpretation
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/absence of allergen DNA into the sample. See below an example of the graphics obtained for
a positive (Fig. A) and a negative (Fig. B) samples, respectively for the allergen target amplification (blue
line) and for the IAC amplification (green line).
After setting the baseline and threshold analyze the plots. The PCR session is valid if the following
conditions are all met:
Sample
Target (FAM)
IAC (HEX)
Positive Control
+
+
Negative Control
-
+
If the session is valid then the possible results for an unknown sample are explained in the following table:
Target
(FAM)
+
IAC
(HEX)
+/-
Unknown sample is negative
-
+
Unknown sample inhibits PCR (**)
-
-
Sample
Unknown sample is positive
(**) In case of inhibition, DNA isolation and purification of the sample need to be improved or you may
dilute your sample 1:10 and perform a new test. Refer to the Troubleshooting paragraph (section 8) for
further suggestions.
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
6 – Inclusivity Panel
The following fish species were tested on DNA extracts:
Alaska Pollock (Gadus chalcogrammus (synonym: Theragra chalcogramma))
Mediterranean Horse Mackerel (Trachurus mediterraneus)
Anchov y (Engraulis encrasicolus)
Megrim (Lepidorhombus whiffiagonis)
Argentine Hake (Merluccius hubbsi)
Narrownose Smooth-Hound (Mustelus schmitti)
Argentine Seabass (Acanthistius brasilianus)
Nile Perch (Lates niloticus)
Atlantic Bluefin Tuna (Thunnus thynnus)
North Pacific Hake (Merluccius productus)
Atlantic Cod (Gadus morhua)
Northern Pike (Esox lucius)
Atlantic Mackerel (Scomber scombrus)
Oceanic Sole (Synaptura spp.)
Atlantic Pomfret (Brama brama)
Pacific Cod (Gadus macrocephalus)
Atlantic Salmon (Salmo salar)
Pandora (Pagellus erythrinus)
Atlantic Sturgeon (Acipenser sturio)
Patagonian W hiphake (Macruronus nov aezelandiae)
Bearded Brotula (Brotula barbata)
Peruv ian Anchov y (Engraulis ringens)
Bigeye Tuna (Thunnus obesus)
Pink Cusk-Eel (Genypterus blacodes)
Big-Scale Sand-Smelt (Atherina boyeri)
Pink Salmon (Oncorhynchus gorbuscha)
Blackspotted (Protonibea diacanthus)
Pink Snapper (Pagrus auratus (synonym: Chrysophrys auratus))
Blue Shark (Prionace glauca)
Rainbow Trout (Oncorhynchus mykiss)
Brill (Scophthalmus rhombus)
Red Mullet (Mullus barbatus)
Brown Meagre (Sciaena umbra)
Redfish (Sebastes marinus (synonym: Sebastes norv egicus))
Canary Drum (Umbrina canariensis)
Rock Grenadier (Coryphaenoides rupestris)
Capelin (Mallotus v illosus)
Saithe (Pollachius v irens)
Coho Salmon (Oncorhynchus kisutch)
Sand Steenbras (Lithognathus mormyrus)
Common Sole (Solea v ulgaris)
Sapphirine Gurnard (Trigla lucerna (synonym: Chelidonichthys lucerna))
Deep-W ater Cape Hake (Merluccius paradoxus)
Seabass (Dicentrarchus labrax)
Derbio (Trachinotus ov atus)
Senegalese Tonguesole (Cynoglossus senegalensis)
Dev il Anglerfish (Lophius v omerinus)
Shallow-W ater Cape Hake (Merluccius capensis)
Dory (Zeus faber)
Shortfin Mako (Isurus oxyrhincus)
Duskytail Grouper (Epinephelus bleekeri)
Skipjack Tuna (Katsuwonus pelamis)
Eel (Anguilla Anguilla)
Smooth Oreo (Pseudocyttus maculatus)
European Hake (Merluccius merluccius)
Smooth-Hound (Mustelus mustelus)
European Perch (Perca fluv iatilis)
South Pacific Hake (Merluccius gayi)
European Pilchard (Sardina pilchardus)
Spanish Sardine (Sardinella aurita)
European Plaice (Pleuronectes platessa)
Spiny Dogfish (Squalus acanthias)
French Sole (Solea lascaris (synonym: Pegusa lascaris))
Swordfish (Xiphias gladius)
Gilthead Seabream (Sparus aurata)
Thornback Ray (Raja clav ata)
Goatfish (Parupeneus spp.)
