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Food Control 22 (2011) 542e548

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Food Control

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Detecting Satureja montana L. and Origanum majorana L. by means of SCARePCRin commercial samples of Mediterranean oregano

Matteo Marieschi, Anna Torelli, Alberto Bianchi, Renato Bruni*

Department of Evolutionary and Functional Biology, University of Parma, V.le G. P. Usberti 11/A, I-43100, Italy

a r t i c l e i n f o

Article history:Received 7 July 2010Received in revised form5 October 2010Accepted 5 October 2010

Keywords:OreganoSavoryMarjoramMolecular markersAuthenticationFood adulteration

Abbreviations: CTAB, hexadecyltrimethylammovinylpyrrolidone; BSA, bovine serum albumin; EDTAacid.* Corresponding author. Tel.: þ39 0521 906004; fa

E-mail address: renato.bruni@unipr.it (R. Bruni).

0956-7135/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.foodcont.2010.10.001

a b s t r a c t

A recent pharmacognostic survey of the European market highlighted a high frequency of adulteration ofcommercial Mediterranean oregano with species of the Lamiaceae family (Satureja montana L. andOriganum majorana L.) having a similar appearance and a superimposing essential oil profile. Whenperformed by skilled adulterators, this dilution can only be detected by pharmacognostic assays orphytochemical analyses, which can be either extremely laborious, expensive and time consuming whenmultiple batches of oregano have to be evaluated, or can produce inconclusive results. Sequence-Char-acterized Amplified Regions markers (SCARs) specific for these adulterants were developed fromRandom Amplified Polymorphic DNA (RAPD) markers, in order to support results of pharmacognostic orchemical analyses and/or to speed up the primary screening of oregano batches and allow the pre-emptive rejection of suspect samples, thus narrowing the number of samples to be subjected to furthertests. The SCAR primers gave rise to the amplification of specific bands of expected sizes which allowedthe detection of down to 0.5% of adulterating plants, despite the small genetic distance between thespecies involved. A specific protocol for DNA extraction was set up. In addiction, the relatively small sizeof these amplicons is suitable for the analysis of potentially degraded DNA obtained from dried andstored commercial material.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Fraud is a common practice carried out in the spice trade sincethe very beginning of commerce and such proclivity cannot beoverlooked nowadays, in the framework of complex, global supplychains that steadily emphasize the gap between producers,suppliers and consumers (Muggeridge, 2004). The impropercommercial practices that can be detected with modern technol-ogies are manifold, but even when the techniques available forquality control are top-notch, unfair traders can always find grey-zones in the analytical process to take advantage of, thus urging thedevelopment of new or upgraded monitoring tools (Cordella,Moussa, Martel, Sbirrazzuoli, & Lizzani-Cuvelier, 2002). Spices areusually composed of dried, processed plant material endowed ofa specific taste or aroma due to the presence of essential oils, whoseabundance and chemical profile are reputed as main qualitymarkers. However, the tailored addition of non-aromatic plants or

niumbromide; PVP, poly-, ethylenediaminetetraacetic

x: þ39 0521 905403.

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plant drugs having a very similar fragrance may be difficult to spotif carefully and suitably performed (Fedec & Kolodziejczyk, 1998).The addition of extraneous, inert matter not being recognizable atfirst sight is one of the most common kinds of adulteration known.Further possibilities may be encountered, with the deliberateaddition of different plants of lower commercial value but withsimilar aromatic profile and belonging to the same botanical family.The spices that are the objects of fraud are either those with highcommercial value and concurrent low availability (like saffron) orthose marketed in large volumes, in which the leverages ofeconomy of scale may multiply small percentages of adulterantsinto large fraudulent gains (like black pepper, chili, cinnamon andoregano) (Chakrabarti & Roy, 2003; Marieschi, Torelli, Poli,Sacchetti, & Bruni, 2009; Sampathu, Shivashankar, & Lewis, 1984;Tremlova, 2001; Weiss, 2002). The spice commercially known asMediterranean oregano is obtained from dried leaves and floralparts of few species and subspecies of the Origanum genus, namelyOriganum vulgare L. subsp. hirtum (Link) Ietsw. (syn. O. her-acleoticum auct. non L.) and O. onites L., Oregano Monograph inEuropean Pharmacopoeia, (2005), (Ph. Eur.). Impurities up to 2%(thereby including different plant organs from those specified inthe drug definition, as stems) can be considered acceptable andsimilar values are tolerated by American Spice Trade Association

