Journal of Cereal Science 56 (2012) 733e740

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Journal of Cereal Science

journal homepage: www.elsevier.com/locate/jcs

A reliable assay for the detection of soft wheat adulteration in Italian pasta is
based on the use of new DNA molecular markers capable of discriminating
between Triticum aestivum and Triticum durum
Anna Paola Casazza a, *, Caterina Morcia b, Elena Ponzoni a, Floriana Gavazzi a, Stefano Benedettelli c,
Diego Breviario a
a
b
c

Istituto di Biologia e Biotecnologia Agraria e IBBA, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milan, Italy
Genomics Research Centre, CRA, Via S. Protaso 302, 29017 Fiorenzuola dArda (PC), Italy
Universit degli Studi di Firenze, Dipartimento di Scienze delle Produzioni Vegetali, del Suolo e dellAmbiente Agroforestale, Piazzale delle Cascine 18, 50144 Florence, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 13 July 2012
Received in revised form
24 August 2012
Accepted 27 August 2012

Italian pasta must be prepared using exclusively durum wheat. According to current Italian rules, only
a maximum of 3% Triticum aestivum is allowed to account for cross-contamination that may occur during
the agricultural process. Efcient methods for the detection of accidental or intentional contamination of
common wheat to durum wheat products are therefore required. This article describes a novel approach
for the detection and quantication of soft wheat adulteration in whole grain durum ours and dried
pasta. The assay relies on the presence of intron-specic DNA length polymorphisms in the plant
b-tubulin gene family, which can be highlighted through the PCR-based TBP (Tubulin-Based Polymorphism) method. In wheat, the TBP method produces species-specic amplication products, which
can be either directly used as new DNA molecular markers capable of discriminating between T. aestivum
and Triticum durum or analyzed at the sequence level for the design of species-specic probes. The latter
approach allowed the development of new sequence-specic targets that can be exploited in RT-PCR
assays for a rapid and accurate quantication of soft wheat adulteration in durum wheat pasta.
2012 Elsevier Ltd. All rights reserved.

Keywords:
b-Tubulin introns
DNA molecular markers
Soft and durum wheat
Pasta adulteration

1. Introduction
Pasta is the Italian national dish, not only because it is the staple
daily food for the majority of the population (in 2011 the annual per
capita consumption of dry pasta amounts to 26 kg) but also because
it is part of Italys identity and self-image. Italy is the world leader in
pasta production covering a market share of 26% with 3.25 million
tons produced in 2010, half of which sold in Italy and the remaining
half exported either to other European countries (70%) or outside
Europe, mainly to the USA and Japan (ISTAT, 2012). Italian pasta is
normally made from 100% durum wheat (Triticum durum Desf.,
tetraploid AABB) semolina and is considered a product of superior
quality with respect to those containing common wheat (Triticum
aestivum L., hexaploid AABBDD) or just made from soft wheat. The

Abbreviations: EPIC-PCR, exon-primed intron-crossing PCR; gDNA, genomic

DNA; HPLC, high-performance liquid chromatography; ILP, intron length
polymorphism; LCeMS, liquid chromatographyemass spectroscopy; PCR, polymerase chain reaction; RT-PCR, real-time PCR; TBP, tubulin-based polymorphism.
* Corresponding author. Tel.: 39 (0) 2 2369 9406; fax: 39 (0) 2 2369 9411.
E-mail address: casazza@ibba.cnr.it (A.P. Casazza).
0733-5210/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcs.2012.08.015

