Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification

Abstract

The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg(-1) range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg(-1). The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.

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Simultaneous detection of DNA from 10 food allergens by ligation-dependent
probe amplification
Alexandra Ehlert a; Anja Demmel a; Christine Hupfer b; Ulrich Busch b; Karl-Heinz Engel a
a Department of General Food Technology, Technische Universität München, D-85350 Freising-
Weihenstephan, Germany b Bavarian Health and Food Safety Authority, Germany
Online Publication Date: 01 April 2009
To cite this Article Ehlert, Alexandra, Demmel, Anja, Hupfer, Christine, Busch, Ulrich and Engel, Karl-Heinz(2009)'Simultaneous
detection of DNA from 10 food allergens by ligation-dependent probe amplification',Food Additives & Contaminants: Part A,26:4,409
— 418
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Food Additives and Contaminants
Vol. 26, No. 4, April 2009, 409–418
Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification
Alexandra Ehlert
a
, Anja Demmel
a
, Christine Hupfer
b
, Ulrich Busch
b
and Karl-Heinz Engel
a
*
a
Department of General Food Technology, Technische Universita
¨tMu
¨nchen, D-85350 Freising-Weihenstephan, Germany;
b
Bavarian Health and Food Safety Authority, Veterina
¨rstrasse 2, D-85764 Oberschleißheim, Germany
(Received 27 August 2008; final version received 1 November 2008)
The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe
amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay.
Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts,
sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with
DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic
ingredients in the low mg kg
1
range. The limit of detection (LOD) for single allergens in different food matrices
was 5 mg kg
1
. The novel analytical strategy represents a useful tool for the surveillance of established legislation
on food allergens within the European Union.
Keywords: food allergen; tree nuts; peanut; sesame; detection; ligation; PCR
Introduction
In industrialized countries, 1–2% of adults and up to
8% of children and adolescents are affected by food
allergies (Sicherer and Sampson 2006). The symptoms
may range from skin irritations to severe anaphylactic
reactions with fatal consequences. Approximately 90%
of the adverse reactions observed have been associated
with eight food groups: cow’s milk, eggs, fish,
crustaceans, peanuts, soybeans, tree nuts and wheat.
In addition to these major food allergens, a broad
spectrum of fruits, vegetables, seeds, spices and meats
have been reported to possess allergenic potential
(Breiteneder and Ebner 2000; Ellman et al. 2002;
Poms et al. 2004).
Taking into account the recommendations of the
Codex Alimentarius Commission (2007), the European
Commission amended the European Food Labelling
Directive 2000/13/EC with a list of ingredients to be
labeled (EC 2000). Currently, Annex IIIa of Directive
2007/68/EC (EC 2007) comprises gluten-containing
cereals, crustaceans, molluscs, fish, peanuts, soybeans,
eggs, milk and dairy products (including lactose), nuts,
celery, mustard, sesame seeds, lupine, sulfite and
products thereof. To protect the health of consumers,
the declaration of these ingredients has been made
mandatory, regardless of their amounts in the final
product.
Allergens are proteins whose routine food analysis
is based on immunological detection using either
specific IgE from human serum or antibodies raised
in animals. Major challenges are the requirement to
check for the presence of food allergens at extremely
low levels and to detect trace amounts of hidden
allergens in composite and processed foods (Poms et al.
2004). PCR-based methods amplifying specific DNA
sequences offer alternative tools for the detection of
allergenic or marker proteins for the species (Goodwin
2004; Poms and Anklam 2004). In most cases, DNA
presents a more stable analyte compared to proteins
and is less affected by denaturation (Poms and Anklam
2004). In addition, species-specific sequences allow the
discrimination of closely related organisms.
Appropriate PCR assays for the detection and
identification of individual food allergens have been
developed for cashew nuts (Brzezinski 2006; Ehlert et al.
2008a), celery (Dovicovicova et al. 2004; Stephan et al.