M. Marieschi et al. / Food Control 22 (2011) 542e548 543

(ASTA, 2006) and European Spice Association (ESA, 2007). On theother hand ISO/FDIS 7925 (1999) standard for oregano allows theuse of any Origanum species, with the sole exception of Origanummajorana L., but lowers the limits of extraneous matter to 1%. Arecent survey of the European market from 2000 to 2007 revealedthat, more often than not, products labeled and sold as trueMediterranean oregano were not corresponding to what wasdeclared, evidence of an established practice consisting of theaddition of: I) plants lacking a clearly detectable essential oilprofile, added as bulk extraneous material (Rubus sp., Cistus incanusL., Rhus coriaria L.) and, II) essential oil bearing plants with oregano-like flavor, mostly belonging to Lamiaceae family (Satureja montanaL., O. majorana L.) (Marieschi et al., 2009). Newmethods for the fastdetection of non-aromatic adulterants, based on molecularmarkers, have been thus developed and successfully evaluated(Marieschi, Torelli, Bianchi, & Bruni, 2010; Marieschi et al., 2009).The detection of an adulteration (intended as the deliberate addi-tion of foreignmaterial or its incidental presence above the allowedlevels) is basically a methodological problem, whose solution ismore difficult the more the addedmaterial is similar is with respectto the starting one. In fact, when species of the same family or evenbelonging to the same genus are added to dilute the original spice,their presence is more difficult to spot as most prominentmorphological or phytochemical traits may be extremely similar. Inthese cases the recourse to traditional microscopical inspectionpresents some shortcomings in terms of reliability and accuracyand cause a heavy dependence on the specific experience of theexaminers and on their subjective interpretation.

On the other side, while providing less subjective and moreautomatized results, analytical chemistry presents some restric-tions too. For instance, the unequivocal definition of chemicalmarkers able to discriminate chemotaxonomically close speciesand the attribution of unambiguous ratios between thesesubstances may be hampered by natural variability (Figueredo,Barroso, Pedro, & Scheffer, 2008; Kintzios, 2002). Furthermore,a dilution purposely performed in order to take advantage of theshortcomings of the techniques most commonly used can gounnoticed, for instance by adding plant material devoid of volatilesubstances to samples screened only by means of GCeMS, which isat present the technique of choice (Baranska, Schulz, Krüger, &Quilitzsch, 2005; Bernath, Novak, Szabo, & Seregely, 2005; Huie,2002; Moller, Catharino, & Eberlin, 2007; Nhu-Trang, Casabianca,& Grenier-Loustalot, 2006; Novak, Zambori-Nemetn, Horvath,Seregely, & Kaffka, 2003; Schultz, Quilitzsch, & Kruger, 2003).Thus, the reliability of pharmacognostic and analytical methodscould be limited, making the recourse to different approachesa valuable option.

DNA markers have recently become an increasingly popularmeans for the identification and authentication of a steadilyincreasing range of food products and medicinal and aromaticplants, given their species-, subspecies- or even cultivar-specificresults (Dhanya & Sasikumar, 2010; Joshi, Chavan, Warude, &Patwardhan, 2004; Marmiroli, Peano, & Maestri, 2003; Tautz,Arctander, Minelli, Thomas, and Vogler (2003); Sucher & Carles,2008). If compared to phytochemical analysis, they offer someadvantages in terms of being less affected by the age of the startingplant material, by environmental and post-harvest factors and forbeing almost non-tissue specific. The low price of the requiredequipment and the possibility of screening large batches in a singlerun are further strengths. Among the different strategies enforce-able, RAPDePCR is maybe the easier choice but reliability andreplicability are major shortcomings. However, the polymorphicbands generated from RAPDs can be converted to more specificSequence Characterized Amplified Regions (SCAR) in order toimprove the reproducibility and overcome these constraints. These

improved markers have several advantages over RAPDs: i) by usinglonger and sequence-specific PCR primers, they improve thereproducibility of band polymorphism; and ii) because theirannealing temperatures are more stringent, only specific loci can bedetected by using each set of primers. SCARePCR is thus emergingas an attractive choice for quality control in foods, in particularwhen traditional techniques pose time constraints as in case ofmonitoring of several batches (Marmiroli et al., 2003).

