dna marker

Trends in Food Science & Technology 26 (2012) 43e55

Review

* Corresponding author.

0924-2244/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2012.01.009

Advances in

vegetable oil

authentication by

DNA-based markers

Joana Costa, Isabel Mafra* and
M. Beatriz P.P. Oliveira
REQUIMTE, Laborat�orio de Bromatologia

e Hidrologia, Faculdade de Farm�acia,

Universidade do Porto, Rua An�ıbal Cunha,164, Porto 4099-030, Portugal

(Tel.: D351 222078902; fax: D351 222003977;

e-mail: [email protected])

The suitability of DNA markers in providing unequivocal iden-

tifiers for authentication and traceability of food has been a sub-

ject of an increasing number of reports. Even in complex food

matrices such as vegetable oils, the use of molecular markers

as diagnostic tools has been exploited. Considering the wide

variety of vegetable oils available for consumers and the differ-

ences in prices, especially among premium olive oil and other

oils, species adulteration leading to economic losses and loss

of consumer confidence can arise. In this review, the advances

of DNA extraction protocols are emphasised as a crucial step to

overcome. Specific identification of several plant oils as poten-

tial adulterants of olive oil has been a subject of very recent

progresses. When the oilseed crops are the source for vegetable

oil production, additional concerns due to the presence of

genetically modified organisms have prompted to further

improvements in DNA analysis. In the specific case of olive

oil, the use of genetic markers has provided analytical tools to

assess authenticity regarding cultivar identification as indepen-

dent markers from environmental fluctuations.

IntroductionIn the recent years, a great interest has been devoted to

the use of vegetable fats in human diet, especially regarding

olive oil and other vegetable edible oils. Since these oilshave a vast connotation for human health, mainly due totheir nutritional properties, they have been a source of sev-eral studies. Over the last decade, a number of studies haveestablished that most olive oils and some vegetable oils,which are naturally rich in monounsaturated fatty acids, an-tioxidants (vitamin E) and phytosterols, may contribute tohealth benefits such as prevention of coronary diseasesand possibly some forms of cancer and other diseases(Giugliano & Esposito, 2005).

Vegetable oils are of utmost significance for human con-sumption, not only from the nutritional point of view, butalso for their use as technical components in chemical,pharmaceutical and cosmetic industries. Recently, theyhave also been used as raw material for renewable energy.The importance of vegetable oils to the global economybecomes clear when considering the amount of vegetableoils produced and consumed worldwide.

The increased attention to food safety has stimulated theinterest in food authentication (Consolandi et al., 2008).Certification of the origin of food, feed and ingredientshas become of primary importance for the protection ofconsumers, in particular for fraud prevention (Woolfe &Primrose, 2004). In the global economy, traceability canbe defined as the ability to track any food, feed, food-producing animal or substance that will be used for con-sumption, along all steps of production, processing anddistribution (http://ec.europa.eu/food/food/foodlaw/traceability/index_en.htm).

Various vegetable oils have been reported as adulterantsof olive oil, namely hazelnut, almond, maize, palm and sun-flower oils (Arvanitoyannis & Vlachos, 2007; Krankel,2010). Although in most cases, the adulteration of vegetableoils does not pose a threat to the consumer’s health, in thespecific cases of potentially allergenic foods such as hazel-nut, its use might represent a risk for sensitised individuals(Arlorio et al., 2010). The use of adulterants also impliesan economical fraud, a disloyal competition among pro-ducers and it violates the consumer’s right to make informedchoices regarding the products they acquire. In the specialcase of olive oil, as a food commodity that can reach pre-mium prices, two main issues should be considered, namelythe profitable adulteration by blending it with lower valuevegetable oils, and the fraudulent mislabelling regardingthe information about the geographical origin, the cultivarsand/or the production methodology (Bell & Gillat, 2010).

mailto:[email protected]

http://ec.europa.eu/food/food/foodlaw/traceability/index_en.htm

http://ec.europa.eu/food/food/foodlaw/traceability/index_en.htm

http://dx.doi.org/10.1016/j.tifs.2012.01.009



Table 1. Summary of DNA extraction protocols used for vegetable oil samples.

DNA extractionprotocol

Oil matrix Starting amount Pre-concentration DNA yield DNA markers and/orPCR method

References

CTAB-based method Monovarietal virgin olive oil Not declared Centrifugation (1 g pellet) Not declared RAPD Muzzalupo and Perri (2002)Monovarietal extra virginolive oil

50e100 mL Double centrifugation.Pellet (�0.5 g) frozenin liquid nitrogen andimmersed in 65 �C bath.

Very low yields AFLP Busconi et al. (2003)

Filtered and unfilteredmonovarietal olive oil

2e40 g No w10 ng/mL SSR-CE, nested PCR Testolin and Lain (2005)

Monovarietal and commercialolive oil

6 mL No 7.75 ng/mL RAPD, ISSR and SSR Martins-Lopes et al. (2008)

Commercial monovarietalolive oil

1 mL No Not declared Real-time PCR Gim�enez et al. (2010)

Monovarietal olive oil 100 mL No 40 ng/mL SNP markers, multiplexPCR with LDR universalarray

Consolandi et al. (2008)

Canola, cotton, maize, soybean,peanut, sunflower, sesame,palm commercial oils

16 mL No 0.1 fmol Species-specific PCR withthin-filmbiosensor chips

Bai et al. (2011)

CTAB-Hexane method Commercial monovarietalolive oil


CTAB-Hexane-Chloroformmethod

Commercial monovarietalolive oil


Hexane method Monovarietal olive oil 2 mL No 27 ng/mL SNP markers, multiplexPCR with LDR universalarray




Monovarietal olive oil 1 mL No Not declared Real-time PCR Gim�enez et al. (2010)Hexane based methodusing guanidinethiocyanate

Crude soybean oil 2 g, 5 g No 1.86 mg/g ofcrude oil

Species-specific PCR Gryson et al. (2002)

Crude and degummedsoybean oil

75 g crude No Not declared Species-specific PCR andreal-time PCR

Gryson et al. (2004)365 g degu.