production and sale of milled products and pasta is strictly regulated

by the current Italian law (Presidente della Repubblica, 2001): only
a maximum of 3% soft wheat can be tolerated in dry pasta to account
for cross contamination that may occur during growing, harvesting
and handling practices. This restriction is limited to products
manufactured for sale in Italy, but surprisingly not for those
destined to export, provided that they are correctly labeled. The
limit of 3% leads to quality control problems for manufacturers, who
strongly need and request reliable methods able to distinguish
between durum and non-durum wheat. Moreover there is
a growing demand among consumers for accurate information
concerning the origin, authenticity and quality of products.
Available analytical methods for the assessment of soft wheat
adulteration in pasta can be roughly divided into two main categories: a) electrophoretic, chromatographic or immunological techniques targeted to the detection of particular protein fractions,
mainly albumins and gliadins (Barnwell et al., 1994; Bonetti et al.,
2004; Cantagalli et al., 1969; Garcia-Faure et al., 1969; McCarthy
et al., 1990; Resmini, 1968; Stevenson et al., 1994) or friabilin
(Durotest kit, R-Biopharm AG, Germany); b) DNA-based techniques
that utilize the polymerase chain reaction (PCR) to amplify D-genome

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A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

specic regions, like the Dgass44 highly repeated sequence (Bryan

et al., 1998; Kelly and Bhave, 2007), the puroindoline-b gene (Alary
et al., 2002; Arlorio et al., 2003; Kelly and Bhave, 2007), gliadin and
glutenin genes (Terzi et al., 2003) or microsatellite regions (Sonnante
et al., 2009). Additional techniques involve the separation and
quantication of sterol palmitates by HPLC (Sarwar and McDonald,
1993), analysis of alkylresorcinol composition by HPLC and LCeMS/
MS (Kndler et al., 2010) and near-infrared spectroscopy (Cocchi
et al., 2006).
The principal drawbacks of most protein-based methods are the
inuence of environmental growth conditions on marker proteins
expression prole (Blumenthal et al., 1993) and the effect of
denaturation, resulting from the widespread utilization of hightemperature drying processes (>70  C) during pasta production,
which makes accurate quantication of the adulteration level
problematical (Aktan and Khan, 1992). Pasta dried at high or ultra
high temperatures is superior (less sticky and more compact) than
pasta dried at low temperatures, due to the behavior of gluten
proteins that form large aggregates limiting the water absorption of
starch and positively affecting pasta cooking quality (Lamacchia
et al., 2007; Wagner et al., 2011). Compared to proteins, DNAbased methods have several advantages: a) DNA exhibits greater
thermal resistance than proteins so that temperature induced
degradation still leaves fragments of sufcient length and integrity
for PCR analysis, b) very small amounts of DNA are required for the
accurate detection of even low levels of adulteration, c) PCR reactions are generally simple to set up and allow the handling of
numerous samples at a time.
This article is aimed at validating the TBP (Tubulin-Based Polymorphism) method as a reliable and alternative source for new DNA
specic markers capable of discriminating between T. aestivum and
T. durum. Originally developed for assaying genetic diversity in
plants, TBP is an Exon-Primed Intron-Crossing (EPIC) PCR tool that
relies on the presence of intron-specic DNA polymorphisms (ILP,
Intron Length Polymorphism) in the members of the plant b-tubulin
gene family. First validated in rice (pending patent PCT/IT99/00415),
the TBP e 1st intron strategy was successfully applied for assessing
intra- and interspecic relationships and differences in oilseed rape
(Brassica napus), coffea and lotus subspecies and varieties (Bardini
et al., 2004). Thereafter, the TBP e 2nd intron approach was used
as an additional source of DNA polymorphism (cTBP, combinatorial
TBP) to assess species/varieties relationships in Eleusine and Arachis
(Breviario et al., 2007) and in common bean (Breviario et al., 2008).
Recently, a new version of the method, called hTBP (horse TBP),
that simultaneously analyzes the polymorphism of the 1st and 2nd
introns through the amplication of the genomic region that
straddle them, has been used for the rapid isolation and characterization of the multiple members of the b-tubulin gene family in
Camelina sativa (L.) Crantz (Galasso et al., 2011). Moreover, the TBP
method and its new developments have been validated as reliable
analytical tools for determining the composition of blends of plant
origin (Casazza et al., 2011).
The results presented here demonstrate that the TBP method
can be either directly used for a simple and fast discrimination
between T. aestivum and T. durum, or applied for the design of
species-specic probes that can be exploited in quantitative assays
for the analysis and quality certication of Italian pasta.
2. Materials and methods