Turbot (Psetta maxima (synonym: Scophthalmus maximus))
Goldstripe Sardinella (Sardinella gibbosa)
Tusk (Brosme brosme)
Greater Amberjack (Seriola dumerili)
W hiting (Merlangius merlangus)
Grey Mullet (Mugil cephalus)
W inter Skate (Leucoraja ocellata)
Halibut (Hippoglossus hippoglossus)
W itch Flounder (Glyptocephalus cynoglossus)
Herring (Clupea harengus)
W ithmouth Croaker (Micropogonias furnieri)
Icefish (Neosalanx taihuensis)
Yellow Goosefish (Lophius litulon)
Keta Salmon (Oncorhynchus keta)
Yellowfin Sole (Limanda aspera)
Lumpfish (Cyclopterus lumpus)
Yellowfin Tuna (Thunnus albacares)
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
The following species are predicted to be detected on the basis of in-silico analysis
Abram is bram a
Anguilla m egastom a
Acanthocybium solandri
Anguilla m ossam bica
Acanthopagrus latus
Anguilla nebulosa nebulosa
Acanthurus lineatus
Anguilla obscura
Achirus lineatus
Anguilla reinhardtii
Acipenser baerii
Anguilla rostrata
Acipenser brevirostrum
Anodontostom a chacunda
Acipenser dabryanus
Anoplopom a fim bria
Acipenser fulvescens
Aphia m inuta
Acipenser gueldenstaedtii
Arctogadus glacialis
Acipenser m edirostris
Argyrosom us japonicus
Acipenser m ikadoi
Atherina hepsetus
Acipenser naccarii
Atherina presbyter
Acipenser nudiventris
Auxis rochei
Acipenser oxyrhynchus oxyrhynchus
Auxis thazard
Acipenser persicus
Bahaba taipingensis (synonym : Otolithes lini)
Acipenser ruthenus
Balistes capriscus
Acipenser schrenckii
Balistes polylepis
Acipenser sinensis
Balistes vetula
Acipenser stellatus
Barbatula nuda
Acipenser transm ontanus
Belonophago hutsebouti
Aetobatus flagellum
Beryx decadactylus
Alburnus alburnus
Beryx m ollis
Alepocephalus bairdii
Beryx splendens
Allocyttus niger
Bodianus m esothorax
Alosa alosa
Bodianus rufus
Alosa pseudoharengus
Boleophthalm us boddarti
Alosa sapidissim a
Boops boops
Aluterus scriptus
Botia alm orhae
Am eiurus m elas
Botia histrionica
Am philophus citrinellus
Botia kubotai
Am phiprion clarkii
Botia lohachata
Am phiprion percula
Botia rostrata
Am phiprion polym nus
Botia striata
Anabas testudineus
Branchiostegus albus
Anarhichas lupus
Branchiostegus argentatus
Anarhichas m inor
Branchiostegus japonicus
Anguilla australis
Callorhinchus callorynchus
Anguilla australis schm idti
Callorhinchus capensis
Anguilla bengalensis labiata
Callorhinchus m ilii
Anguilla bicolor bicolor
Cam pylom orm yrus com pressirostris
Anguilla bicolor pacifica
Carangoides arm atus
Anguilla celebensis
Carangoides m alabaricus
Anguilla dieffenbachii
Caranx ignobilis
Anguilla interioris
Caranx m elam pygus
Anguilla japonica
Carassius auratus
Anguilla luzonensis
Carassius carassius
Anguilla m algum ora
Carassius cuvieri
Anguilla m arm orata
Carassius gibelio
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SPECIALfinder Allergens Assays Rev. 0 04/01/2016
Catla catla
Cyclocheilichthys enoplos
Centrolophus niger
Cynoglossus abbreviatus
Cephalopholis argus
Cynoglossus bilinearis
Cephalopholis sexm aculata
Cynoglossus lineolatus
Cephalopholis sonnerati
Cynoglossus puncticeps
Channa argus
Cynoglossus sem ilaevis
Channa m aculata
Cynoglossus sinicus
Channa m arulius
Cynoscion arenarius
Chanos chanos
Cynoscion nebulosus
Cheim erius nufar
Cynoscion regalis