Based on DNA extractionfrom paraffin withmodifications

Monovarietal olive oil 2 mL No Not declared AFLP Pafundo et al. (2005)Monovarietal olive oil 2 mL No Not declared SCAR from AFLP Pafundo et al. (2007)

Nucleospin� plant kit(MachereyeNagel)



Monovarietal olive oil atdifferent times of storage

Not declared Not declared Not declared AFLP Pafundo et al. (2010)

Nucleospin� food kit(MachereyeNagel)

Monovarietal olive oil 2 mL No 24 ng/mL SNP markers, multiplexPCR with DR universalarray



4.2 mL No 23 ng/mL RAPD, ISSR and SSR Martins-Lopes et al. (2008)

Crude and refined (neutralised,washed, bleached and

50 g crude Centrifugation 28.3 ng/mL crude Species and event-specificRR soybean PCR and

Costa et al. (2010b)200 g steps 3.90e32.5 ng/mL

44

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deodorised) soybean oil quantitative real-time PCRof refining steps of refiningBlended and refined oils 200 g Centrifugation 3.6 ng/mL Species-specific PCR and

real-time PCRCosta et al. (2010a)

QIAamp DNA Stool kit(Qiagen)

Monovarietal olive oil 200 mL No 5e10 ng/mL SSR-CE Ayed et al. (2009)Filtered and unfilteredmonovarietal olive oil


Destoned oil and oil withpits olive oil

100, 200,300 mL

No 3.67e15.0 ng/mL SSR Muzzalupo et al. (2007)

Filtered olive oil spiked withlDNA

50 mL Centrifugation 10 mg lDNA lDNA specific PCR Spaniolas, Bazakos, Nturouet al. (2008)

Plant oils 50 mL Centrifugation Very low yields PCR of chloroplast trnLintron polymorphismswith CE

Spaniolas, Bazakos, Awadet al. (2008)

DNeasy Plant mini kit(Qiagen)





Gene Elute plant kit(Sigma)

Filtered and unfilteredmonovarietal and binaryvarietal olive oils

50 mL unfilteredoil

Centrifugation(50 mL pellet)

Not declared SSR Pasqualone et al. (2007)

200 mL filteredoil

Monovarietal olive oil 200 mL Centrifugation (pelletof cellular residues)

Not declared AFLP Montemurro et al. (2008)

Monovarietal olive oil 250 mL Centrifugation (pelletof cellular residues)

5 ng/mL SSR-CE Alba et al. (2009)

Hydroxyapatite Biogel(Sigma)

Commercial olive oils(PDO, PGI)

400 mL No Not declared SSR Breton et al. (2004)

Silica kit (Sigma) Commercial olive oils(PDO, PGI)


Wizard� Magneticpurification systemfor food (Promega)

Commercial olive oils(PDO, PGI)




Monovarietal olive oil 120 mL No 34 ng/mL SNP markers, multiplexPCR with LDR universalarray


Sunflower and maizecommercial oils

Not declared No Not declared Species-specificreal-time PCR

Doveri and Lee (2007)

Crude and degummedsoybean oil

5 mL No 3.2 mg crude Species-specific PCR Bogani et al. (2009)1.7 mg, degummed

Olive, maize, rapeseed,sesame, soybean, peanut andsunflower oils

160 mL No 0.1 pg/uL olive oil PCR-CE-SSCP Wu et al. (2011)

Commercial LBLink-Bioteck ExtMan



Official Swiss methodfor lecithin and oilDNA extraction

Monovarietal olive oil 2.5 mL No Low yield SSR Doveri et al. (2006)

(continued on next page)

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Table

1(continued

)

DNAex

traction

protoco

lOilmatrix

Startingam

ount

Pre-conce

ntration

DNAyield

DNAmarke

rsan

d/or

PCRmethod

Referen

ces

TEA

based

buffer

(Tris,ED

TAan

dasco

rbic

acid)method

Sedim

ents,unfiltered

and

filtered

monovarietal

olive

oil

25e500g

Cen

trifugation

(pellet1g)

0.2e2ng/goil

sedim

ent

SCARmak

ersfrom

RAPD

dela

Torreet

al.(2004)

0.1e0.5

pg/goil

TNEbased

buffer

(Tris,NaC

l,ED

TA,SD

S)methodwithWizard

DNAClean

-upresin

(Promega)

Olive

drupes

withno

amplificationforolive

oil

250mL

Cen

trifugation

Notdeclared

ISSR

andSS

RPasqualoneet

al.(2001)

TNEbased

buffer

(Tris,

NaC

l,ED

TA,SD

S)method

Cold-pressed

andrefined

rapesee

doil

200mL

Cen

trifugationan

dco

ncentrationby

mini-co

nce

ntrators

(pellet200e300mL)

Notdeclared

Spec

ies-spec

ificPCR,

nestedPCR

Hellebrandet

al.(1998)

WizardDNAEx

trac

tion

method(Promeg

a)Cold-andwarm-pressed

soyb

eanoilpriorto

filtration

300mL

No

Notdeclared

Spec

ies-spec

ificPCR,

nestedPCR

Pauliet

al.(1998)

NIABProtoco

lBan

dGen

eClean

IIIkit,

Bio

101(combined

kits)

Sunflower

andmaize

refined

oils

1mL

No

Notdeclared

Spec

ies-spec

ific

real-tim

ePCR

Doverian

dLe

e(2007)

Petroleum

ether/PBS

extrac

tion

Crudepalm

oil

30mL

No

LOD

10pg

Spec

ies-spec

ific

real-tim

ePCR

Zhan

get

al.(2009)

46 J. Costa et al. / Trends in Food Science & Technology 26 (2012) 43e55

Considering the importance of authentication and trace-ability for food processors, regulatory authorities and con-sumers, different analytical methodologies have beenproposed over the last years. Instrumental techniques basedon chromatographic analysis of different families of com-pounds are among the most suggested and used approachesfor monitoring the quality and authenticity of vegetableoils. Other methodologies mainly based on spectroscopictechniques, such as near-infrared spectroscopy and nuclearmagnetic resonance (NMR) spectroscopy have also beenproposed to assess vegetable oil identity (Casale, Casolino,Ferrari, & Forina, 2008; Cunha, Amaral, & Oliveira, 2011;Luykx & van Ruth, 2008). Compared to chromatographic,spectroscopic techniques are considered faster, simpler andless expensive. Regarding olive oil, since its chemical com-position may differ among seasons and growing area,depending on the environmental conditions, the use of chem-ical markers for authenticity assessment of olive cultivar isnot effective in this case (Gim�enez, Pist�on, Mart�ın, &Atienza, 2010).