Eureka [11], Frassineto [12], Gentil Bianco [13], Gentil Rosso aristato
[14], Gentil Rosso mutico [15], Inallettabile [16], Marzuolo dAqui 3
[17], Marzuolo Val Pusteria [18], Mieti [19], Palesio [20], Postarello
[21], San Francisco [22], Sieve [23], Terricchio [24], Verna [25]) and
seven durum wheat varieties (cv. Ancomarzio [26], Arcangelo [27],
Claudio [28], Iride [29], Triticum turanicum [30], Levante [31], Urria
[32]) were obtained from the University of Florence. Soft wheat cv.
Aubusson [A] and durum wheat cv. Claudio [C] kernels were
provided by CRA, Genomic Research Centre, Fiorenzuola dArda.
The selected wheat panel comprises modern varieties commonly
cultivated in Italy, as well as old ones grown in the early 20th
century, to have a representative sample of the wheat genome.
Besides, the old varieties, in the last period, are interesting for their
functional quality and for their yield capacity in organic growing
(Dinelli et al., 2009).
Fourteen commercial samples of Italian pasta [1/14] manufactured by eleven different brands (indicated with a lower-case
letter following the sample number) were purchased from
various Italian food shops: 100% durum wheat pasta dried at high
(Vermicellini [2b], Gnocchi [3b]) or low temperature (Mezze
Maniche Rigate [1a], Toffarelle [4c], Mezzi Rigatoni [5d], Farfalle
[6e], Gnocchi Sardi [7f], Linguine [8g]); 100% durum wheat egg
pasta (Maltagliati [9b], Lasagne [10d] and [11h], Tagliatelle [12i], all
containing 19-19, 36% fresh hen-eggs Category A); 100% soft wheat
pasta for infants (Stelline [13l]) and 100% emmer wheat (Triticum
dicoccum AABB) pasta (Ditalini [14m]).
Wheat kernels and pasta specimens were reduced respectively
to whole grain our or ne powder using the vibration mill TissueLyser (Qiagen) with stainless steel grinding jars (Retsch). For the
preparation of dened our blends, the durum wheat cultivar
Ancomarzio or Claudio and the common wheat cultivar Bilancia or
Aubusson were used as indicated. Whole grain durum our was
blended with different percentages of common wheat our and
genomic DNA (gDNA) was extracted using the GenElute Plant
Genomic DNA Miniprep kit (SigmaeAldrich). gDNA from pasta
specimens was extracted using the Speedtools Food DNA Extraction
kit (Biotools). DNA quality was assessed by agarose gel electrophoresis and EtBr staining, while DNA quantication was performed spectroscopically using the Qubit (1.0) Fluorometer
(Invitrogen). For each sample, gDNA extraction was repeated on the
same material at least three times.
2.2. DNA fragments isolation, cloning and sequencing
TBP amplication fragments, electrophoretically separated on
polyacrylamide gels and visualized by silver staining as described
previously (Breviario et al., 2007), were excised from the gel matrix
using a sharp blade and immediately rehydrated with deionized
water in order to collect the gel slide. DNA was directly extracted
from the gel matrix with the QIAEII Gel Extraction Kit (Qiagen)
and cloned using the TOPO TA-Cloning Kit (Invitrogen). Positive
transformants were selected by colony PCR (see Section 2.3) and
plasmid DNA isolated using the Nucleospin Plasmid Kit
(Macherey-Nagel).
Sequencing of cloned fragments was performed by the ABI
PRISM 310 Genetic Analyzer, using the BigDye Terminator v3.1
Cycle Sequencing Kit (Applied Biosystems). Nucleotide sequences
have been bioinformatically analyzed by performing BLAST (Basic
Local Alignment Search Tool, Altschul et al., 1997) searches on NCBI
(http://www.ncbi.nlm.nih.gov/).