A specific experience acquired in setting up protocols for DNAextraction, RAPDePCR analysis and SCAR markers development inoregano samples were thus put into practice in order to obtaina specific protocol enforceable to detect contaminants of Mediter-ranean oregano made of other species of the same Lamiaceaefamily orOriganum genus. The objective of the present paper is thusto develop robust SCAR markers derived from RAPD fragments forS. montana and O. majorana, in order to confirm their presence asadulterants, simplify and speed up the primary screening ofcommercial Origanum batches. The final goal is to obtain a fast andreliable diagnostic tool for the pre-emptive rejection of suspectsamples, thus reducing the number of samples to be evaluated bymeans of pharmacognostic analyses and providing useful data forfurther molecular diagnostic tools. Themethodwas optimized withspecific respect to an easy and robust application on plant materialof commercial grade in order to complement existing pharma-cognostic and chemical methods.

2. Material and methods

2.1. Plant material

Fresh oregano samples were obtained from seeds of differentOriganum taxa (O. onites L., O. vulgare L., O. vulgare L. subsp. vulgare,O. vulgare L. subsp. hirtum Ietswaart, O. heracleoticum L, O. vulgare L.subsp. virens Ietswaart, O. vulgare L. subsp. gracile Ietswaart,O. vulgare L. subsp. viride Hayek, O. majorana L., O. vulgare subsp.viride � O. majorana) and cultured on silty soil in greenhouse(Botanical Garden of the University of Parma, Italy). Seeds werekindly provided by Giardino delle Erbe Officinali di Casola Valsenio(Ravenna, Italy), North Central Regional Plant Introduction Station(Ames, Iowa, USA) and Genbank Dept., Leibniz Institute of PlantGenetics and Crop Plant Research (Gatersleben, Germany). Plantmaterial for S. montana L., and Satureja hortensis L. was kindlyprovided by Giardino delle Erbe Officinali di Casola Valsenio (Rav-enna, Italy). Fresh plant material was collected and immediatelyfreeze-dried in liquid nitrogen and stored at�80 �C until molecularanalysis. Dried material of each species was prepared from freshmaterial by drying at 40 �C for one week. Mediterranean oreganodried samples were purchased from international wholesaletraders as previously reported (Marieschi et al., 2009). Onceacquired and in order to simulate real commercial conditions,samples have been stored in dry and dark conditions at 5 �C untilperforming molecular analysis.

2.2. DNA extraction

Genomic plant DNA was isolated from fresh and dried materialas previously described with some modifications (Marieschi et al.,2009; Tibbits, McManus, Spokevicius, & Bossinger, 2006), toincrease the yield and the purity of the DNA extracted and reducethe inhibitory effects of carbohydrates and polyphenols in thesubsequent PCR reactions. About 50e100mg of plantmaterial wereplaced in 2 ml tubes with 900 ml of CTAB extraction buffer in thepresence of activated charcoal and PVP (Marieschi et al., 2009). Theextraction was conducted overnight, at room temperature undermild agitation. After the extraction the tubes were centrifuged at

M. Marieschi et al. / Food Control 22 (2011) 542e548544

2000�g for 10 min at room temperature to remove cellular debris.The supernatants were collected and transferred into new 2 mlreaction tubes, fresh CTAB extraction buffer was added to bring thefinal volume to 800 ml. To each sample were added 110 ml ofa mixture of NaCl 4.54M and BSA 3.63% and 2 ml of RNAse 20mg/mland the tubes were incubated at 37 �C for 30 min. After incubationextraction with 1 vol of chloroform:isoamyl alcohol (24:1) wasperformed twice. The supernatants were then transferred into new1.5 ml reaction tubes, precipitated with 1 vol of propan-2-ol 100%and dissolved in 50 ml of sterile distilled water or TE (10 mMTriseHCl and 1 mM EDTA, pH 8.0). DNA quality, concentration andintegrity were evaluated both by spectrophotometric analysis(A260/A280 and A260/A230 were evaluated with a spectrophotometerUvikon 930, Kontron Instruments) and by visual comparison withDNA standards, of known concentration, in ethidium bromide-stained agarose/TAE (TriseacetateeEDTA) gels, and adjusted toapproximately 10 ng/ml with sterile distilled water or TE. Agarosegels were analyzed and quantified with a Kodak DC40 camera(Kodak) using the Kodak digital science 1D Image analysis software(Eastman Kodak Company, Rochester, NY, USA). DNA extracted wasoften strongly contaminated by carbohydrates and polyphenols,this possible hindrance to PCR reaction was overcome through theuse of PCR additives (Tibbits et al., 2006; Marieschi et al., 2010) (seeabove).

