[1] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

VERYfinder Soft/Durum Wheat Contamination Quantitative Assay Real-Time PCR quantification kit

Cat. # PSV02Q

User Manual

Via San Geminiano, 4 41030 San Prospero (MO)- Italy : +39 059 8637161 : +39 059 7353024 : laboratorio@generon.it : www.generon.it

[2] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

Pasta is normally made from 100% durum wheat due to its special features to obtain a good final product.

Pasta production and sale is strictly regulated by the current Italian law: only a maximum of 3% of soft wheat

can be tolerated in dry pasta as cross contamination that may occur during growing, harvesting and

handling practices.

Cereal composition is a key factor in the quality and safety of food and feed. In particular, durum wheat is

considered the election wheat to make pasta, given its unique properties as relatively high yellow pigment

content and high protein content favourable for good cooking quality. The dough made from durum

wheat has rheological properties ideally suited for pasta manufacturing process. Due to an accidental

contamination occurring during either wheat harvest or storage and transport, pasta is only officially

regarded as impure when the common wheat level exceeds 3%, as stipulated by the Italian Regulation

(D.l. 187, 09/02/2001).

VERYfinder Soft/Durum Wheat Contamination Quantitative Assay allows the quantitative detection in

samples like flour, semolina and pasta of common wheat (Triticum aestivum) DNA in durum wheat (Triticum

durum) DNA.

VERYfinder Soft/Durum Wheat Contamination Quantitative Assay is based on DNA detection that is more

stable than other components even when subjected to technological processes used in the food industry.

The DNA can be amplified and detected with high sensitivity and specificity, constituting an excellent

molecular marker of the presence of soft wheat in the product.

VERYfinder Quantitative assay contain DNA from standard powders pre-extracted using Generon ION Force

DNA Extractor FAST (Cat. # EXD001) called as Standard Points for both SOFT WHEAT -fragment and

ENDOGENE -fragment detection.

Both SOFT WHEAT- and ENDOGENE- standard points must be analyzed on the same reaction plate of the

unknown samples. Double SOFT WHEAT- and ENDOGENE- standard curves and two negative control

reactions are recommended to be setup for each PCR run according with the good laboratory practice.

Assays performances

The VERYfinder Soft/Durum Wheat Contamination Quantitative Assay will detect the Soft Wheat DNA

following the recommended protocols and reagents are storing properly.

This assay uses the polymerase chain reaction (PCR) to amplify a genetic target typical of the soft wheat

(Triticum aestivum) and durum wheat (Triticum durum).

The absolute LOD of Real-Time PCR is comprised between 1 and 10 DNA copies. The sensitivity of the assay

was determined using purified genomic DNA from soft wheat, 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.

1 - Introduction

[3] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

This Real-Time PCR assay detects a specific DNA sequence in the DNA of target in less than 1.5 hours. The

use of a specific fluorescence-labeled probe (FAM) measure the amplification of the target sequence.

2.1 - Assay Content 100 + 100

Box 1 Box 2

Description N.vials Volume (μl) Description N.vials Volume (μl)

VERYfinder Event-Specific OLIGO Mix * 2 150 VERYfinder Endogene OLIGO Mix * 2 150

Negative Control 1 300 Negative Control 1 300

STD Soft Wheat ** 5 200 STD Endogene ** 4 200

GENERase Mastermix 2 750 GENERase Mastermix 2 750

* Reagents are supplied with an 5% of extra volume.

** See the specific Control Quality document for the % contamination level for Soft Wheat and Endogene Standards both.

2.2 - Storage & 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.