2.1. Materials and DNA extraction

2.3. Primer design and amplication conditions
Kernels of twenty-ve soft wheat varieties (cv. Abbondanza [1],
Andriolo [2], Autonomia A [3], Autonomia B [4], Benco [5], Bianco
Nostrale [6], Bilancia [7], Bolero [8], Canove [9], Carosello [10],

Species-specic primers were designed on the basis of intron

sequence alignments, obtained using the CLUSTAL W software

A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

(Thompson et al., 1994), on unique nucleotide stretches (as shown

in Fig. 4 and Fig. A.4, Supplementary data). Oligonucleotides specic
for T. durum: Tdu_106F 50 -AATGTACGCCATAATAGTGTTGAG-30 ,
Tdu_106R 50 -ATGGGTGGATTGCAT-GGATTGC-30 . Oligonucleotides
specic for T. aestivum: 1st pair, Tae_109F 50 -GTCAGGAAGGATGATGCCGTG-30 and Tae_109R 50 -TGATTTCAAGTGGACACTGGACA30 ; 2nd pair, 4FDf 50 -GTTGTCAGGAAGGATGATGC-30 and Tae_109a-r1
50 -CACAAGATATGGAAGCAATGA-GAGT-30 . PCR conditions were as
follows: 20 ml reaction volume, 20 ng gDNA as template, 0.5 mM each
primer, 0.15 mM each dNTP, 2.5 mM Mg2, 0.6 U Taq DNA Polymerase (Eppendorf) and corresponding buffer. PCR program: 3 min
initial denaturation at 94  C followed by 40 amplication cycles
(94  C for 40 s, 68  C for 40 s, 72  C for 40 s) and nal extension step
for 1 min at 72  C. As for gDNA extractions, all PCR reactions were
repeated at least three times.
For TBP analysis, template amount (gDNA) was in the range of
15e30 ng, while primer sequences, PCR conditions, polyacrylamide
gel electrophoresis and subsequent silver staining were as previously described (Breviario et al., 2007).
For colony PCR, bacterial colonies were picked from the plates,
resuspended in a small volume of water (10e20 ml, depending on
the colony dimension), boiled for 5 min and used as template (1 ml)

735

for the PCR reaction (10 ml total volume). Reaction mixtures contained: 0.5 mM each primer (M13-Reverse and T7-Promoter,
universal primers), 0.15 mM each dNTP, 2.5 mM Mg2, 0.6 U Taq
DNA Polymerase (Eppendorf) and corresponding buffer. PCR
program: 2 min initial denaturation at 94  C followed by 25
amplication cycles (94  C for 1 min, 55  C for 1 min, 72  C for
1 min) and nal extension step for 7 min at 72  C.
PCR products were separated by agarose gel electrophoresis and
visualized by EtBr staining. DNA molecular weight markers
(SHARPMASS50, pUC Mix and GeneRuler 1 kb Plus DNA
Ladders) were purchased from EuroClone and Fermentas,
respectively.
The method that leads from TBP to the isolation of plant specic
diagnostic probes is under pending patent protection (No.: PCT/
IB2011/000146 deposited on the 31st of January 2011).
2.4. Real-time PCR and data analysis
Reactions for the RT-PCR using SYBR Green detection consisted of
12.5 ml of SYBR Green PCR Master Mix (Applied Biosystems), 900 nM
forward and reverse primers, 100 ng of DNA template and water to
25 ml. All PCR samples and controls were prepared in duplicate. The

Fig. 1. TBP amplication proles (1st intron, hTBP and 2nd intron approach) of ve selected wheat varieties. T. aestivum cultivars: [7] Bilancia; [14] Gentil Rosso aristato and [17]
Marzuolo dAqui 3. T. durum cultivars: [26] Ancomarzio and [28] Claudio. Diagnostic bands and groups of bands (numbered in ascending order from low to high molecular weight
and labeled with A or B for 1st and 2nd intron amplication fragments, respectively) are highlighted by boxes. The insert on the left side shows a magnication of the boxed
portion of the 1st intron prole. m, molecular weight ladder (fragments lengths are indicated in base pair).