2.3. RAPD markers for conversion

S. montana, and O. majoranawere comparedwith nine Origanumtaxa mentioned above to develop RAPD markers (Marieschi et al.,2009). PCR reactions were conducted on approximately 20e40 ngof DNA template and were performed in 25 ml volume containing67 mM TriseHCl (pH 8.8), 16.6 mM (NH4)2SO4, 0.01% Tween 20,2 mM MgCl2, 1U SubTherm Taq DNA Polymerase (Fisher MolecularBiology, Trevose, PA, USA), 25 pmol specific primers. Amplificationwas performed as follows: 94 �C for 5min, 40 cycles of 94 �C for 40 s,36 �C for 40 s, 72 �C for 2 min, followed by one cycle of 72 �Cfor 10 min (PTC-100, MJ Research Inc.). One RAPD fragment,OPA09Sm193, specific for S. montana was obtained from the ampli-fication with the primer OPA09 and converted in the SCAR markerScSm169. From the RAPD amplicon OPA07670, present in the profilesof all the consideredOriganum taxa following amplificationwith theprimer OPA07, two SCAR markers were designed by constructingthefirst primer pair on the regions ofmaximal homology among thesequences of four Origanum species and the second on the regionsofmaximal heterogeneity between the sequence ofO.majorana andthe grouped sequences of the other three Origanum taxa. The firstcouple of primers amplifies an Origanum genus specific SCARmarker (ScOspp382e385), suitable as positive control to check thePCR availability of the DNA extracted from dried commercialsamples. The second primer pair defines instead a further SCARmarker (ScOm349) used for the detection of O. majorana. Theselected marker bands were excised from 2% agarose gels, purifiedusing JET-Sorb Gel Extraction Kit (Genomed, Löhne, Germany),cloned in pGEM T-easy vector (Promega Corporation, Madison, WI,

Table 1Contaminant-specific SCAR markers and primer specifications.

Species Orginal RAPDamplicon

SCARmarker

SCAR primer sequence

Origanum spp. OPA07Ospp670 ScOspp382e385 For TGACACTGAACRev GGGTGGGAAAA

Origanum majorana OPA07Om667 ScOm349 For GCTCAGTATTCTRev CGACGCTCATT

Satureja montana OPA09Sm193 ScSm169 For AAGAAGATGTARev TTAACTAAGAC

USA). The transformed bacterial colonies were screened throughPCR colony and clones carrying correctly sized inserts weresequenced.

2.4. Sequence analysis

Sequencing of the positive clones was carried out following theprotocol “CEQ 2000 dye terminator cycle sequencing” (BeckmanCoulter, Fullerton, CA, USA), by means of the automatic sequencerCEQ 2000 (Beckman Coulter). Database searches of sequencehomology were performed using the programs BlastN, BlastX, andPSI-BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al.,1997) set to standard parameters. The sequence alignment wasconducted with ClustalW 1.82 (http://www.ebi.ac.uk/cgi-bin/clustalw/) (Chenna et al., 2003).

2.5. SCAR design and analysis

The sequence data were used for the SCAR primer design andhomology searches by the BLAST program. SCAR primers at least 20nucleotides long were designed for stringent conditions ofannealing temperature (about 60 �C) and did not contain thesequence of the original RAPD primers (Table 1). The ScOm349reverse primer, which was designed in a region high in GC content,was 19 nucleotides long. The annealing temperature was firstcalculated as 4-fold the number of GC plus 2-fold the number of AT.Each primer pair was tested up to 5 �C over and under its annealingtemperature to obtain the optimum.