2004; Hupfer et al. 2006), cereals (wheat, barley, rye)
(Dahinden et al. 2001; Sandberg et al. 2003; Hernandez
et al. 2005), peanuts (Hird et al. 2003; Stephan and
Vieths 2004), pistachios (Barbieri and Frigeri 2006) and
tree nuts (walnut, pecan, hazelnut) (Holzhauser et al.
2000; Herman et al. 2003; Brezna et al. 2005; Germini
et al. 2005; Arlorio et al. 2007; Brezna and Kuchta
2007). Conventional and real-time PCR methods for the
detection of soybean, sesame, mustard, peanut, hazel-
nut and almond have recently been compared (Pancaldi
et al. 2005). At present, only a few duplex-PCR systems
are known allowing the simultaneous detection of
peanut and hazelnut or wheat and barley (Rossi et al.
2005; Ronning et al. 2006).
*Corresponding author. Email: k.h.engel@wzw.tum.de
ISSN 1944–0049 print/ISSN 1944–0057 online
ß2009 Taylor & Francis
DOI: 10.1080/02652030802593529
http://www.informaworld.com
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The aim of this study was to develop and validate
a multi-target method for the simultaneous detection
of allergens in different food matrices. The application
of a ligation-dependent probe amplification (LPA)
technique for the simultaneous detection of DNA
from peanut, cashew nut, pecan nut, pistachio nut,
hazelnut, sesame seeds, macadamia nut, almond,
walnut and brazil nut in a single reaction is described.
Ligation-dependent PCR was originally introduced to
allow the detection of nucleic acid sequences (Carrino
1996; Hsuih et al. 1996; Belgrader et al. 1997). First
applications in the field of medical diagnostics allowed
the detection and the relative quantification of up to 40–
50 target sequences in a single assay (Gille et al. 2002;
Schouten et al. 2002; Eldering et al. 2003; Hogervorst
et al. 2003; Taylor et al. 2003). The suitability of this
method for the event-specific detection and relative
quantification of DNA from two genetically modified
organisms (GMO) has been demonstrated using com-
mercially available maize and soya standards (Moreano
et al. 2006). The technique does not amplify the target
sequences, but is rather based on the amplification
of products resulting from the ligation of bipartite
hybridization probes. The use of this analytical strategy
results in a flexible system that can be complemented
with further hybridization probes to broaden the range
of target sequences to be detected. This approach has
been realized by the development of a modular system
allowing the simultaneous detection of several GMO
targets corresponding to different levels of specificity in
a one-tube assay (Ehlert et al. 2008b).
Materials and methods
Materials and samples
Nut materials, sesame seeds, ingredients of self-
prepared walnut cookies and commercial food samples
were purchased from local grocery stores. DNA plant
and animal materials used to test the specificity of the
method and spiked samples of chocolate, cookies and
pesto used to determine sensitivity were obtained from
the Bavarian Health and Food Safety Authority
(Oberschleißheim, Germany). Chocolate samples had
been spiked with peanuts (100, 10, 5, 1 and 0.5 mg kg
1
)
and hazelnuts (20, 10 and 5 mg kg
1
). Cookies spiked
with peanuts contained 100, 10, 5, 1 and 0.5 mg kg
1
of
peanuts and pesto spiked with cashew nuts contained
100, 20, 10, 5, 2 and 1 mg kg
1
of cashew nuts. In all
cases samples of the unspiked material were included
in the analysis.
Preparation of walnut cookies spiked with
different nuts
Nuts ground with a Thermomixer (Vorwerk,
Wuppertal, Germany) as well as the other cookie
ingredients were analyzed by LPA to ensure the purity
of the starting materials. Two doughs containing 25%
wheat flour, 25% sugar, 25% butter and 25% ground
nuts (either walnuts only or walnuts spiked with 10% of
peanut, hazelnut, pecan and macadamia) were prepared
using a food processor (Braun, Germany). The refer-
ence cookies containing only walnuts and the cookies
with all five nuts were baked separately at 180C for
10 min and ground afterwards. The concentrations of
peanut, hazelnut, pecan and macadamia were adjusted
in the spiked cookies to 10,000, 1000, 100, 10 and
1mgkg
1
, respectively, by mixing the corresponding
amounts of ground spiked cookies and ground walnut
reference cookies in a food processor. The mixtures
became fluid due to the high fat contents; however, the
procedure resulted in visually homogeneous
dispersions.