In the last years, there has been a growing interest towardsthe application of methodologies based on the analysis ofDNA regarding food authentication (Mafra, Ferreira, &Oliveira, 2008). DNA analysis presents several advantagessuch as, a high durability of DNA molecules compared toother compounds such as proteins, associated to their ubiq-uity in cells. These advantages make the use of DNAmarkersas effective targets independent from geographical, climaticor agronomical factors. Most DNA-based methods rely onthe high specific amplification of one ormoreDNA fragmentsby means of polymerase chain reaction (PCR). A number ofpapers is available reporting the application of different DNAfingerprinting methods to olive oil traceability and cultivaridentification (Alba, Sabetta, Blanco, Pasqualone, &Montemurro, 2009; Breton, Claux, Metton, Skorski, &Berville, 2004; Martins-Lopes, Gomes, Santos, & Guedes-Pinto, 2008; Montemurro, Pasqualone, Simeone, Sabetta, &Blanco, 2008; Pafundo, Agrimonti, & Marmiroli, 2005).

While olive oils have been essentially a subject of authen-ticity and traceability studies due to their high economicvalue, other vegetable oils are further focus on geneticallymodified organism (GMO) detection, beyond species identi-fication. With the increasing commercial use of GM oilseedcrops, DNA has also been considered as a preferable targetfor the traceability of transgenic material in vegetable oils.In this context, the aim of the present work is to provide anupdated overview of the DNA-based methods applied tovegetable oil matrices.

DNA extraction from vegetable oil matricesFor the effective application of PCR techniques, a critical

step to overcome in the case of complex and highly processedfood matrices is the DNA extraction and purification. Ade-quate strategies are required to ensure efficient recovery ofnucleic acids and removal of PCR inhibitors. Substancessuch as polysaccharides, phenolics and others are not entirely

47J. Costa et al. / Trends in Food Science & Technology 26 (2012) 43e55

removed during classical extraction protocols, remaining ascontaminants in the final DNA preparations. These inhibitorscan interfere with the reaction at some levels, causinga decrease or even a complete inhibition of DNA polymeraseactivity. In the specific case of vegetable oils, besides theproblem of being a lipidic matrix containing minor amountsof DNA, another difficulty to overcome is the low integrity ofDNA as a consequence of refining treatment, needed in mostvegetable oils (Costa, Mafra, Amaral, & Oliveira, 2010b).Regarding the extraction of DNA from olive oil, it hasbeen considered a hard task due to its low amount and integ-rity caused by DNA nucleases present in the olive oil(Muzzalupo & Perri, 2002).

Numerous methods have been attempted for DNAextraction from vegetable oils, mainly olive oils, whichare summarised in Table 1. It can be noted that the classicalCTAB-based protocol with or without modifications (cetyl-trimethylammonium bromide) is one of the most reportedmethods for DNA extraction from olive oil, although itsapplication on refined vegetable oils has not been describedas successful (Costa, Mafra, Amaral, & Oliveira, 2010a).From the other reported methods, emphasis is given tothe kit Wizard Magnetic purification system for food thatwas successfully applied to several types of vegetableoils. Nucleospin kits were also effective in the extractionof DNA from monovarietal olive oils and refined soybeanoil, while QIAamp DNA Stool kit was successful in filteredolive oils and other plant oils. When the extraction regardsolive oil, a wide range of starting amounts, from low(100 mL) to relatively high amounts (500 g), with or with-out pre-concentration step, has been reported. This is possi-ble because olive oil is the oily juice mechanicallyseparated from the other components of fruit pulp, beingunique among common vegetable oils since it can be con-sumed in the crude form. The extraction of olive oilinvolves a set of steps that correspond mainly to fruit clean-ing (removal of leaves and washing), crushing (lacerationof cells to access the fat content), malaxation (enhancementof the effect of crushing to make the paste uniform), press-ing and centrifugation (to separate different liquid densi-ties) (Petrakis, 2006).

Still regarding DNA extraction from olive oil, one impor-tant issue to consider is the storage period after milling.According to Pafundo, Busconi, Agrimonti, Fogher, andMarmiroli (2010), for a good traceability of olive oil, thesample should be as fresh as possible to avoid oxidation dam-ages to DNA and to obtain good repeatability and reliabilityof results. Their study showed that AFLP profiles of someolive oil varieties remained similar until a maximum periodof onemonth. Tomonitor the DNA fragmentation in olive oilduring storage, Spaniolas, Bazakos, Ntourou et al. (2008)used lDNA as a marker. The amplification of 415 and 691bp amplicons was not successful for samples stored longerthan 20 and 10 days, respectively, while the 107 bp ampliconwas obtained for all the samples regardless of both concen-tration of spiking lDNA and storage period.

To extract other refined plant oils, it becomes evident theneed of higher starting amounts to overcome the low DNAintegrity, beyond the low yield. Thus, reports stating theneed for relatively high oil amounts that were subjected toa pre-concentration step prior to DNA extraction area more frequent practice in the case of refined oils. Theheat treatments, the use of activated clays and charcoal,and the pH variations during refining may affect the quantityand quality of the DNA that remains in the fully refined oil(Gryson et al., 2002). In spite of the difficulties, some studieshave evidenced positive results for the DNA detection incrude oils, although in refined oils the number of studies isstill limited, especially when compared to olive oil (Table 1).

Hellebrand, Nagy, and M€orsel (1998) reported theextraction of DNA from cold-pressed and refined rapeseedoil from initial start samples of 200 mL. After extractingthe oil with water, the aqueous phase was separated andconcentrated to a volume of 4 mL, which was further con-centrated by centrifugation using mini-concentrators. Theresidue of about 200e300 mL was then incubated at37 �C overnight using TNE buffer, containing sodiumdodecyl sulphate and proteinase K. After digestion, thesample was extracted using phenol and trichloromethanebased solvents and the DNA purified by cold ethanol pre-cipitation overnight. Although this protocol enabled the iso-lation of detectable amounts of DNA, it was laboriousneeding at least two working days.

In another work concerning theDNA extraction from soy-bean oil (Pauli, Liniger, & Zimmermann, 1998), the WizardDNA extraction kit produced amplifiable DNA from a sub-sample of 300 mL (starting sample of 5mL) of crude soybeanoil achieved by cold-pressing. However, when applied tocommercial refined oils no amplifiable DNA was obtained,concluding that the first step of the refining process couldhave removed the DNA to an extent below the limit of detec-tion. Gryson et al. (2002) and Gryson, Messens, andDewettinck (2004) reported the possibility of getting DNAfragments from crude soybean oil samples based on the useof hexane and guanidine thiocyanate as the extraction buffer.Like Pauli et al. (1998), those authors were not able to pro-duce amplifiable DNA from samples submitted to chemicalrefining process after the degumming step, indicating thatalmost all DNA was transferred to lecithin water solution.Even, in samples obtained from physical refining process,they failed to produce amplifiable DNA after the referredstep (Gryson et al., 2002). However, increasing the amountof degummed oil to 365 g, Gryson et al. (2004) verifiedthat the degumming step does not remove DNA completelyfrom the crude oil and higher amounts of test sample couldlead to positive amplifications.