Chelidonichthys capensis
Cyprinus carpio
Chelon labrosus
Cyprinus carpio carpio
Chitala blanci
Cyprinus carpio color
Chitala ornata
Cyprinus carpio haem atopterus
Chondrostom a lem m ingii
Cyprinus carpio wananensis
Cirrhinus m olitorella
Cyprinus carpio wuyanensis
Cirrhinus m rigala
Cyprinus carpio xingguonensis
Citharinus latus
Cyttus australis
Clupea pallasii
Dasyatis akajei
Cobitis choii
Dasyatis bennetti
Cobitis elongatoides
Diagram m a pictum
Cobitis granoei
Diplodus sargus
Cobitis lutheri
Dissostichus eleginoides
Cobitis sinensis
Dissostichus m awsoni
Cobitis striata
Distichodus affinis
Cobitis takatsuensis
Distichodus decem m aculatus
Coilia ectenes
Distichodus fasciolatus
Coilia grayii
Distichodus hypostom atus
Coilia lindm ani
Distichodus noboli
Coilia m ystus
Distichodus sexfasciatus
Coilia nasus
Drepane africanus
Coilia reynaldi
Echelus m yrus
Colisa lalia
Echelus uropterus
Colistium nudipinnis
Echeneis naucrates
Coregonus clupeaform is
Echiichthys vipera
Coregonus lavaretus
Eleginops m aclovinus
Coregonus nasus
Engraulis japonicus
Coreoperca whiteheadi
Epinephelus akaara
Coryphaena equiselis
Epinephelus am blycephalus
Coryphaena hippurus
Epinephelus areolatus
Cottus aleuticus
Epinephelus awoara
Cottus cognatus
Epinephelus bruneus, (synonym : E. m oara)
Cottus gobio
Epinephelus clippertonensis
Cottus hangiongensis
Epinephelus coeruleus
Cottus nozawae
Epinephelus coioides
Cottus poecilopus
Epinephelus daem elii
Cottus reinii
Epinephelus epistictus
Crossocheilus latius
Epinephelus fasciatom aculosus
Ctenopharyngodon idella
Epinephelus fuscoguttatus
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Epinephelus goreensis
Gobius cobitis
Epinephelus howlandi
Gobius cruentatus
Epinephelus lanceolatus
Gobius niger
Epinephelus latifasciatus
Gobius paganellus
Epinephelus m acrospilos
Gudusia chapra
Epinephelus m erra
Gym nodiptychus pachycheilus
Epinephelus m iliaris
Harpadon m irochir
Epinephelus polyphekadion
Harpadon nehereus
Epinephelus quoyanus
Heteropneustes fossilis
Epinephelus septem fasciatus
Heterotilapia buttifokeri
Epinephelus sexfasciatus
Hilsa kelee
Epinephelus spilotoceps
Hippoglossoides platessoides
Epinephelus stictus
Hippoglossus stenolepis
Epinephelus striatus
Hoplias m alabaricus (synonym : Macrodon ferox)
Epinephelus trim aculatus
Hoplostethus japonicus
Epinephelus tukula
Hucho bleekeri
Epinephelus undulosus
Huso dauricus
Escualosa thoracata
Huso huso
Esox niger
Hyperoglyphe japonica
Esox reichertii
Hyporham phus sajori
Ethm alosa fim briata
Ictalurus balsanus
Etm opterus baxteri
Ictalurus furcatus
Etm opterus fusus
Ictalurus m eridionalis
Etm opterus granulosus
Ictalurus pricei
Etm opterus lucifer
Ictalurus punctatus
Etm opterus polli
Ictalurus punctatus
Etm opterus princeps
Istiophorus albicans
Etm opterus pseudosqualilolus
Istiophorus platypterus
Etm opterus unicolor
Johnius belangerii
Etm opterus virens
Johnius gripotus
Etroplus m aculatus
Kuhlia m ugil
Eugnathichthys m acroterolepis
Labeo angra
Euthynnus alletteratus
Labeo bata
Gadus ogac
Labeo calbasu
Gam busia affinis
Labeo chrysophekadion
Garra flavatra
Labeo cyclorhynchus
Garra m ullya
Labeo lineatus
Garra orientalis
Labeo pierrei
Garra rufa
Labeo rohita
Garra spilota
Labeo senegalensis
Gasterochism a m elam pus