DNA extraction
Each sample (2 g) was mixed with 10 ml
CTAB–extraction buffer [2% (w/v) cetyltrimethylam-
moniumbromide, 1.4 M NaCl, 20 mM EDTA, 100 mM
Tris–OH/HCl] and 30 ml proteinase K (20 mg ml
1
)in
a 50 ml Falcon tube and incubated at 65C overnight.
After 5 min centrifugation at 5000 g, 1000 ml super-
natant was transferred to a 1.5-ml tube and centrifuged
again at 14,000 gfor 5 min. Then, 500 ml chloroform/
isoamylalcohol (Ready Red
TM
; MP Biomedicals,
Heidelberg, Germany) was mixed with 700 ml super-
natant and centrifuged at 16,000 gfor 15 min. A 500 ml
aliquot of supernatant was added to 500 ml isopropanol
(stored at 20C) and incubated at room temperature
(RT) for 30 min. After 15 min centrifugation at
16,000 g, the supernatant was removed, the pellet
washed with 500 ml ethanol (70%; stored at 20C)
and centrifuged for 5 min at 16,000 g. After removal of
ethanol, the pellet was dried for 1 h at 50C and
afterwards diluted in 100 ml TE–buffer (1).
Additionally, the DNA extracts were purified using
spin filter columns (Qiagen, Hilden, Germany).
DNA concentrations were determined fluorome-
trically using PicoGreenÕdsDNA quantification
reagent (Invitrogen, Karlsruhe, Germany) on a Tecan
GENios
TM
plus reader (Ma
¨nnedorf, Switzerland).
Probes and primers
Probes used for the detection of peanut, cashew nut,
pecan nut, pistachio, hazelnut, sesame, macadamia nut,
almond, walnut and brazil nut were designed
using Beacon Designer 4.0 software (Premier Biosoft
Int., Palo Alto, CA, USA) and FastPCR software
(University of Helsinki, Finland). The synthesis of
the probes was done by Biolegio B.V. (Nijmegen,
The Netherlands). Primers are included in the
410 A. Ehlert et al.
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MLPA reagents kit (MRC-Holland, Amsterdam,
The Netherlands). Sequences of LPA probes and
primers, as well as GenBank accession numbers of the
selected targets, are listed in Table 1.
Ligation-dependent probe amplification
The LPA reaction was essentially carried out as
described by Schouten et al. (2002). Hybridization
was performed overnight in 0.5 ml reaction vessels
using a thermocycler (Mastercycler Gradient;
Eppendorf, Hamburg, Germany) and 100 ng DNA
sample. After 5 min of DNA denaturation at 98C,
1.5 ml of MLPA buffer and 1.5 ml of a mixture of the
synthesized probes containing 1 fmol of each LPA
probe for the detection of peanut, cashew, pecan nut,
pistachio, hazelnut, sesame, macadamia nut, almond,
walnut and brazil nut, respectively, were added and
held at 60C for 16 h. Ligation reaction was performed
at 54C for 15 min after adding 3 ml Ligase-65 buffer A,
3ml Ligase-65 buffer B, 25 mlH
2
O and 1 ml Ligase-65
(MRC-Holland). After ligation, reaction mixes were
heated for 5 min at 98C to inactivate the enzyme.
For amplification of the ligation products, 10 ml
polymerase mixed with primers, dNTPs, buffer and
polymerase enzyme of the MLPA kit (MRC-Holland)
were added to 40 ml ligation reaction mixture at 60C.
Thirty-five amplification cycles at 95C for 30 s, 60C
for 30 s and 72C for 60 s were followed by a final step
of 20 min at 72C and cooled down to 4C.
Fragment length analysis
Fragment length analysis was performed on an ABI
PRISMÕ310 Genetic Analyzer using capillaries
(47 cm) and polymer (POP-6
TM
performance optimized
polymer); reagents were obtained from Applied
Biosystems (Forster City, CA, USA). One ml of the
PCR product (pure or diluted) was mixed with 0.3 ml
size standard (GeneScanÕ-500 [TAMRA]
TM
) and
14.7 ml Hi-Di
TM
formamide. Prior to analysis, DNA
was denatured at 94C for 3 min and cooled on ice.