PCR reactions were conducted on approximately 20 ng of DNAtemplate and were performed in 25 ml volume containing 67 mMTriseHCl (pH 8.8), 16.6 mM (NH4)2SO4, 0.01% Tween 20, 2 mMMgCl2, 1U SubTherm Taq DNA Polymerase (Fisher MolecularBiology),12.5 pmol specific primers. Unless not otherwise specified,amplification was performed as follows: 94 �C for 5 min, 35e40cycles of 94 �C for 40 s, 55 �C for 40 s, 72 �C for 40 s, followed by onecycle of 72 �C for 10 min (PTC-100, MJ Research Inc.). An aliquot(10 ml) of the amplification product was resolved by electrophoresison 1.5% agarose gel and detected by ethidium bromide staining.

2.6. Validation of the SCAR markers

DNA samples of the two contaminant species were analyzedwith the specific primer pair defined for each species. After this firstvalidation the primer pairs were tested on different species ofOriganum to exclude possible misinterpretation of the results. Theprimer pair constructed on the S. montana sequencewas also testedon DNA extracted from S. hortensis. Once marker specificity wasconfirmed, SCAR analysis was conducted on genomic DNA extrac-ted both from mixtures of Origanum containing 0.5, 1, 2 and 5% ofthe contaminant species prepared by dried material and from driedcommercial samples. The PCR reactions conducted on DNA fromdried material were strongly impaired by the presence of largeamount of both carbohydrates and polyphenols co-precipitatedwith DNA during the extraction. This hindrance was avoided by

(50/ 30) Calculated annealingtemperature (�C)

Working annealingtemperature (�C)

Ampliconlenght (bp)

GTCACG 52 55 382e385GAAGAG 52 55CAGCCTCC 62 62 349

TCAGTCCC 62 62AAGCATTCTGC 60 58 169AAGAACCGAGG 62 58

M. Marieschi et al. / Food Control 22 (2011) 542e548 545

enhancing the PCR amplification through the addition of BSA 0.4%and a non-ionic detergent like Triton X-100 0.5% or Tween 20 0.5%(Demeke & Adams, 1992; Monteiro et al., 1997).

3. Results and discussion

3.1. Development of SCAR markers

We have previously analyzed 210 RAPD profiles obtained byamplification with 20 primers on samples from 10 taxa belongingto the genus Origanum and the species S. montana, both belongingto Lamiaceae family. Only two of the analyzed Origanum taxa areallowed for their use as oregano by Ph. Eur. (O. onites, O. vulgaresubsp. hirtum), while ISO standard broaden this denomination to allthe species of the genus Origanum with the only exception of O.majorana, which is considered as adulterant in every respect (ISO/FDIS 7925, 1999; Ph. Eur., 2005). A previous pharmacognosticsurvey indeed indicated that O. majorana and S. montana are two ofthe contaminants most frequently detected in Mediterraneanoregano commercial samples, often in elevated percentages and insome cases exceeding 90% (w/w), in most of cases well above thelimits allowed by both Ph. Eur. and ISO standard (Marieschi et al.,2009). The polymorphic bands to be cloned were selected on thebasis of their reproducibility, the amount of DNA obtained ina single RAPD reaction and the absence of other bands in theproximity to avoid contamination.

Fig. 1. ClustalW analysis of the homologous RAPD fragments OPA07Ospp670 obtained fromstars below the alignment indicate that the nucleotides are identical. Gray boxes highlight tfor the amplification of the Origanum genus specific SCAR marker (ScOspp382e385). BlacO. majorana and the grouped sequences of the other Origanum species, these regions were seO. majorana specific SCAR marker (ScOm349).

A RAPD amplicon specific for S. montana (OPA09Sm193), absentin the RAPD profiles of the considered Origanum taxa, was clonedand sequenced. The sequence was deposited into dbGSS/GenBank(accession number GS885206). The BLAST of the nucleotidesequences did not have homology with any coding sequences ofstructural genes. This discriminating RAPD band was subsequentlyconverted into the SCAR marker ScSm169.