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A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

Fig. 2. TBP 1st and 2nd intron analysis of gDNA extracted from T. durum (cv. Ancomarzio [26]), T. aestivum (cv. Bilancia [7]) and blends of durum wheat whole grain our containing
different amounts of soft wheat (1, 3, 6, 12, 24 and 50% w/w). Diagnostic bands A1 and B4 are highlighted by arrows. Asterisks indicate samples where bands A1 and B4 are still
clearly detectable, i.e. the detection limit of the approach (see Section 3.2). m, molecular weight ladder (fragments lengths are indicated in base pair).

PCR mixture was held at 50  C for 5 min and denatured at 95  C for

10 min. After this initial step, 40 amplication cycles were carried
out at 95  C for 15 s followed by 68  C for 1 min. The PCR reactions
were subjected to a heat dissociation protocol and analyzed by the
AB7300 software for melting curve analysis. Following the nal PCR
cycle, the reactions were heat-denatured at 0.03  C/s over a 35  C
temperature gradient from 60 to 95  C. The RT-PCR data were
plotted as the DRn uorescence signal versus cycle number.
A standard curve was obtained by using gDNA extracted from
durum wheat (cv. Claudio [C]) whole grain our mixtures containing increasing amounts (2, 3, 5, 10, 50, 100% w/w) of soft wheat
(cv. Aubusson [A]) and developed by plotting the Ct (cycle
threshold) versus the Log10 of the T. aestivum amount (ng). The
quantication of soft wheat in pasta specimens was derived by
interpolating the obtained Ct values in this regression line.

3. Results and discussion

3.1. TBP analysis on T. aestivum and T. durum cultivars
The introns of plant b-tubulins constitute a remarkable source of
DNA polymorphisms that can be used as molecular markers for the
genetic barcoding of plant species (Breviario et al., 2007). In order
to highlight species-specic (and perhaps varieties-specic)
DNA polymorphisms in wheat, the TBP method and its further
developments have been applied on 25 different varieties of
T. aestivum and 7 varieties of T. durum (1st intron approach and
hTBP results are shown in Figs. A.1 and A.2, Supplementary data).
The TBP proles of the hexaploid soft wheat always contain
a higher number of amplication products with respect to those
of the tetraploid durum wheat, but on the whole the degree of

A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

737

conrmed by analyzing all the available varieties of soft and durum

wheat (data not shown). In order to verify whether bands A1 and
B4 could be used as molecular markers for detecting the presence
of soft wheat in durum wheat our and pasta, whole grain our
blends of durum wheat with increasing amounts of soft wheat
have been prepared and analyzed (Fig. 2) together with a selection
of Italian pasta specimens (Fig. 3) comprising 100% durum wheat
pasta dried at low or high temperature, egg pasta and 100% soft
wheat pasta (for TBP 1st and 2nd intron proles of all analyzed
pasta samples see Fig. A.3, Supplementary data). Fig. 3 clearly
shows that pasta dried at temperatures higher than 60  C (samples
[2b] and [3b]) cannot be analyzed with the TBP approach. These
drying conditions heavily affect the integrity of total DNA allowing
the amplication of only short size products. On the contrary, pasta
dried at lower temperatures (samples [1a], [10d] and [13l]), from
which genomic DNA of good quality is routinely recovered (data
not shown), shows a TBP 1st and 2nd intron prole that fully
resembles that of T. durum (samples [1a] and [10d]) or T. aestivum
(sample [13l]), respectively. On the whole, bands A1 and B4
represent new molecular markers for a simple and rapid discrimination of ours made up of T. aestivum or T. durum. However, they
cannot be used to uncover soft wheat adulteration in pasta, since
the TBP analysis performed on quantitatively dened blends
(Fig. 2) indicates in 6% (w/w), the lowest detectable amount of soft
wheat in durum wheat our when scoring the A1 marker, while
such a limit increases to 12% (w/w) for B4 (as highlighted by an
asterisk in Fig. 2). Both values are well above the current limit of 3%
set by the Italian law.
3.3. From length- to sequence-polymorphism analysis