2 - VERYfinder Soft/Durum Wheat Quantitative Assay

[4] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

3.1 – Extraction(1)

Material/Equipment Source

Extraction Kit

Generon ION FORCE DNA Extractor FAST (Cat. # EXD001)

or Generon Ion Spin DNA Prep One-for-All (Cat. # EXD018)

Chemicals: n-Hexane Lab Suppliers

Tubes, 50 ml and 15 ml

Generon or other Lab Suppliers

Adapters for columns and syringes (EXD010-A)

Plastic spoons

Technical balance or similar, sensitivity: 0.01 g

10 ml syringes, without needle, and cellulose acetate filters, 0.45 μm pore size

Graduated cilinder, 50 ml

Moulinex homogenizer or equivalent

Centrifuge with rotors for 1.5-2 ml microtubes and 50 ml tubes (range within 500 – 14000 rpm)

Thermal Water Bath or Block heated up to 85° C ± 2° C

Pipette sets

Pipette tips (Barrier)

Tube rack for 1.5 ml tubes

2.0 and 1.5 ml micro-tubes

DNA Extraction VACUUM BOX + Vacuum pump or Venturimeter

Perform the sample preparation (grinding, transferring, weighing, etc.) according to GLP to minimize

chance of cross-contamination between samples. 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 allows processing up to 20 grams of starting material in

order to maximize sample’s lot representation.

Weight the pulverized/homogenized sample and extract the appropriate amount, according to DNA

extraction method. Refer to manufacturer user manual for extraction procedure details.

3 – Materials and equipment needed

[5] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

3.2 – Detection via Real-Time PCR

Material/Equipment Source

Real-Time PCR System (2) Generon or other Lab suppliers

VERYfinder Soft/Durum Wheat Quantitative Assay Generon Cat. # PSV02Q

Optical Adhesive Seal or Optical Caps Generon or other Lab suppliers

Optical reaction plate or Optical Tube Strips Generon or other Lab suppliers

Micropipette sets Generon or other Lab suppliers

(1) Equipment necessary only when ION Force DNA Extractor FAST (Cat. # EXD001) is used. (2) The assay was validated on Bio-Rad CFX Deep-Well. The kit is compatible with other Real-time PCR instruments excluded Roche Light Cyclers I and II.

[6] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

Allow the reagents to thaw (GENERase Mastermix, VERYfinder OLIGO MIX for the SOFT WHEAT-target and for

ENDOGENE-target, 5 SOFT WHEAT-Standard Points and 4 ENDOGENE-Standard Points). Vortex tubes when

thawed.

Reaction set-up for SOFT WHEAT-Target

I. Mix 150 µL of VERYfinder SOFT WHEAT-target OLIGO Mix with 750 µL of GENERase Mastermix to

prepare VERYfinder Working Mastermix (WMX-GM) (label vials with SOFT WHEAT-Target name).

II. Vortex shortly and spin down in order to homogenize the mix.

III. Transfer 18 µL of WMX-SW into PCR plate wells according to the number of unknown samples,

number of SOFT WHEAT-Standards Points and number of acting well as negative control.

IV. Add 12 µL of negative control into wells acting as negative control.

V. Add 12 µL of each sample to wells testing the unknown samples.

VI. Add 12 µL of SOFT WHEAT-Standards Points to wells to create a standard curve for SOFT WHEAT-

target.

Close wells and ensure no bubbles are present at the bottom of the wells.

Reaction set-up for ENDOGENE- Target

I. Mix 150 µL of VERYfinder ENDOGENE-target OLIGO Mix with 750 µL of GENERase Mastermix to

prepare VERYfinder Working Mastermix (WMX-ER) (label vials with ENDOGENE-Target name).

II. Vortex shortly and spin down in order to homogenize the mix.

III. Transfer 18 µL of WMX-ER into other PCR plate wells according to the number of unknown samples,

number of ENDOGENE- Standards Points and number of acting well as negative control.

IV. Add 12 µL of negative control into wells acting as negative control.

V. Add 12 µL of each sample to wells testing the unknown samples.

VI. Add 12 µL of ENDOGENE-Standards Points to wells to create a standard curve for ENDOGENE-target.

Close wells and ensure no bubbles are present at the bottom of the wells.

4 – Real-Time PCR detection

PINK=Wells aimed for SW-STANDARD POINTS BLUE=Wells aimed for ER-STANDARD POINTS ORANGE=Wells aimed UNKNOWN SAMPLES BLACK=Wells aimed for NEGATIVE CONTROL

E.g.: example of plate setup for a proper quantitative analysis where either SW- and ER- standard points and unknown samples are in triplicates with a double negative control for both targets.