Electrokinetic injections were performed at 15 kV for
5 s. Electrophoretic separations were run at 60C
and 15 kV.
Sequencing
Ligation products were generated and amplified
separately prior to sequencing. Primers used for
sequencing were identical to those listed in Table 1.
FAM-labeling did not interfere with cycle sequencing.
Amplified products were cleaned up using a PCR
purification kit (QIAquickfflQiagen GmbH, Hilden,
Germany) and used as template for the sequencing
PCR. This reaction was performed using a BigDye
Terminator v1.1 cycle sequencing kit (Applied
Biosystems). One reaction mix (20 ml) contained 2 ml
5buffer, 4 ml RR-mix, 2 ml primer (10 pmol), 8 ml
H
2
O and 4 ml template. Reaction conditions were as
follows: initial denaturation (1 min at 96C), 30 cycle
denaturation steps (10 s at 96C) and primer annealing
(5 s at 56C), and a final step (4 min at 60C).
Purification of the PCR products was carried out
following amplification. PCR products (10 ml) were
mixed with 16 mlH
2
O, 4 ml Na-acetate (3 M) and 50 ml
EtOH (100%) in a 1.5 ml reaction vessel and centri-
fuged at 15,000 rpm for 15 min. EtOH was removed
carefully without damaging the precipitated DNA
pellet. The pellet was vortexed with 50 ml EtOH and
centrifuged at 15,000 rpm for 5 min. After carefully
removing EtOH, the pellet was allowed to dry at 50C
for 1 h. Finally, DNA was dissolved in 20 mlH
2
O.
Sequencing of the diluted PCR products (10 ml
H
2
Oþ6ml purified DNA) was carried out on an ABI
PRISMÕ310 genetic analyzer (Applied Biosystems)
using 47 cm capillaries and POP-6
TM
performance
optimized polymer. Electrokinetic injections were
performed at 2 kV for 30 s. Runs were carried out at
50C and 15 kV.
Real-time PCR and ELISA assays
SureFoodÕallergen kits for the qualitative detection
of DNA from hazelnut and peanut from Congen
Biotechnology GmbH (Berlin, Germany) were used for
real-time PCR analysis. The limits of detection for
both kits were 10 copies of genomic DNA (Congen
2006a,b). For the detection of cashew nut, a recently
described real-time PCR system was used (Ehlert et al.
2008a). RidascreenÕenzyme immunoassays for the
quantitative analysis of hazelnut and peanut were
obtained from R-Biopharm AG (Darmstadt,
Germany). The limit of detection as indicated by the
manufacturer were 2.5 mg kg
1
for both hazelnut and
peanut (R-Biopharm 2006; 2007).
Results and discussion
Design of LPA probes and choice of target sequences
The principle of ligation-dependent probe amplifica-
tion reaction, as well as suggestions and rules for the
design of LPA probes, have been specified in detail
previously (Schouten et al. 2002; Moreano et al. 2006,
MRC Holland). The detection of each target sequence
requires the design of two probes containing the
respective target-specific hybridization sites as well as
identical primer binding sites (PBS) at their 50-or
30-ends. The LPA system designed in this study uses the
advantages of synthetic oligonucleotides for hybridiza-
tion, as described by Moreano et al. (2006).
Food Additives and Contaminants 411
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Table 1. Probes and primers.