Spaniolas, Bazakos, Awad, and Kalaitzis (2008) com-pared three different methods to extract DNA from sun-flower and olive oil: the DNAExtractor Fat kit, theWizard Magnetic DNA Purification System for Food kitand the QIAamp DNA Stool mini kit. The results showedthat the QIAamp DNA Stool kit gave the stronger PCR


amplification signal compared to the DNAExtractor Fat kit,whereas the Wizard kit gave only one signal for dilutedDNA. The successful results with QIAamp DNA Stool kitwere obtained extracting the pellet after centrifugation of50 mL of refined sunflower oil. Doveri and Lee (2007)referred that using the kit Wizard Magnetic DNA Purifica-tion System for Food and a combination between two dif-ferent methods (NIAB Protocol B and Gene Clean III kit,Bio 101) it was possible to extract amplifiable DNA frompure sunflower and maize oils. Although they did not statethe initial oil amount used for the first method, 1 mL of oilsample was extracted with the combination of the tworeferred methods. Bogani et al. (2009) obtained amplifiableDNA from crude and degummed soybean oils when theyincreased the sample amount from 500 mL to 5 mL usingthe Wizard Magnetic DNA Purification System for Food,with a total of DNA extracted of 3.20 and 1.70 mg,respectively.

To obtain amplifiable DNA from commercial samples offully refined edible oils, Costa et al. (2010a) tested the per-formance of four DNA extraction methods: two commer-cial kits (Nucleospin food and Wizard Magnetic DNAPurification System for Food) and two in-house basedmethods (CTAB and Wizard). The results showed thatonly the Nucleospin food kit was able to extract amplifiablesoybean DNA from all refined oil samples. The sameauthors performed another study where oil samples werecollected at an industrial refining unit comprising crude,degummed/neutralised, washed, bleached and deodorisedsoybean oil, as final product (Costa et al., 2010b). Theydemonstrated that the detection of amplifiable DNA in allstages of the soybean oil refining using the selected Nucle-ospin food kit from 50 g of crude oil and 200 g of neutral-ised, washed, bleached and refined (deodorised) oil waspossible. The successful DNA amplification was attributedto the combination of the pre-concentration step of a rela-tively high amount of oil samples (200 g), the extractionprotocol based on the use of DNA adsorption to silica col-umns and guanidine reagent, and the amplification of smallDNA fragments (103e106 bp).

Identifying species of origin in vegetable oilsThe main sources of vegetable oil production in the

world are oilseeds, totalising more than 85% in 2010,from which soybean was the major contributor (58%), fol-lowed by rapeseed, cottonseed and sunflower (SoyStats,2011). According to the same source of information, soy-bean oil was the second most consumed vegetable oil,accounting for 29%, after palm oil with 33%, while oliveoil accounted with only 2% of the world vegetable oil con-sumption in 2010. Olive oil is undoubtedly one of the oilsmore prone to fraudulent practices as it commands a higherprice than other vegetable oils. The peculiar organolepticcharacteristics of olive oil associated to its proved benefi-cial health effects have increased its popularity and demandin the last years.

Most vegetable oils cannot be used as crude oils sincethey contain several substances that may contribute toundesirable colour, taste and aroma, limiting their applica-tion and shelf-life. To remove those substances, the oilsmust be submitted to a refining process that can be per-formed either physically or chemically, depending on theoil characteristics. Physical refining is essentially done inoils with low content of free fatty acids and phospholipids(palm and coconut), which includes a distillation step thatis enough to remove these components followed by wash-ing and deodorisation. The chemical refining comprehendsan alkali treatment with NaOH in order to remove the freefatty acids and the phospholipids. This refining process alsoincludes degumming, washing, bleaching and deodorisationstages (Johnson, 2002). By the end of the oil production,the final product is considered suitable for daily dietary.

Authentication of vegetable oils can be carried out bya variety of methods, from the classical physic-chemicaltechniques to more recent chromatographic, spectroscopicand molecular-based methodologies, among others. Instru-mental techniques based on chromatographic analysis of dif-ferent families of compounds are among the most usedapproaches suggested for monitoring the quality and authen-ticity of vegetable oils. High performance liquid chromatog-raphy (HPLC) or gas chromatography (GC) have beenapplied for obtaining qualitative and quantitative dataregarding different compounds such as fatty acids, triacyl-glycerols, phytosterols, tocopherols and tocotrienols, hydro-carbons, phenolic compounds, pigments and volatilecompounds. In general, chemical pre-treatment of the sam-ple prior to the chromatographic analysis is required, makingsome of these methodologies time consuming and labourintensive. Several other alternative approaches mainly basedon spectroscopic techniques, such as near-infrared (NIR),mid-infrared (MIR), Fourier transform infrared (FTIR), frontface fluorescence (FFF) and nuclear magnetic resonance(NMR) spectroscopy, are also being increasingly used forevaluating vegetable oils identity (Cunha et al., 2011).

The rising interest towards the use of DNA for foodauthentication is also patented in the case of vegetableoils. Table 1 presents the resumed DNA-based methods toauthenticate vegetable oils. To overcome the problems ofhigh degradation and minute amounts of DNA present invegetable oils, the amplification of small DNA fragmentshas been a recommended practice. Thus, taking advantageof the favoured real-time PCR kinetics for the amplificationof very small fragments, recent papers have proposed thistechnique for vegetable oil authentication. The 5S spacerof the DNA was successfully used to detect the presenceof DNA in refined vegetable oils by multiplex PCR andreal-time PCR with SYBR Green coupled to melting curveanalysis. Pure sunflower and maize oils produced fragmentsof 108 bp and 75 bp size, respectively, reinforcing the spec-ificity and sensitivity of the DNA analysis and suggestingits usefulness for the identification of vegetable oil adulter-ation (Doveri & Lee, 2007).