Fig. 3. TBP 1st and 2nd intron analysis of gDNA extracted from selected 100% durum
wheat pasta dried at low [1a] or high temperature [2b and 3b], egg pasta [10d] and
100% soft wheat pasta [13l] (more details on pasta specimens can be found in Section
2.1). For reference, the amplication patterns of T. aestivum (cv. Bilancia [7]) and
T. durum (cv. Ancomarzio [26]) are also shown. T. aestivum diagnostic bands A1 and B4
are highlighted by arrows.

polymorphism is rather low. Nevertheless a few differences can be

detected between the two species and in some cases also within
species. Diagnostic bands (or groups of bands) are highlighted in
Fig. 1, which shows the TBP amplication proles of ve selected
varieties presenting the most signicant intra- and interspecic
differences.
3.2. Bands A1 and B4 as molecular markers for T. aestivum
Further analysis was performed by investigating the usefulness
of the most simple 1st intron and 2nd intron amplication proles
comprising bands A1e2e3 and B2e3e4, respectively (see Fig. 1). In
T. aestivum varieties the band A1 is always present and is often
associated with a second band of higher molecular weight, which
can be A2 or A3. In T. durum, the band A1 is always absent, while
the band usually detected is A2 or A3 (see Fig. 1 and Fig. A.1,
Supplementary data); this makes A1 specic for T. aestivum.
With regard to the group of bands B2-3-4, all T. durum varieties
show two bands (B2 and B3), while in T. aestivum, an additional
slower migrating band (B4) is always present. This nding was

Since the TBP analysis, which is based on b-tubulin intron length

polymorphisms, is not as sensitive as required for the reliable
detection of very low amounts of soft wheat in durum wheat our
or pasta, the possibility of exploiting intron sequence polymorphisms has been considered. Starting from the TBP proles
obtained from different varieties of T. aestivum and T. durum,
a systematic work of isolation, cloning and sequencing of putatively
diagnostic bands (mainly those surrounded by the boxes in Fig. 1)
was undertaken.
Nucleotide sequence comparison between isolated b-tubulin
introns shows that:
a) All TBP amplied bands are b-tubulin introns (exon portions of
the amplied regions have been bioinformatically analyzed by
comparison with public nucleotide databases). This further
conrms the effectiveness of the TBP method and its additional
developments.
b) From the exon portion of the amplied fragments, it is possible
to clearly identify isoform-specic introns (for example the
bands B2, B3 and B4 represent the second introns of b-tubulin
isoform 5, GenBank ID: U76896).
c) TBP amplied bands that migrate at different positions in
polyacrylamide gels do not always correspond to intron
sequences that really differ in length. For example the intron of
B4 (see Fig. 1) is actually 488 bp long but migrates much slower
than B2, whose intron is 489 bp (the intron of B3 is 509 bp
long). B4 is therefore a typical case of an anomalously
migrating DNA fragment. Since the degree of sequence identity
between B2 and B4 is very high, the DNA features that determine this phenomenon are currently under investigation
(unpublished results).
d) Introns of the same b-tubulin isoform share highly homologous
sequences that may differ only by means of very short insertions or deletions.

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A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

Fig. 4. Sequence alignment of the b-tubulin rst introns corresponding to the isolated bands A7, A8 and A9. Positions of the T. aestivum specic primers are underlined (Tae_109F
and Tae_109R) or highlighted in gray (4FDf and Tae_109a-r1), respectively.