Target/GenBank
Accession No. Left probe Right probe
Ligation products
(retention time) [nt]
Peanut/L77197 50-GGGTTCCCTAAGGGTTGGAGCGAG
GCAGCAGTGGGAACTC-30
P-50-CAAGGAGACAGAAGATGCCAGAG
CCTCTAGATTGGATCTTGCTGGCAC-30
88 (87.10 0.13)
Cashew/AY081853 50-GGGTTCCCTAAGGGTTGGACTTATTA
GATTAATTCACTGGACTGC-30
P-50-CATGAAGTGAAGCAGTAGTAGAAGTCT
AGATTGGATCTTGCTGGCAC-30
92 (91.16 0.17)
Pecan nut/DQ156215 50-GGGTTCCCTAAGGGTTGGACACAATC
CCTACTACTTTCACTCCCAGGGA-30
P-50-CTCAGGTCGAGACATGAGTCCG
GGTCTAGATTGGATCTTGCTGGCAC-30
96 (94.67 0.17)
Pistachio nut/Y07600 50-GGGTTCCCTAAGGGTTGGACCTGAA
CACGGCGAGCACAAAG-30
P-50-AGGGACTGGTGGAGAAGATCAAAGAC
AAgtgtgtgtTCTAGATTGGATCTTGCTGGCAC-30
100 (99.37 0.18)
Hazelnut/AF136945 50-GGGTTCCCTAAGGGTTGGAGATCACC
AGCAAGTACCACACCAAGG-30
P-50-GCAACGCTTCAATCAATGAGGAGGA
GAtgtgtgtgtTCTAGATTGGATCTTGCTGGCAC-30
104 (102.75 0.12)
Sesame seeds/
AF240006
50-GGGTTCCCTAAGGGTTGGAgtgtgttGA
AGGGAGAGAAAGAGAGGAGGAGCAA-30
P-50-GAAGAACAGGGACGAGGGCGGATtg
tgtgtgtTCTAGATTGGATCTTGCTGGCAC-30
108 (108.11 0.13)
Macadamia nut/
AF161883
50-GGGTTCCCTAAGGGTTGGACTTA
ATCAACCGAGACAACAACGAGAGG-30
P-50-CTCCACATAGCCAAGTTCTTACAGACCA
TtgtgtgtgtgtgtTCTAGATTGGATCTTGCTGGCAC-30
112 (111.07 0.14)
Almond/X65718 50-GGGTTCCCTAAGGGTTGGAgtgtgtgtgt
CCATTACAAGTCTCCACCACCACCAC-30
P-50-CTTCTCCTACTCCTCCAGTCTACTCACC
ACCgtgtgtgTCTAGATTGGATCTTGCTGGCAC-30
116 (114.84 0.12)
Walnut/AF066055 50-GGGTTCCCTAAGGGTTGGAgtgtgtgtgtGG
CACAATCCCTACTACTTTCACTCCCAGAG-30
P-50-CATTAGGTCGAGACATGAGTCCGAGGA
AGGtgtgtgtTCTAGATTGGATCTTGCTGGCAC-30
120 (119.80 0.11)
Brazil nut/M17146 50-GGGTTCCCTAAGGGTTGGAgtgtgtgtgtgtgt
gtgGAGGAGGAGAACCAGGAGGAGTGTC-30
P-50-GCGAGCAGATGCAGAGACAGCAGgtgtgt
gtgtgtgtgtgTCTAGATTGGATCTTGCTGGCAC-30
124 (124.84 0.09)
Primer R – unlabeled 50-GTGCCAGCAAGATCCAATCTAGA-30
Primer F – labeled FAM-50-GGGTTCCCTAAGGGTTGGA-30
Capitals: plant DNA.
Bold capitals: primer binding site.
Lowercase fonts: spacer DNA.
412 A. Ehlert et al.
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Different tree nuts (macadamia, cashew, pecan,
walnut, brazil nut, pistachio, almond and hazelnut),
as well as peanut and sesame, were chosen as examples
for the detection of allergenic components in foods by
LPA. They are often present as hidden allergens in
pastries, candies or chocolate, and pose potential
health risks for allergic individuals.
Details of the selected target sequences and the
GenBank accession numbers of the LPA primers and
probes are listed in Table 1. The probes mostly detect
genes encoding for plant food proteins of the cupin and
prolamin superfamily, which are known to cause IgE-
mediated allergic reactions. The simultaneous detec-
tion of all targets is shown in Figure 1.
The primer sequences described by Schouten et al.
(2002) for MLPA analysis were also used in this study.