Wu et al. (2008) reported the successful application ofa real-time PCR assay to distinguish olive oil from otherplant oils. By the use of olive-specific primers targeting thecDNA encoding the Olea europaea plasma intrinsic proteinand a novel fluorescent dye (EvaGreen) for real-time PCR,they were able to establish a sensitive method to detect theadulteration of olive oil with other plant oil species.

Gim�enez et al. (2010) determined the relative quantity oftarget molecules by real-time PCR using the universal SYBRGreen dye of nuclear and chloroplastidial DNA. Indepen-dently of the DNA isolation protocol, they verified that theamount of amplicons around 80 bp was much higher thanthose of 200 bp, suggesting that the determination of optimalamplicon size should be considered for olive oil authentica-tion. Moreover, regarding the DNA type, the quantity ofchloroplastic DNAwas higher than the nuclear DNA for allthe test extraction protocols, with the exception for hexanemethod. Thus, considering the results presented, more atten-tion should be paid when choosing the DNA target for oliveoil authentication. Beyond differences in quantity, cytoplas-matic DNA ismaternal inherited, offering several advantagesover nuclear DNA.

According to Spaniolas, Bazakos, Spano, Zoghby, andKalaitzis (2010), the use of DNA makers to authenticateplant oils requires polymorphic and high copy analytical tar-gets such as the plastid region of the trnL (UAA) intron thathas been used for discriminating several plant species. Thevariability in length of the chloroplast trnL intron amongplant species was exploited to identify ten oil producing spe-cies with extra emphasis on olive oil (Spaniolas, Bazakos,Awad et al., 2008). DNA templates from olive, sunflower,soybean, sesame, hazelnut, maize, cotton, walnut, almondand avocado were mixed prior to PCR amplification, result-ing in the detection of 6 peaks, based on the combinatorialuse of a PCR assaywith a lab-on-a-chip capillary electropho-resis system. However, olive, sesame and avocado producedamplicons with similar electrophoretic mobility makingtheir discrimination not feasible. To further improve the res-olution of PCR products from trnL (UAA) intron anddevelop a reliable analytical method with emphasis on thedetection of sesame in olive oil, the same researchers useda capillary DNA sequencer system (Spaniolas et al., 2010).Eleven plant species could be discriminated on the basis ofdifferential length amplicons, except olive and avocado. Inthe same study, a complementary approach based on SNPdetection technology enabled the discrimination of the tworeferred species. Although the approaches of Spaniolas,Bazakos, Awad et al. (2008) and Spaniolas et al. (2010)seem very promising for plant oil authentication, their appli-cability was mainly tested on leafs and seeds, with the draw-back of using high length PCR amplicons (300e400 bp).

To identify several vegetable oils blended in olive oil,a PCR assay coupled to capillary electrophoresis andsingle-strand conformation polymorphism (CE-SSCP)method targeting the rbcL gene of chloroplast genomewas proposed (Wu et al., 2011). The method presented an

absolute LOD of 0.1 pg/mL of olive DNA, a relative LODof less than 10% DNA from other plant oils and a practicalLOD of 30e50% soybean oil. Applicability to commercialoils was demonstrated, but further improvements are re-quired to increase sensitivity to vegetable oils for its effec-tive use on olive oil authentication.

Because palm oil is readily available and sold at low pri-ces, adulteration of higher valued oils such as peanut oiland soybean oil with palm oil is currently a widespreadpractice (Zhang et al., 2009). Based on the MT3-Bsequence, conventional and real-time PCR assays wereestablished to detect palm oil contamination by amplifyingan amplicon of 109 bp. The methods were able to detectfive haploid copies of palm DNA (10 pg) and were effec-tively applied to commercial edible vegetable oils, indicat-ing the presence of unlabelled palm oil.

Bai et al. (2011) developed a thin-film biosensor chip-based analytical device to rapidly authenticate eight vegeta-ble oils. The method used primers and probes to specificallydetect canola, cotton, maize, soybean, peanut, sunflower,sesame and palm, relying on the hybridization of biotinylatedPCR fragments with covalently attached probes to a thin-filmsilicon biosensor chip in a specific array. The method couldspecifically detect trace levels of oil DNA down to0.1 fmol. However, information about the sources of vegeta-ble oils is missing, as well as the DNA extraction method,which after contacting the corresponding author we wereinformed it was based on CTAB method (Table 1).

Tracing GMO in vegetable oilsThe International Service for the Acquisition of Agri-

biotechApplications estimates that in 2010, 15million farmerscultivated genetically modified (GM) crops over 148 Mhaspread across 29 countries (James, 2010). The major GMcrop species are soybean,maize, cotton and canola, whose cul-tivation is concentrated in the developed countries, dominatingglobal trade of these commodities. Soybean is the main genet-ically modified crop, corresponding to 81% of total plantedsoybean and to 50%ofglobal biotech area (James, 2010).Con-sequently, the use of crops for oil production has been rapidlyincreasing. Recent data have revealed that in 2010, 29% of theworld’s vegetable oil consumption was from soybean, witha major contribution arising from GM seeds since the mainexporting countries (USA, Argentina and Brazil) adoptedmainly this kind of seeds (SoyStats, 2011).

The increase of novel food production and the lack ofinformation and confidence within society regardingGMO have led to the establishment of specific traceabilityguidelines and compulsory labelling requirements by someregulatory agencies. The EU regulations, based on precau-tionary principles, established both the legal basis for theapproval procedure of GMO and the post market traceabil-ity and labelling requirements for GMO and GMO-derivedfood and feeds (Regulations (EC) No. 1829/2003, 1830/2003). Accordingly, any food containing more than 0.9%GM content should be labelled as such.


To guaranty the implementation of these regulations,analytical methodologies allowing accurate determinationof GMO are demanded. Specifically, in blended edible oilsprepared with mixtures of two or more different oils, it isimportant to verify the labelling statements, not only con-cerning their constituents, but also to assess the presenceof GM material since soybean, maize or canola oils are fre-quently used. The most accepted analytical methods forGMO detection are based on DNA techniques, since theprotein-based methods are not reliable for highly processedfood analysis. However, in the specific case of refined vege-table oils, as already mentioned, it is very difficult to obtainamplifiable DNA once it is present at very low amounts and,after refining, it is further reduced and degraded, affectingfinal quantity and integrity (Costa et al., 2010a, b).