The SCAR marker for O. majorana was derived from a mono-morphic RAPD band (OPA07Ospp670) obtained in all the comparedOriganum taxa from the amplificationwith the RAPD primer OPA07(Marieschi et al., 2009). The bands, nearly 670 bp long, obtainedfrom O. onites, O. vulgare, O. vulgare subsp. hirtum and O. majoranawere cloned and sequenced. The sequences were deposited intodbGSS/GenBank (accession numbers GS923389, GS885204,GS885205, GS885201) and the BLAST of their nucleotide sequencesdid not have homology with any coding sequences of structuralgenes. ClustalW analysis (Fig. 1) shown the almost complete iden-tity of the OPA07Ospp670 nucleotide sequences and allowed theidentification of two regions of scarce homogeneity between thesequences of O. majorana and the grouped sequences of the otherthree Origanum taxa. SCAR primer pairs specific for O. majorana(ScOm349) and generic for Origanum spp. (ScOspp382e385) were thusrespectively constructed on the regions of maximal heterogeneity(Fig. 1, black boxes) and maximal homology (Fig. 1, gray boxes). Thesequences amplifiable with these two SCAR primer pairs arepartially overlapped and the sequence obtainable with the SCAR

four Origanum taxa: O. vulgare subsp. hirtum, O. vulgare, O. onites and O. majorana. Thehe regions of high sequence homology selected to design the primer pair (gray arrows)k boxes highlight the regions of maximal heterogeneity between the sequences oflected to design forward and reverse primers (black arrows) for PCR amplification of the

Fig. 2. SCAR markers specificity assay. A- PCR performed with the Origanum genus specific ScOspp382e385 primer pair; B- Reactions performed with ScOm349 primer pair specific forthe contaminant species Origanum majorana; C- Reactions performed with ScSm169 primer pair specific for the contaminant species Satureja montana. - (negative control),amplification with no template DNA; O.h., DNA from O. vulgare subsp. hirtum; O.o., DNA from O. onites; O.m., DNA from O. majorana; S.h., DNA from Satureja hortensis; S.m., DNA fromS. montana; M, 100 bp DNA ladder.

M. Marieschi et al. / Food Control 22 (2011) 542e548546

primer pair ScOspp382e385 for O. majorana results 3 bp shorter thanthose obtainable from the other analyzed Origanum taxa. In orderto increase the amplification specificity, with the exception ofScOspp382e385 reverse primer which contains a 5 bp partialsequence of the original OPA07 primer, all the SCAR primers weredesigned internally to the sequence of the original amplicons anddid not contain the sequences of the RAPD primers (OPA09 andOPA07, respectively for OPA09Sm193, and OPA07Om670), thus givingrise to amplification products shorter than the respective RAPDbands. The relative short dimension of the amplification targetsoffers a further advantage, being these SCARmarkers more suitablefor the analysis also when DNA is partially degraded. This event isextremely likely to occur in dried plant material stored at roomtemperature often for a rather long time, a fact that representsroutine in the herbs and spices market. The primer sequences, theirannealing temperature and the dimensions of the amplicons arelisted in Table 1.

In our previous work the use of BSA and Tween 20% in PCRreactions demonstrated to be highly effective in improving PCRsuccesses without requirement of DNA purification and thusreducing the time required for each analysis (Marieschi et al., 2010).These two additives were thus added in PCR reaction allowinga reliable amplification close to the designed annealing tempera-ture and in the case of ScOspp382e385 even 3� above it, without

Fig. 3. ScOm349 and ScSm169 SCAR markers sensitivity assay. Analyses were conducted onhirtum and each contaminant species. Dried leaves of every single contaminant species wereof Origanum, before extracting genomic DNA. A- Limiting PCR (30 cycles of amplification)control), DNA from O. vulgare subsp. hirtum; O.m. (positive control), DNA from O. majorana100 bp DNA ladder. B- Limiting PCR (33 cycles of amplification) performed with ScSm169 primhirtum; S.m. (positive control), DNA from S. montana; 0.5%, 1%, 2%, 5%, DNA from mixtures

losing specificity as a single band of the expected length wasobtained for each reaction.