They were tested regarding their suitability for analyz-
ing foods by database enquiry via NCBI GenBank and
PCR using DNA of different plant species. The use of
spacer sequences between PBS and hybridization sites
rendered ligation products with lengths characteristic
for each target DNA. Differences of four nucleotides
(nt) in length are sufficient for unequivocal
determination of the amplification products using
POP-6
TM
polymer (Ehlert et al. 2008b). Simple repeats
of GT bases were used for the spacer sequences to
avoid intra- and intermolecular hybridizations.
Using this approach, all target signals were
sufficiently separated. The signal obtained for pista-
chio nut overlapped one of the standard signals
(100 nt); however, owing to the use of different
fluorescent dyes for standard and target probes,
a discrimination of the two signals is possible.
Evaluation of target specificity
To avoid cross homologies, the specificities of the
probe target sequences were first evaluated by
a BLAST search within the NCBI GenBank using
Beacon Designer software. Additionally, each probe
was checked with DNA extracted from the other target
species. The performance of the LPA system was
examined using DNA extracts of the 10 targets
adjusted to 20 ng ml
1
and different mixtures prepared
to simulate composite food products with different nut
proportions. No unspecific signals were observed for
almond, peanut, pecan, pistachio, hazelnut, sesame,
macadamia, walnut or brazil nut probes. Furthermore,
differentiation between the phylogenetically closely
related tree nuts, pecan and walnut could be achieved.
Published sequencing data were used to design the
ligation probes with only four different DNA bases
(Brezna et al. 2005; Brezna and Kuchta 2007). Cross-
reactivity of the cashew probes with other members
of the Anacardiaceae family, specifically pistachio and
mango, could be eliminated by detailed characteriza-
tion of the target and a new design of the probes.
Different pairs of primer were designed to amplify the
target region of the 2s albumin gene (ana o3 allergen).
PCR analysis using one of the primer pairs and DNA
of cashew, mango and pistachio resulted in specific
amplification of a 103 bp DNA section of cashew
DNA (Ehlert et al. 2008a). No signals were observed
when DNA of pistachio or mango was used in the PCR
reaction (data not shown). The region that was also
used for specific detection of cashew DNA by real-time
PCR analysis (Ehlert et al. 2008a) was, therefore,
chosen as hybridization site for the cashew LPA
probes.
Subsequently, the specificity of the method was
tested by analyzing DNA from organisms related to
the selected targets and from other organisms that are
expected to be ingredients of composed foods. Non-
coding regions of chloroplast DNA were amplified for
verification (Taberlet et al. 1991). The species tested
with all LPA probes are listed in Table 2. No false
positive signals were observed for peanut, cashew,
pecan, pistachio, hazelnut, sesame, macadamia, walnut
or brazil nut. The probes developed for the detection of
92nt Cashew
92nt Cashew
100 nt Pistachio nut
104 nt Hazelnut
104 nt Hazelnut
108 nt Sesame
112 nt Macadamia nut
96nt Pecan nut
88nt Peanut
88nt Peanut
116 nt Almond
116 nt Almond
120 nt Walnut
124 nt Brazil nut
Signal intensitySignal intensity
(a)
160100 nt
Tar
g
et len
g
th
(b)
50 75 139 150
Figure 1. (a) Electropherogram of a sample containing
a DNA mixture of peanut, cashew, pecan nut, pistachio
nut, hazelnut, sesame, macadamia nut, almond, walnut and
brazil nut. (b) Analysis of the commercial sample ‘‘organic
mixed nut butter’’. Electropherogram showing the detection
of four tree nuts contained in the sample. Non-assigned
signals correspond to the used length standards.
Food Additives and Contaminants 413
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almond DNA also yielded positive results in the
presence of DNA from apricot, nectarine, peach and
plum due to their phylogenetical relationship. Further
characterization of the target by sequencing will be
necessary to increase the specificity of the almond
detection.
Slight peaks at the n1 and n2 nucleotides are
caused by impurities of the synthesized oligonucleo-
tides, which could be reduced by a new synthesis but
could not be eliminated completely (data not shown).