One of the first attempts of tracing DNA from vegetableoils for GMO detection was reported by Hellebrand et al.(1998). Those authors were able to amplify DNA from crudeand refined rapeseed oil based on nested PCR to increasespecificity and sensitivity with two separate set of primersin two sequentially PCR amplifications. They concludedthat the amplification of short fragments of DNA (first step350 bp and second step 248 bp) was more successful thanthose of longer length (1005 bp and 439 bp). These resultsindicate that oils might contain amplifiable DNA and PCRcould be used for the detection of GM oilseeds, adulterationof oils and/or identification of species. In opposition to that,Pauli et al. (1998) declared that no signal was observedapplying the same technique (nested PCR) to refined soy-bean oils. They considered that a simple centrifugationstep, which represented the degumming step of the industrialsoybean oil refining, was sufficient to purify crude soybeanoil at least by a factor of 10,000 with respect to DNA. There-fore, according to these authors, oil from GM soybeans didnot need to be labelled as containing GMO. Later on, otherresearchers tried to amplify DNA from crude and degummedsoybean oil (Gryson et al., 2002). The end-point PCR resultsconfirmed that the DNA from crude oil was highly concen-trated (1.86 mg/g of crude soybean oil), presenting a highquantity of fragments of 118 bp even after diluting theDNA extracts by a factor of 5000. After degumming, noamplifiable DNA was observed, thus, in samples from neu-tralised, bleached and deodorised oil, DNAwas not detected,suggesting that it remained in the water fraction (Grysonet al., 2002). Nevertheless, further studies conducted bythe same researchers showed the possibility of detectingPCR fragments after the degumming step, if the DNA wasextracted from a test portion with sufficiently high volume(w365 g of degummed soybean oil) (Gryson et al., 2004).

Bogani et al. (2009) were able to amplify PCR frag-ments from crude and degummed soybean oil until thesize of 470 bp using construct-specific primers of RoundupReady� (RR) soybean. However, no data was presented forthe other steps of refining, until the fully refined oil.

Considering the difficulties addressed to amplify traceamounts of degraded DNA in refined vegetable oils, Costa

et al. (2010a, b) used PCR primers to target small DNA frag-ments. In contrast to previous reports (Gryson et al., 2002,2004; Pauli et al., 1998), Costa et al. (2010a) succeeded todetect DNA fragments of 103 bp targeting the soybean lectingene in samples of refined oils, including blended vegetableoils and soybean oil. For the event-specific detection of RRsoybean, primers specifically designed to target the plant ge-nome and the NOS terminator junction zone produced PCRfragments of 106 bp, which were then verified by real-timePCR with TaqMan probes. The same authors (Costa et al.,2010b), when applying the same PCR protocols, achievedfor the first time the detection soybean DNA in all the stepsof industrial refining, including crude, neutralised, washed,bleached and deodorised oil samples. Moreover, the produc-tion of soybean oil with GM seeds along the total refiningprocess was confirmed by end-point PCR. Amplificationby real-time PCR with specific TaqMan probes reinforcedall the results and proved that it is possible to detect andquantify GMO in the fully refined soybean oil. However,much more efforts are still needed to increase the levels ofDNA detection for quantitative purposes considering thelabelling requirements imposed by EU regulations.

DNA markers for olive oil cultivar identificationThe determination of cultivar(s) of origin can be a deci-

sive aspect regarding the authenticity of olive oil. Olive(O. europaea L.) has a considerable number of differentcultivars, which present differences concerning chemicalcomposition and sensorial characteristics. Moreover,among different countries and even in different regions ofthe same country, genetically identical cultivars are some-times designated by different names (Matos et al., 2007).The chemical composition and sensorial descriptors outlin-ing each cultivar are also affected by climatic and agro-nomic aspects, together with olive ripeness and the oliveextraction system (Arvanitoyannis & Vlachos, 2007). Toprotect foods with unique characteristics, the EU has cre-ated legislation to establish and protect olive oils awardingthem with certification brands PDO (Protected Designationof Origin) and PGI (Protected Geographical Indication),ensuring both consumers’ rights and fair commercial trade.In this context, the determination of olive cultivar(s) used inolive oil production is of high importance for the final prod-uct authentication as, depending on the PDO olive oil, onlycertain cultivars are allowed to be used. Therefore, severalanalytical techniques have been suggested to ensure PDOolive oil authentication regarding the cultivar. The analysisof different chemical components of olive oil coupled tochemometric techniques for data exploitation has beenreported by several authors as a possible approach.

Recently, DNA-based markers have been successfullyapplied to overcome problems associated with differencesdue to environmental conditions of growth and to functionas a diagnostic tool for food authenticity and traceability ofa variety/type composition of complex food matrices in anincreasing number of worldwide projects (Consolandi


et al., 2008; Martins-Lopes et al., 2008; Montemurro et al.,2008; Pafundo et al., 2010).

After overcoming the task of successfully extractingamplifiable DNA from olive oil, a number of markerssuch as simple sequence repeats (SSR), amplified fragmentlength polymorphism (AFLP), random amplified polymor-phic DNA (RAPD), inter-simple sequence repeats (ISSR)and single nucleotide polymorphism (SNP) have been pro-posed to identify olive cultivars present in olive oil samples.It is important to emphasise that the olive oil may be pro-duced, not only from monovarietal fruits, but also frommultiple cultivars, increasing even more the complexityand the usefulness of DNA-based techniques for their dis-tinction. In Table 1 are resumed the application of DNAmakers for olive oil cultivar identification.

AFLPAFLP allow the simultaneous screening of a large number

of lociwithout any need of preliminary sequence knowledge.They are the most efficient markers in revealing many poly-morphic bands in a single assay. When DNA extraction isdifficult to achieve such as in the case of oil samples, the abil-ity of these markers to provide many bands in a single anal-ysis results efficiently (Montemurro et al., 2008). For theiradvantages and high reliability, AFLP have been widelyused for genotyping in a large number of crops and wild spe-cies including olive (Angiolillo, Mencuccini, & Baldoni,1999; Sanz-Cort�es et al., 2003). Busconi et al. (2003) wereable to demonstrate that the DNA extracted frommonovarie-tal olive oil could be used for AFLP analysis, whose profilerevealed high correspondence to the DNA from the leaves ofthe same cultivar. This finding allowed cultivar identificationused for olive oil production by means of the fingerprint ofAFLP analysis. Pafundo et al. (2005) emphasised the DNAextraction as a critical step for the success of AFLP analysis,referring its influence on reproducibility. They reacheda maximum correspondence between AFLP profiles in fourcultivars and respective olive oils of 70%. Nevertheless,while AFLP profiles of DNA from plants were at longer frag-ments, their coincidence in oils was restricted to the shorterfragments below 250 bp. To improve the traceability of oliveoil, Pafundo et al. (2005) suggested that the development ofsequence-characterised amplified region (SCAR) markersderived from reproducible fragments obtained throughAFLP fingerprinting of monovarietal oil would be more use-ful. Thus, the same research group (Pafundo, Agrimonti,Maestri, & Marmiroli, 2007) converted an AFLP fragmentto a robust and specific-single locus PCR-based marker toextend the use of molecular makers to complex foodmatrices. The amplification of the chloroplast fragmentCP-rp116T was considered a SCAR marker, which allowedthe classification of 56 olive oil cultivars in four groups.Though, in general, when fragments were small they couldbe retrieved in oil DNA, being very difficult to amplify frag-ments longer than 300 bp.