On the basis of these evidences, the bands B1, B2, B3, B4 and A1
(see Fig. 1) have been discarded from the pool of putatively useful
fragments and further investigation was restricted to the 1st intron
amplied fragments highlighted in the insert of Fig. 1. Bands A4, A5
and A6 all correspond to the rst introns of b-tubulin isoform 2
(GenBank ID: U76745). Band A4 is present in both T. aestivum and
T. durum, while A5 and A6 are specic for T. aestivum and T. durum,
respectively. Bands A7, A8 and A9 correspond to the rst introns of
b-tubulin isoform 5, with both A7 and A8 common to T. aestivum
and T. durum, while A9 was specic for T. aestivum. In the T. aestivum
varieties Bilancia [7] and Mieti [19], the band A8 has a short
insertion of 15 nucleotides and therefore migrates very close to A9
(see Fig. 1 and Fig. A.1, Supplementary data). After an extensive
work of sequence analysis and comparison, a few nucleotide
stretches which are highly specic for T. durum and for T. aestivum
could be identied. Fig. 4 shows the alignment obtained for the
intron sequences derived from bands A7, A8 and A9. The positions
where T. aestivum specic primers (see Section 2.3) have been
designed are underlined (Tae_109F and Tae_109R) or highlighted in
gray (4FDf and Tae_109a-r1), respectively. The same procedure has
been followed for bands A4, A5 and A6 (sequence alignment is
shown in Fig. A.4, Supplementary data) in order to design T. durum
specic primers (Tdu_106F and Tdu_106R, see Section 2.3).

3.4. Species-specic probes and pasta analysis

The species-specicity of the designed primers was rst tested
by performing a standard endpoint PCR reaction on all the available
varieties of soft and durum wheat, obtaining a single amplicon of
the expected size: 240 bp for T. durum, 257 bp with the 1st pair and
111 bp with the 2nd pair of primers for T. aestivum (see Fig. A.5,
Supplementary data, for probe validation on two selected varieties). The specicity of the probes was then tested by performing an
RT-PCR (based on the SYBR Green approach) and subsequent
dissociation curve analysis, on gDNA extracted from durum/soft
wheat whole grain our mixtures of known composition. A single
peak, indicating specic amplication, is obtained with the
T. durum primers set and with the 2nd pair of T. aestivum primers. In
contrast, the 1st pair of T. aestivum primers forms dimers,
a tendency already visible in endpoint PCR (see Fig. A.5,
Supplementary data) that increases when the amount of common
wheat in the template decreases (data not shown), rendering these
primers not appropriate for quantitative detection of low amounts
of soft wheat. Further analysis was therefore focused on the 2nd
T. aestivum specic probe (4FDf and Tae_109a-r1, see Section 2.3
for oligonucleotide sequences) in order to verify its usefulness
for the detection of soft wheat adulteration in durum wheat pasta.

A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

739

for accurate quantication of T. aestivum percentages below 3%.

RT-PCR analysis was then performed on gDNA extracted from all
the available Italian pasta specimens (Fig. 5B) and the amount of
soft wheat that is present in the different samples was derived by
interpolating the obtained Ct values in the regression line described
above. As reported in Table 1, all samples are consistent with the 3%
limit of soft wheat xed by the Italian legislation, with the only
exception of specimens [2b], [3b] and [6e]. In addition, specimen
[14m], which is declared as 100% emmer wheat pasta, undoubtedly
contains more than 3% soft wheat. The results obtained clearly
demonstrate that the T. aestivum specic probe (4FDf and
Tae_109a-r1), designed in the present study, can be exploited in
quantitative RT-PCR assays for the rapid and reliable detection of
soft wheat adulterations in durum wheat pasta.
4. Conclusion

Fig. 5. RT-PCR analysis using the T. aestivum 4FDf/Tae_109a-r1 specic probe.

(A) Standard curve obtained on DNA samples (three replicates) extracted from blends
of durum wheat (cv. Claudio [C]) whole grain our containing different amounts (2, 3,
5, 10, 50 and 100% w/w) of soft wheat (cv. Aubusson [A]). Ct, cycle threshold.
(B) Amplication plots (DRn uorescence signal versus cycle number) of pasta samples.
NTC, no template control.