WMX for SW-Target

WMX for ER-Target

[7] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

4.2 – Instrument setup

With GENERase Mastermix set the following parameters on your thermocycler:

I. Total Reaction volume: 30 µL

II. Fluorophores/Quenchers: SOFT WHEAT- target and ENDOGENE- target (FAM/BHQ1-NFQ);

III. Thermal profile:

Step T (°C) Duration Loops

UNG 50 2 min 1

Taq Activation 95 10 min 1

DNA Denaturation 95 15 sec 45

Annealing/Extension + Plate Reading 60 60 sec

[8] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

% SOFT WHEAT Target = [(SW Target %) / (ER Target %)] x 100

Results evaluation must be done according to the analysis software recommended by the Real-Time PCR

instrument manufacturer.

After running the samples on the instrument, data are analyzed using the software to produce Cq values of

each reporter dyes for each sample run. These values are then used to determine the presence and,

afterwards, quantify the amount of SOFT WHEAT material in each sample.

The amount of SOFT WHEAT must be normalized to the amount of endogenous plant material (ENDOGENE-

target) detected in each sample. SOFT WHEAT-target and ENDOGENE-target Cq of unknown samples are

interpolated on the respectively calibration/standard curve obtained through Cq values of the SOFT

WHEAT-target concentration Standard Points and ENDOGENE- target concentration Standard Points,

respectively.

After interpolation, operator obtains the corresponding specific SOFT WHEAT- and ENDOGENE- percentage

for each unknown sample. Obtaining the % SOFT WHEAT-target and % ENDOGENE-target results for each

unknown sample, operator is able to simply calculate the % SOFT WHEAT-matrix in the sample.

After setting the baseline, the analysis outcome for the unknown samples should be evaluated following the

indications below.

Target

Endogene

E=109%; R2=0,998

E=110%; R2=0,995Endogene

Target

5 – Data interpretation

E.g.: % SW target presence in unknown sample: % SW target resulted from Real-Time PCR run: 15% % ER target resulted from Real-Time PCR run: 75%

% SW target presence in unknown samples = (15/75)*100 = 20% SW target.

[9] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

The method presents 100% specificity as none of the following species DNA extracts shows a positive result in a 30 μl total reaction volume (120 ng DNA).

Allergens:

Almond (Prunus dulcis), Barley (Hordeum vulgare), Brazil Nut (Bertholletia excelsa), Cashew (Anacardium occidentalis), Celery (Apium graveolans), Hazelnut (Corylus avellana), Lupine (Lupinus albus/angustifolius/luteus), Macadamia nut (Macadamia integrifolia), Mustard (Brassica nigra/juncea/alba), Oats (Avena sativa), Peanut (Arachis hypogaea), Pecan nut (Carya illinoiensis), Pistachio (Pistacia vera), Rye (Secale cereale), Sesame (Sesamum indicum), Soy (Glycine max), Walnut (Juglans regia).

Products of plant origin:

Apple (Malus domestica), Apricot (Prunus armeniaca), Aubergine (Solanum melongena), Banana (Musa acuminata), Basil (Ocimum basilicum), Bean (Phaseolus vulgaris), Black Cabbage (Brassica oleracea var. palmifolia ), Black pepper (Piper nigrum), Blackberry (Rubus idaeus), Broccoli (Brassica oleracea var. ita), Brussels sprouts (Brassica oleracea var. gemmifera), Buckwheat (Fagopyrum esculentum), Cabbage White (Brassica oleracea var. capitata f. alba), Cacao (Theobroma cacao), Caraway (Carum carvi), Cardamom (Elettaria cardamomum), Carrot (Daucus carota), Cauliflower (Brassica oleracea var. b), Champignon (Agaricus campestris), Chard (Beta vulgaris var. cicla), Cherry (Prunus avium), Chestnut (Castanea vulgaris), Chickpeas (Cicer arietinum), Chinese cabbage (Brassica rapa subsp. narinosa), Cinnamon (Cinnamonium verum), Clementina (Citrus x celementina), Cloves (Syzygium aromati), Coconut (Cocos nucifera), Coriander (Coriandrum sativum), Corn (Zea mays), Cubeb pepper (Piper cubeba), Cucumber (Cucumis sativus), Cumin (Cuminum cyminum), Currant (Ribes nigrum), Ethiopian mustard (Brassica carinata), Fennel (Foeniculum vulgare), Fig (Ficus carica), Flax (Linum usitatissimum), Garlic (Allium sativum), Ginger (Zingiber officinale), Grapefruit (Citrus x paradisi), Japanese spinach (Brassica rapa var. perviridis), Kale (Brassica rapa var. campestris), Kiwi (Actinidia chinensis), Laurel (Laurus nobilis), Lemon (Citrus limonia), Lentil (Lens culinaris), Lives (Vitis vinifera), Mahaleb (Prunus mahaleb), Mandarin (Citrus reticulata), Mango (Mangifera indica), Marjoram (Origanum majorana), Nutmeg (Myristica fragrans), Olive (Olea europaea), Onion (Allium cepa), Orange (Citrus aurantium), Origan (Origanum vulgare), Parsley (Petroselinum crispum), Pea (Pisum sativum), Peach (Prunus persica), Pear (Pyrus communis), Pepper (Capsicum annuum), Pimento (Pimenta dioica), Pine seed (Pinus pinea), Pineapple (Ananas comossus), Pink berries (Schinus molle), Plum (Prunus domestica), Pomelo (Citrus maxima), Poplar (Populus spp.), Poppy (Papaver rhoea), Potato (Solanum tuberosum), Radish (Raphanus sativus), Rape (Brassica napus), Raspberry (Rubus idaeus), Red Cabbage (Brassica oleracea convar. capitata var. capitata), Rice (Oryza sativa), Rocket salad (Eruca sativa), Saffron (Crocus sativus), Savoy cabbage (Brassica oleracea var. sabauda), Sea kale (Crambe maritima), Shepherd's purse (Capsella bursa-pastoris), Spinach (Spinacia oleracea), Strawberry (Fragaria x ananassa), Sunflower (Helianthus anuus), Tarragon (Schinus molle), Thyme (Thymus vulgaris), Tomato (Solanum lycopersicon), Turnip greens (Brassica rapa sylvestris), Zucchini (Cucurbita pepo).

Products of animal origin:

Rabbit (Oryctolagus cuniculus), Bovine (Bos Taurus), Horse (Equus caballus), Donkey (Equus asinus), Chicken (Gallus gallus), Goat (Capra hircus), Swine (Sus domesticus), Wild boar (Sus scrofa), Sheep (Ovis aries), Buffalo (Bubalus bubalis), Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), Goose (Anser anser), Quail (Coturnix coturnix), Dog (Canis familiaris), Cat (Felis catus), Tuna (Thunnus albacares), Mackerel (Scombrus scombrus), Anchovy (Engraulis encrasicolus), Sardine (Sardina pilchardus), Cod (Merluccius merluccius), Salmon (Onchorthynchus kisutch), Trout (Salmo trutta), Clam (Venus gallina), Lobster (Nephrops norvegicus), Mussel (Mytilus edulis), Prawn (Penaeus vannamei), Sea bream (Sparus aurata), Sepia (Sepia officinalis), Squid (Loligo edulis).

6 – Inclusivity and Exclusivity Panel

[10] User Manual - VERYfinder Soft/Durum Wheat Quantitative Assay - Rev. 1 - 14/10/2016

I. Concomitant no target or endo amplification, or amplification plots grossly abnormal. Possible

causes and corrective actions:

• An excess of DNA in the target might inhibit the reaction and endo 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.

II. Standard Points reactions failed to amplify, but other reactions appear correct (e.g. the endo is

amplified):

• Standard Points DNA was not added to the reaction wells. PCR run should be repeated.

III. Negative Control reactions are positive:

• Contamination of the negative control vial or the VERYfinder PCR mix with VERYfinder

Endogene-positive DNA. Use more care to prevent contamination while handling assay

reagents and setting up assays.

In case support is needed contact Generon at: laboratorio@generon.it

Generon S.p.A. guarantees the buyer exclusively concerning the quality of reagents and of the

components used to manufacture the product. Generon S.p.A. 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.

7 – Troubleshooting

8 – Disclaimers