Montemurro et al. (2008) demonstrated the possibility ofusing AFLP markers to detect the varietal origin of theolive oil. However, the AFLP profiles from oil were less in-tense and defined than those obtained with the DNAextracted from the corresponding leaves, with a partialcoincidence restricted to fragments under 350 bp, as foundby Pafundo et al. (2005).

RAPDRandom amplified polymorphic DNA makers are widely

applied to plant research such as phylogenetic studies,genome mapping, population genetic studies, as well as incultivar identification such as in olive. The advantages ofthis technique rely on the simplicity of use, low cost andthe need of a small amount of plant material. Muzzalupoand Perri (2002) reported the possibility of using RAPD toanalyse DNA from sediments of olive oil, previously treatedwith proteinase K during the oil production (malaxation).Those authors verified some differences between the leavesand the oil profiles, assigning this discrepancy to the cross-pollination process observed in olive-trees.

Busconi et al. (2003) also applied RAPD to obtain fin-gerprint profiles from 15 olive cultivars, from which theyselected two fragments that after cloning and sequencingwere transformed in more reliable SCAR markers. A simi-lar approach by the development of specific SCAR markersfor olive-tree produced amplification for olive sediments,filtered and unfiltered olive oil (de la Torre, Bautista,C�anovas, & Claros, 2004). The differences found in sixSCAR patterns of three olive oil cultivars were consideredas characteristic fingerprint. Nonetheless, whenever possi-ble, the use of DNA from sediments is recommended.

Martins-Lopes et al. (2008) tested eleven RAPD primersfrom which two produced reproducible bands in all olive oilsamples under study. Among RAPD markers obtained, sevenof nine bands were considered as polymorphic, reportinga mean level of polymorphism of 78%. This finding is ingood agreement with the general lack of reproducibilityattributed to RAPD makers (Jones et al., 1997), which in ouropinion are not adequate markers for olive oil fingerprinting.

ISSRInter-simple sequence repeat markers are generated by

PCR amplification of DNA regions situated between adja-cent and inversely oriented 16e18 bp length simplesequence repeats. Since microsatellite loci are abundantin plant genomes, ISSR primers often produce multiplebands that may be useful for genotyping and mapping,and also for developing SSR markers. Although this tech-nique is less used than other methods, some studies basedtheir work in this type of DNA markers. Pasqualone,Caponio, and Blanco (2001) used ISSR markers for the dis-tinction of drupes from different cultivars combining twosets of primers that were the most polymorphic. In spiteof their unsuccessful application to olive oil, they suggestedISSR as a potential useful tool for varietal identification


purposes when milling the drupes for the production ofPDO oils. Later on, ISSR markers were tested by otherauthors (Martins-Lopes et al., 2008) in leaves and oliveoil material, obtaining a total of 18 reproducible ISSR frag-ments from the two most informative sets of primers. Com-paring with RAPD markers, they consider the ISSRtechnique more informative.

MicrosatellitesMicrosatellites or simple sequence repeats are among

the most reliable markers since they are characterised bya high polymorphic level due to variations of the numberof repeats. Several studies have reported the successfuluse of the microsatellites (Table 1). Pasqualone,Montemurro, Caponio, and Blanco (2004) obtained accept-able levels of amplification from olive oil DNA with pat-terns identical to those of leaves and drupes of the samecultivar. Of the seven primer sets used for SSR markers,six proved to be polymorphic, yielding fragments of differ-ent lengths for each oil type under study. They concludedthat DNA microsatellites were able to distinguish virginolive oils from different cultivars, with good discriminatingability and that this technique might be applicable to mix-tures of 3e4 cultivars, such as those usually adopted inPDO oils. Testolin and Lain (2005) considered the use ofmicrosatellite polymorphism for olive oil cultivar identifi-cation a promising tool. From six SSR primer sets tested,all gave DNA amplicons of the expected sizes for unfilteredoil samples. In case of low DNA yield, nested PCRimproved the amount of amplified DNA. Although DNAfrom olive oil was found degraded, they were consideredto be long enough to allow making copies of fragmentsup to 188 bp, enabling consistent amplification of SSRfrom low starting amounts of oil.

Doveri, O’Sullivan, and Lee (2006) investigated the con-tribution of paternal alleles to the DNA content of olive oilby the use of SSR markers, verifying that care should betaken when interpreting DNA profiles from olive oil.DNA extracted from maternal tissues (leaves and olivepulp) revealed identical genetic profiles by means of SSRmarkers. However, those authors found additional allelesin embryos (stone), also found in the paste obtained bycrushing whole fruits and from oil pressed from this mate-rial. These results demonstrate that the DNA profile fromolive oil is likely to represent a composite profile of thematernal alleles juxtaposed with alleles contributed by var-ious pollen donors. In opposition to that, Muzzalupo,Pellegrino, and Perri (2007) showed that DNA purifiedfrom olive oil can be used for microsatellite analysis andthat the profile of DNA purified from monovarietal oil cor-responded to the profile of DNA from the leaves of thesame cultivar. Ayed, Grati-Kamoun, Moreau, and Reba€ı(2009), when assessing the potential applicability of micro-satellites to trace Tunisian olive oil cultivars, compared thegenetic profiles from DNA extracted from oil and leaves oftwo cultivars. For some SSR markers, they were able to

identify alleles of the pollinators in oil samples and distin-guish them from alleles of tree somatic tissues, suggestingtheir reliability for olive oil traceability. Alba et al. (2009)went even further reporting that the low concentration andhigh degradation of DNA, and the possible presence ofadditional paternal alleles in oils from entire drupes, shouldbe taken in consideration when comparing SSR profilesfrom leaves with the corresponding oils for varietal trace-ability purposes. Those authors amplified the SSR frag-ments with 85.7% rate of success and evidenced that 90%of their experiments showed identical patterns betweenleaves and oil DNA. The use of capillary electrophoresisby an automatic sequencer facilitated the identification ofspecific alleles, even for weak signals, confirming thatDNA microsatellites were able to distinguish and identifyolive oils from different cultivars.