3.2. Validation of selected markers

SCAR marker specificity was validated by testing each primerpair on plants other than the target species on which primersequences were designed. The primer pair ScOspp382e385 func-tioned as a genus specific marker giving, as expected, amplificationwith all the considered Origanum taxa (Fig. 2, panel A) producinga 385 bp fragment in all the tested Origanum taxa and a 382 bpfragment in O. majorana, whereas ScOm349, allowed the amplifi-cation of a species specific SCAR marker, giving an amplificationband of the expected size only with DNA extracted from O. major-ana (Fig. 2, panel B). The tested primer pair specific for S. montanadid not generate any amplification product when applied todifferent taxa of Origanum (O. onites, O. vulgare subsp. hirtum) norwhen tested on DNA extracted from S. hortensis, revealing to beScSm169 a species specific SCAR marker as well (Fig. 2, panel C).

After checking marker specificity, the method was evaluated inorder to define its sensitivity in adulterant detection. In Fig. 3 arereported the results obtained with DNA extracted from artificialmixtures (see Section 2.6) containing O. vulgare subsp. hirtum and0.5, 1, 2 or 5% of O. majorana (Fig. 3, panel A) or S. montana (Fig. 3,

DNA extracted from individual dried leaves and mixtures of Origanum vulgare subsp.mixed in three different percentages (shown at the top of the figure) with dried leavesperformed with ScOm349 primer pair specific for Origanum majorana: O.h. (negative; 0.5%, 1%, 2%, 5%, DNA from mixtures of O. vulgare subsp. hirtum and O. majorana; M,er pair specific for Satureja montana: O.h. (negative control), DNA from O. vulgare subsp.of O. vulgare subsp. hirtum and S. montana; M, 100 bp DNA ladder.

Table 2Composition of Mediterranean oregano samples identified by microscopic pharmacognostic assay.

Sample Botanical species (w/w %)

Mediterranean oreganoa Origanum majorana Satureja montana Cistus incanus Rubus sp. Rhus sp. Olea europaea Other

1 60.1 2.9 9.7 0.0 0.0 0.0 26.7 0.62 86.4 8.7 4.9 0.0 0.0 0.0 0.0 0.03 67.9 6.4 7.8 16.4 0.0 0.0 0.0 1.44 95.9 0.0 3.3 0.0 0.0 0.7 0.0 0.15 5.0 95.0 0.0 0.0 0.0 0.0 0.0 0.06 6.7 3.0 5.4 0.0 0.0 83.0 0.0 1.97 73.7 11.7 0.0 0.0 0.0 0.0 14.6 0.0

a Origanum species and subspecies, excluding O. majorana L., in accordance with the International Standard for processed and semi-processed dried oregano ISO/FDIS 7925(1999).

M. Marieschi et al. / Food Control 22 (2011) 542e548 547

panel B) subjected to amplification with the primer pairs forScOm349 and ScSm169 respectively. The selected SCAR markersallowed an easy detection down to 0.5% of each contaminant, asevidenced by the amplification of an individual specific bandabsent in the lanes of Origanum (Fig. 3, lanes indicated as O. h.) andpresent both in the reactions with the individual contaminantspecies (Fig. 3, panel A, lane O. m.; panel B lane S. m.) and with itsmixtures with Origanum (Fig. 3, panel A: lanes 0.5, 1, 2, 5%; panel B:lanes 0.5, 1, 2, 5%). As shown in Fig. 3, when PCR is not conducted tothe end-point, by reducing the number of reaction cycles (30 cyclesfor ScOm349, 33 cycles for ScSm169), the intensity of the amplifica-tion bands is related to the initial amount of DNA template, thusallowing a relative estimate of the degree of adulteration.

Fig. 4. SCAR markers validation on Mediterranean oregano commercial samples ofknown composition (Table 2). A-End-point PCR (40 cycles of amplification) performedwith ScOspp385 primer pair specific for Origanum genus in order to certify the integrityand PCR availability of DNA; B- End-point PCR (40 cycles of amplification) performedwith ScOm349 primer pair specific for Origanum majorana; C- End-point PCR (40 cyclesof amplification) performed with ScSm169 primer pair specific for Satureja montana. �(negative control), amplification with no template DNA; O.h., DNA from O. vulgaresubsp. hirtum; O.m., DNA from O. majorana; S.m., DNA from S. montana; 1e7, DNA fromMediterranean oregano commercial samples of known composition; M, 100 bp DNAladder. The percentage (w/w) of the contaminant species target of the amplification ineach commercial sample, as evaluated by pharmacognostic examination, is listed at thebottom of the panels B and C.