Labiobarbus lunaris
Gasterosteus aculeatus
Labrus m erula
Gasterosteus wheatlandi
Labrus viridis
Genypterus capensis
Laem onem a barbatulum
Glossogobius celebius
Laem onem a goodebeanorum
Glossogobius circum spectus
Lagocephalus inerm is
Glossogobius olivaceus
Lagocephalus spadiceus
Glyptocephalus zachirus,
Larim ichthys crocea
Gobius bucchichi
Larim ichthys polyactis
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Lateolabrax japonicus
Microstom us pacificus
Lates calcarifer (synonym : Lates calcifer)
Misgurnus anguillicaudatus
Lem onem a longipes
Mogurnda adspersa
Lepidocybium flavobrunneum
Molva m olva
Lepidopsetta bilineata
Monotrete leiurus
Lepom is cyanellus
Morone chrysops
Lepom is hum ilis
Morone saxatilis
Lepom is m acrochirus
Mugil curem a
Leptobotia m icrophthalm a
Mullus surm uletus
Leptobotia rubrilabris
Muraena augusti
Lethrinus lentjan
Muraena helena
Lethrinus obsoletus
Muraena lentiginosa
Leuciscus rutilus
Mycteroperca acutirostris
Leuciscus waleckii
Mycteroperca bonaci
Liza affinis
Mycteroperca interstitialis
Liza aurata
Mycteroperca jordani
Liza ram ado
Mycteroperca m icrolepis
Lophius am ericanus
Mycteroperca olfax
Lophius budegassa
Mycteroperca prionura
Lophius piscatorius
Mycteroperca rosacea
Lota lota
Mycteroperca tigris
Lutjanus argentim aculatus
Mycteroperca venenosa
Lutjanus bengalensis
Mystacoleucus m arginatus
Lutjanus johnii
Nannaethiops bleheri
Lutjanus kasm ira
Nannaethiops unitaeniatus
Lutjanus m alabaricus
Nem acheilus rueppelli
Lutjanus rivulatus
Nem atalosa nasus
Lutjanus russellii
Neocyttus rhom boidalis
Lutjanus sebae
Neolebias trilineatus
Luvarus im perialis
Neolebias unifasciatus
Macquaria australasica
Nibea albiflora
Makaira indica (synonym : Istiom pax indica)
Notopterus notopterus
Makaira m azara (synonym : M. nigricans)
Odontesthes argentinensis
Mastacem belus arm atus
Odontesthes bonariensis
Mastacem belus favus
Odontesthes hatcheri
Mastacem belus m astacem belus
Odontesthes incisa
Melanogram m us aeglefinus
Odontesthes sm itti
Merluccius albidus
Odontobutis sinensis
Merluccius angustim anus
Oncorhynchus clarkii henshawi (synonym : Salm o clarkii henshawi)
Merluccius australis
Oncorhynchus m asou m asou
Merluccius senegalensis
Oncorhynchus tshawytscha
Mesoborus crocodilus
Oreochrom is m ossam bicus, (synonym : Tilapia m ossam bica)
Metzia m esem brinus
Oreochrom is niloticus
Microm esistius poutassou
Osm erus eperlanus
Microphysogobio tafangensis
Osm erus m ordax
Micropterus dolom ieu
Oxyeleotris m arm orata
Micropterus floridanus
Oxyeleotris urophthalm oides
Micropterus salm oides salm oides
Padogobius nigricans
Microstom atichthyoborus bashforddeani
Pagellus bogaraveo
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Pagrus auriga
Reinhardtius everm anni (synonym : Atheresthes everm anni)
Pagrus m ajor
Reinhardtius hippoglossoides
Pagrus pagrus
Rexea solandri
Pam pus argenteus
Rhabdosargus globiceps
Pam pus chinensis
Rhodeus pseudosericeus
Pam pus echinogaster
Rom anogobio tenuicorpis
Pam pus punctatissim us
Ruvettus pretiosus
Paracheirodon axelrodi
Salm o trutta trutta
Paralichthys dentatus
Salvelinus alpinus
Paralichthys olivaceus
Salvelinus fontinalis
Param isgurnus dabryanus
Salvelinus leucom aenis
Paraneetroplus synsipilus