SNPCompared to other genetic markers, single nucleotide

polymorphism are abundant in the genome and geneticallystable, being effectively applied to genotype olive cultivars(Reale et al., 2006). The application of SNP to olive culti-var identification was then performed coupled to a microar-ray based assay by means of ligation detection reaction(LDR) in a universal array (UA) format (Consolandiet al., 2007). The same authors further improved theLDR-UA platform by introducing multiplex PCR to simul-taneously amplify 13 DNA fragments containing 17 SNPloci from leaves and olive oil (Consolandi et al., 2008).The assay provided enough discriminating power to distin-guish 49 olive cultivars, allowing high-throughputcapacities if used as a semi-automated SNP genotypingassay to identify the origin of monovarietal olive oils. Con-sidering the needs for authentication and the great numberof olive oil cultivar, the availability of automated multiplexplatforms seems to represent very promising tools to dis-criminate olive oil cultivars.

Concluding remarksThe growing interest towards the use of DNA makers for

food authentication together with the increasing demandsof tracing GMO have been the driving forces for the appli-cation of molecular methods in food analysis, includingvegetable oil matrices. Concerning the authentication ofolive oil, which is a food commodity that can reach pre-mium prices, the efforts and progress in the application ofmolecular makers are patented in this review. The progresson DNA extraction protocols applied to olive oil has led toenhancements in DNA recovery and quality. Analyticalparameters independent from environmental fluctuations,such as DNA-based markers, have provided useful toolsfor cultivar discrimination, as well as plant species identifi-cation in olive oils. It is pertinent to refer the great impor-tance of small length DNA markers to discriminate olive oilvarieties, mainly due to the difficulties in obtaining DNAextracts with adequate quantity and purity. Considering

Table 2. Summary of pros and cons of the main molecular markers for vegetable oil authentication.

Pros Cons

PCR � Species identification in crude and refined vegetable oils� Detection of one or more DNA fragments, including GMO,� Detection of vegetable oil adulterations,� Application of real-time PCR technique as a potentialquantitative tool

� DNA extraction is a critical step for its applicationto refined vegetable oils

� Unable to discriminate olive cultivars

SNP � Easy performance and interpretation with the possibility ofcombining high-throughput technologies (arrays),

� Application to genotype olive cultivars,� Multiple SNP detection in single DNA analysis,� Ability to distinguish small differences among very similarolive oil cultivars or others oil crop species,

� Apparently allows high correlation between olive leavesand olive oil,

� Application to assess the authenticity of monovarietal olive oils.

� Requires expensive technology (capillary electrophoresis,LDR universal array, multiplex PCR coupled withmicroarray technology),

� More recently applied, less tested in olive oil matrices,� Lack of information concerning the comparison withother single locus markers.

SSR � Highly polymorphic and reliable markers� Easy performance and interpretation� Most employed DNA markers, great discriminatory power� Identification of different olive oil cultivars� Application to assess the authenticity of monovarietal olive oils.

� Amplification of relatively high length DNA fragments� Limited reproducibility when applied to olive oilsdue to the low DNA integrity

AFLP � Highly polymorphic and reliable markers without previousknowledge of the genome sequence

� One of the most used multi-locus DNA markers for theidentification of olive oil cultivars

� Possibility of converting AFLP into SCAR markers� Application to assess the authenticity of monovarietal olive oils.

� High complexity of the technique� Difficult to analyse the numerous bands obtained,so not suitable for varietal oil mixtures

� Limited reproducibility when applied to olive oilsdue to the low DNA integrity


that it was demonstrated the integrity of DNA is reducedwith the storage period of olive oil after milling, the useof small length DNA fragments is highly recommended ifevaluation of commercial samples, with previous storageperiod, is required.

Regarding olive oil markers, SNP seem to be the mostuseful markers since they allow distinguishing small differ-ences among very similar individuals, while SSR markershave been the most widely applied because of the high dis-criminatory power. However, the reproducibility of SSRmarkers is sometimes compromised because some frag-ments, mainly of higher length, are not always amplified.Table 2 summarises the main advantages and disadvantagesof the principal DNA markers used to identify species oforigin of vegetable oils and varietal composition of olive oil.

Concerning the specific identification of several plantoils as potential adulterants of olive oil, recent progresseshave been done for species differentiation. Exploiting chlo-roplast DNA to discriminate plant oil species seem to bea powerful approach taking advantage of the high numberof DNA copies, which is especially beneficial for refinedoils. However, amplification of short DNA fragments isrecommended.

When the oilseed crops, such as soybean, are the sourcefor vegetable oil production, additional concerns due to thepresence of GMO have prompted the improvements inDNA analysis for this target. It became clear and emphas-ised by several authors that one main constraint to over-come in the case of refined vegetable oil matrices is theisolation of acceptable quality DNA. To isolate minute

amounts of DNA present in this kind of matrices, highquantities of starting sample (w200 g) has been recommen-ded mainly for refined vegetable oils. Considering thecapacity for DNA amplification from fully refined oils orsubjected to a first step of refining (degumming), it ishighly advised the use of primers targeting small fragments(w100 bp), such as highlighted in the case of olive oil.

Taking in consideration the unsuccessful first attempts indetecting DNA from refined vegetable oils, the latest devel-opments present promising results regarding the traceabil-ity for the origin of plant oils and GMO detection. Thisis of major importance to verify the labelling compliance,with great potential for application in the food industries.However, future research is still required to increase theamount and quality of template DNA for quantitative anal-ysis by real-time PCR, which is undoubtedly the techniqueof choice for this purpose.

AcknowledgementsThis work has been supported by Fundac~ao para a Ciencia

e aTecnologia (FCT) through grant no. PEst-C/EQB/LA0006/2011. Joana Costa is grateful to FCT PhD grant (SFRH/BD/64523/2009) financed by POPH-QREN (subsidised by FSEand MCTES).

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http://europa.eu.int/eurlex/pri/en/oj/dat/2003/l_268/l_26820031018en00240028.pdf

http://europa.eu.int/eurlex/pri/en/oj/dat/2003/l_268/l_26820031018en00240028.pdf

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