3.3. Analysis of commercial samples

Given its sensitivity in artificial samples, the method describedabove was therefore applied to commercial samples in whichdifferent percentages (w/w) of plant contaminants were evidencedthrough pharmacognostic examination (Table 2). The protocol wasapplied to seven commercial Mediterranean oregano samples forwhich pharmacognostic analysis evidenced different contamina-tion levels of O. majorana (ranging between 0% and 95%) andS. montana (ranging between 0% and 9.7%). These samples wereobtained from wholesale traders during a survey carried outbetween 2001 and 2009 and belong to different harvest years(2009, sample 5; 2008, samples 1 and 2; 2005, sample 4; 2003,samples 6 and 7; 2002, sample 3). The integrity and PCR availabilityof DNA were first checked with the primer pair ScOspp382e385which amplifies a 385 bp fragment in all the commercial samples(Fig. 4, panel A: lanes 1e7) and in the Origanum taxon used aspositive control (Fig. 4, panel A: lane O.h.). This SCAR markerrepresents a very useful tool in the analysis of commercial oreganosamples, allowing the evaluation of the accuracy of the wholeprocedure of DNA extraction and downstream assays and acting asa positive control by preventing the misinterpretation of falsenegatives due to bad quality DNA. The results presented in Fig. 4show that DNA is stable for SCAR markers analysis even incommercial sample 8-year aged. In Fig. 4, panel B and panel C, arereported the results for the end-point amplification of the SCARmarkers ScOm349 and ScSm169 in the same commercial samples asin panel A (lanes 1e7). The percentages of contamination forO. majorana and S. montana, evaluated by pharmacognosticexamination in each sample, are reported at the bottom of eachpictures. The specific primer pairs for O. majorana e S. montanaallowed the detection of the contaminant species in all the samplesin which pharmacognostic assay individuated the contaminantplant species. Even after an end-point PCR, no amplification wasobtained in the samples in which pharmacognostic assay (Table 2)indicated the absence of contamination by O. majorana (Fig. 4,

M. Marieschi et al. / Food Control 22 (2011) 542e548548

panel B: lane 4) or S. montana (Fig. 4, panel C: lanes 5 and 7) thusevidencing the reduced risk of obtaining false positives.

These results further confirmed the sensitivity and the speci-ficity of the selected SCAR markers which can individuate thecontaminant species up to concentrations lower than that allowedby the Ph. Eur., ISO, American Spice Trade Association and EuropeanSpice Association standards for oregano (ASTA, 2006; ESA, 2007;ISO/FDIS 7925, 1999; Ph. Eur, 2005), without inducing misinter-pretation of the results even in complex plant mixtures.

4. Conclusions

From the economic standpoint, product authentication and theavailability of adequate tools for the detection of adulteration andsophistication are key points to avoid unfair competition betweentraders and assure the quality of the spices available for consumers.Applicable methods are not adequate for food control institutionsto protect consumers and honest traders of high-grade products,without inducing excessive constraints in terms of cost and time.An ideal strategy should provide and combine different techniquesin order to cross morphological and genomic datawith physical andchemical information. This would allow to take advantage of theirdifferent approach and at the same time make up for specificshortcomings. Furthermore, such multiple approaches could bothstrengthen the effectiveness of the quest for adulterants and theeventual forensic value of the results.

The described method allows the detection of trace levels ofsavory and marjoram in commercial oregano samples, thus con-firming the applicability of SCARePCR in quality control of driedspices and leading to suggest that a larger recourse to tailoredmolecular methodsmay represent an extremely useful tool in orderto speed up the analysis of a large number of samples. The methodproposed here is essentially qualitative. A relative quantification ofthe contaminant levels can be obtained by reducing the number ofPCR cycles and comparing the amplification intensity of the stan-dard mixture. Absolute quantification however could hardly beobtained since loss in DNA integrity can affect in different extentsdried samples subjected to different post-harvest management.The elevated specificity of SCAR markers helps the transferabilityof the described protocol between different laboratories. Moreover,the small size of the amplification products obtainable with eachprimer pair make these SCAR markers applicable to dried and agedcommercial samples in which DNA degradation likely occur. Theseadvantages warrant a strong improvement to the primary routinescreening of large batches and could provide a helpful support topharmacognostic and phytochemical analyses.

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