A standard quantication curve was assessed from RT-PCR analysis

on gDNA samples extracted from durum wheat (cv. Claudio) whole
grain our mixtures containing increasing amounts (2, 3, 5, 10, 50,
100% w/w) of soft wheat (cv. Aubusson). The obtained curve
(Fig. 5A), showing a slope of 3.319, an intercept of 30.617 and an
R2 value (Pearson coefcient) of 0.9901, results perfectly suitable

Table 1
Mean Cts obtained from real-time PCR analysis of pasta specimens using the
T. aestivum 4FDf/Tae_109a-r1 specic probe and quantication of soft wheat content
(% w/w).
Samplea

Mean Ct

Soft wheat amount (%)

1a
2b
3b
4c
5d
6e
7f
8g
9b
10d
11h
12i
13l
14m
NTCb

32.07
28.05
28.24
32.49
30.66
28.01
30.60
29.21
32.87
29.89
32.30
30.41
23.73
28.07
0

0.4
5.9
5.2
0.3
1.0
6.1
1.0
2.7
0.2
1.7
0.3
1.2
100
5.9
0

a
b

More details on pasta specimens can be found in Section 2.1.

NTC, no template control.

The TBP method was originally developed for assessing genetic

diversity in plants and the results presented here further conrm
its versatility and stress its usefulness for more applicative
purposes. The length polymorphism of b-tubulin introns (ILP), that
can be highlighted by performing a TBP analysis, gives rise to
amplication proles which are highly specic for each analyzed
species. Also in wheat, among all the TBP amplied fragments,
there is always one band, or a combination of bands, that can be
exploited as molecular marker for the rapid discrimination
between T. aestivum and T. durum. However, the relative low
sensitivity of this approach together with the high-temperature
drying processes often applied during pasta production, makes
the TBP method of polymorphisms detection unsuitable for the
direct identication of soft wheat adulteration in durum wheat
pasta. Nonetheless, the TBP method can also be exploited for the
rapid isolation and subsequent cloning of the amplied, polymorphic DNA sequences. In accordance, different b-tubulin intron
sequences isolated from T. aestivum and T. durum have been cloned,
analyzed and compared, allowing the identication of several
species-specic nucleotide stretches on which specic primers can
be designed and used as species-specic probes. In the present
study, a new T. aestivum specic probe has been developed and
validated in RT-PCR assays for the quantitative detection of soft
wheat eventually present in durum wheat pasta, demonstrating the
feasibility of the proposed procedure. With a detection limit around
2%, the new probe could in principle satisfy the requirements of the
Italian law to certify product quality. Once properly validated
through ring tests among different laboratories and compared in
terms of efciency with other PCR-based methods or analytical
systems, the probe could be used as an alternative tool for the
reliable detection of soft wheat adulterations in durum wheat
Italian pasta. This could be done either by the manufacturers in
order to guarantee and certify the quality of their products or by the
competent anti-fraud authorities in order to protect the consumers
from non-genuine products.
In conclusion, it is important to underline that the advantage of
this assay over those already reported, is certainly not given by its
efcacy nor by the single probe per se, which indeed should be
considered as only one example, and perhaps even not the most
efcient one, of the several possible T. aestivum specic probes that
can be designed following the procedure described here. The real
novelty rather relies in the whole process that allows the rapid
isolation of DNA polymorphic sequences and the easy development
of a wide range of new species-specic probes from theoretically
any given plant species. In the present work, wheat has been
chosen as reference system for agronomic crops of great economic
importance and the issue of soft wheat adulteration in Italian pasta
has been addressed, but since the proposed procedure works

740

A.P. Casazza et al. / Journal of Cereal Science 56 (2012) 733e740

independently from any detailed information on the genome under

investigation, its spectrum of applicability is much wider and the
method can in principle be extended to all cereal- and more general
plant-containing food matrices.
Acknowledgments
This work was entirely supported by the Program Risorse
biologiche e tecnologie innovative per lo sviluppo sostenibile del
sistema agroalimentare, WP2, as part of the activities dened
within the Accordo Quadro Consiglio Nazionale delle Ricerche and
Regione Lombardia.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.jcs.2012.08.015.
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