Sander canadensis
Paranthias colonus
Sarda sarda
Paranthias furcifer
Sardinella albella
Paraplagusia bilineata
Sardinella m aderensis
Paraplagusia blochii
Sardinops m elanostictus
Paraplagusia japonica
Saurida m icrolepis
Parastrom ateus niger
Saurida undosquam is
Patagonothen longipes
Scardinius erythrophtalm us
Pelthorham phus novaezeelandiae
Scarus forsteni
Peprilus triacanthus
Scarus frenatus
Perca flavescens
Scarus ghobban
Phago boulengeri
Scarus rubroviolaceus
Phoxinus eos
Scarus schlegeli
Phoxinus oxycephalus jouyi
Schedophilus velaini
Phoxinus percnrus sachalinensis
Schism atorhynchos nutka
Phoxinus percnurus m antschuricus
Schizopyge niger
Phycis blennoides
Schizothorax dolichonem a
Pinjalo pinjalo
Schizothorax esocinus
Platichthys stellatus
Schizothorax labiatus
Plectropom us leopardus
Schizothorax plagiostom us
Plesiom yzon baotingensis
Schizothorax prenanti
Pleurogram m us azonus
Schizothorax progastus
Pleurogram m us m onopterygius
Sciaenops ocellatus
Pollachius pollachius
Scom ber australasicus
Pom atom us saltatrix
Scom ber colias
Pom atoschistus knerii
Scom ber japonicus
Pom atoschistus m inutus
Scom berom orus m unroi,
Pristipom oides m ultidens
Scom berom orus cavalla
Pristolepis m alabarica
Scom berom orus com m erson
Protosalanx chinensis
Scom berom orus m aculatus
Psettodes erum ei
Scom berom orus niphonius
Pseudobram a sim oni
Scom berom orus sem ifasciatus
Pseudorasbora parva
Scophthalm us aquosus,
Puntius sem ifascicolatus
Selar crum enophthalm us
Puntius snyderi
Seriola lalandi
Rachycentron canadum
Seriola quinqueradiata
Rasbora borapetensis
Sicyopterus lagocephalus
Rastrelliger kanagurta
Siganus fuscescens
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Siganus guttatus
Trachurus trachurus
Siganus puellus
Trinectes m aculata
Siganus unim aculatus
Trisopterus esm arkii
Sillago siham a
Trisopterus m inutus
Sillago vincenti
Upeneus m oluccensis
Solea senegalensis
Variola albim arginata
Sphyraena barracuda
Variola louti
Sphyraena japonica
Zenopsis nebulosus
Spicara m aena
Sprattus antipodum
Sprattus m uelleri
Sprattus sprattus
Squalidus argentatus
Squalus m egalops
Stizostedion lucioperca (synonym : Sander lucioperca)
Stolephorus chinensis
Stolephorus waitei
Sym phodus cinereus
Sym phodus m elanocercus
Sym phodus ocellatus
Sym phodus roissali
Sym phodus tinca
Syncrossus beauforti
Syncrossus berdm orei
Syncrossus helodes
Syncrossus hym enophysa
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
Tham naconus tessellatus
Theragra finm archica
Thunnus alalunga
Thunnus m accoyii
Thunnus orientalis
Thunnus tonggol
Thym allus arcticus
Thym allus grubii
Thym allus thym allus
Thynnichthys polylepis
Tinca tinca
Trachinotus blochii
Trachurus japonicus
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
7 – Exclusivity Panel
The following DNA extracts showed no amplification curve in a 20 µl (40 ng DNA) total reaction volume:
Meat
Crustaceans and Molluscs
Beef (Bos taurus)
American Lobster (Homarus americanus)
Buffalo (Bubalus bubalis)
Clam (Tapes semidecussatus)
Donkey (Equus asinus)
Coastal Mud Shrimp (Solenocera crassicornis)
Duck (Anas crecca, Cairina muschata)
Cuttlefish (Sepia officinalis)
Goat (Capra hircys)
Kiddi Shrimp (Parapenaeopsis stylifera)
Goose (Anser spp.)
Lobster (Palinurus spp.)
Horse (Equus caballus)
Lobster (Palinurus spp.)
Poultry (Gallus gallus domesticus)
Mantis Shrimp (Squill mantis)
Quail (Coturnix coturnix)
Northern Prawn (Pandalus borealis)
Rabbit (Oryctolagus cuniculus)
Norway lobster (Nephrops norv egicus)
Sheep (Ov is aries)
Pacific Shrimp (Heterocarpus affinis)
Swine (Sus scrofa domesticus)
Poulpe (Octopus cyaena)
Turkey (Meleagris gallopav o)
Prawn (Penaeus v annamei)
W ild boar (Sus scrofa scrofa)
Red Squat Lobster (Pleuroncodes monodon)
Squid (Loligo edulis)
Allergens
Other Vegetables
Almond (Prunus dulcis)
Arugula (Eruca v esicaria)
Barley (Hordeum v ulgare)
Basil (Ocimum basilicum)
Brasilian W alnut (Bertholletia excelsa)
Bean (Phaseolus v ulgaris)
Cashew (Anacardium occidentalis)
Buckwheat (Fagopyrum esculentum)
Celery (Apium grav eolens v ar. dulce)
Carrot (Daucus carota)
Durum W heat (Triticum durum)
Eggplant (Solanum melongena)
Hazelnut (Corylus av ellana)
Fennel (Foeniculum v ulgare)
Kamut (Triticum turgidum)
Garlic (Allium sativ um)
Lupine (Lupinus albus)
Grape (Vitis v inifera)
Macadamia Nut (Macadamia integrifolia)
Lemon (Citrus limon)
Mustard (Brassica nigra)
Maize (Zea mays)
Oat (Av ena sativ a)
Mango (Mangifrea indica)
Peanut (Arachis hypogaea)
Mushrooms (Agaricus campestris)
Pecan Nut (Carya illinoinensis)
Oliv e (Olea europaea)
Pistachio (Pistacia v era)
Onion (Allium cepa)
Rye (Secale cereale)
Parsley (Petroselinum crispum)
Sesame (Sesamum indicum)
Pepper (Capsicum annum)
Soft W heat (Triticum aestiv um)
Pine Nuts (Pinus pinea)
Soybean (Glycine max)
Plum (Prunus domestica)
Spelt (Triticum monococcum)
Potato (Solanum tuberosum)
W alnut (Juglans regia)
Rapeseed (Brassica napus)
Rice (Oryza sativ a)
Spinach (Spinacia oleracea)
Sunflower (Helianthus annuus)
Tomato (Solanum lycopersicon)
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016
8 – Troubleshooting
I.
Both target and IAC show no-amplification, or amplification plots grossly abnormal. Possible
causes and corrective actions might be:
• An excess of DNA in the target inhibiting 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. Use DNase/RNase Free
Water to prepare dilutions.
• Inadequate sealing of optical caps/film caused sample evaporation. Repeat the analysis using
proper tools and proper optical caps/film to secure perfect sealing.
• Possible use of wrong consumables. Repeat 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. Repeat sample DNA extraction ensuring extraction
method is properly performed. We suggest to perform an extraction using SpyX Fish (Cat. N.
PAV20X) as a reference material.
II.
Positive Control reactions failed to amplify, but other reactions appear correct.
• Positive Control DNA was not added to the reaction wells.
III.
Negative Control reactions are positive:
• Contamination of reagents with positive DNA. Use more care to prevent contamination while
handling assay reagents and setting up assays.
In case support is needed contact Generon at: [email protected]
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User Manual
SPECIALfinder Allergens Assays Rev. 0 04/01/2016