The fragment lengths determined for each target
were highly reproducible (SD 0.2 nt). The observed
slight shifts (1 nt) of the absolute length values
(Table 1) are well-known effects arising from differ-
ences in the mobilities of the length standards and the
analyzed fragments caused by different labeling dyes
and by differences in the structures and base composi-
tions of the DNA (Magnuson et al. 1996; Wenz et al.
1998).
Sensitivity of the LPA system
Due to the lack of appropriate reference materials for
the detection of allergens, different food matrices, in
which the selected plants typically occur, were chosen
to evaluate the sensitivity of the LPA system.
Chocolate was spiked with peanuts and hazelnuts,
pesto with cashew nuts, and cookies with peanuts.
In addition, walnut cookies spiked with peanuts,
hazelnuts, pecan and macadamia nuts were
self-prepared. These examples were selected to simulate
fraudulent labeling or the unintended contamination of
foods with different nuts (Table 3). Starting materials
and ingredients were checked for the absence of any
target of the LPA system.
Two independent DNA extractions of the
spiked samples were analyzed in duplicate by LPA
to determine the limits of detection (LOD).
Fragment-length analysis was performed with the
pure and a 1 : 20-diluted PCR product. The LOD was
assessed as the least concentration for which all results
were consistently positive. For peanut, hazelnut and
cashew nut, limits of detection of 5 mg kg
1
were
determined in the chocolate, cookie and pesto
matrices.
To study the influence of unequal proportions of
allergenic components in a food, i.e. high excess of
one of the LPA targets in the sample, on the
sensitivity of the method, cookies containing 25%
(250 000 mg kg
1
) walnuts were spiked with defined
amounts (1–10 000 mg kg
1
) of four other nuts.
Due to competitive amplification of the probes
during PCR, under these conditions the added
reagents are mainly used to amplify the excess of
walnut ligation products. Consequently, the sensitiv-
ity for the nuts present as trace amounts is reduced,
resulting in LODs of 1000 mg kg
1
for peanut,
hazelnut, pecan, macadamia and 100 mg kg
1
for
hazelnut (Table 3).
This inherent feature of the multi-target LPA
method has to be taken into account when traces of
targets are to be simultaneously detected in the
presence of a high amount of one of the other LPA
targets.
The samples used to determine the limits of
detection of the LPA method were also analyzed by
real-time PCR and ELISA (Table 3). For the
samples analyzed by the commercially available
ELISA kits, the LODs determined for hazelnut and
peanut were slightly higher than those given by the
manufacturer (R-Biopharm 2006, 2007). For the
LODs determined for these targets by real-time
PCR methods, such a comparison was not possible
because the information given by the manufacturer
refers to copies of genomic DNA (Congen, 2006a, b).
The limits of detection observed for peanuts, hazel-
nuts and cashew nuts in chocolate and pesto by LPA
Table 2. Species used for determining the target specificity of the LPA method.
Plants Animals
Anise Garlic Plum (leaf ) Beef
Apple Ginger Pumkin seed Chicken
Apricot (leaf ) Lemon grass Raspberry (leaf and fruit) Duck
Banana (leaf) Linseed Rice Turkey
Basil Maize Rye Pork
Blackberry (leaf ) Mango (pulp and paring) Sour cherry
Cardamom seeds Nectarine (leaf ) Soya Microorganism
Carrot Nutmeg Strawberry (leaf and fruit) Yeast
Cinnamon Oregano Sunflower seed
Coconut Parsley Sultana
Coriander Peach Sweet cherry
Cumin Pear Wheat
Curcuma Pepper (black)
Currant Pimento
Fennel Pine nut
414 A. Ehlert et al.
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were similar to those determined for the tested
ELISA and real-time PCR approaches. The real-
time PCR method applied for the detection of
peanuts showed higher sensitivity; however, the
application of this kit also resulted in positive signals
for the non-spiked walnut reference cookie.
Therefore, the reliability of these results remains
questionable. The hazelnut ELISA test also showed
cross-reactivity to walnut (0.001–0.036%); owing to
the false-positive results, a detection of hazelnut
traces in the presence of walnut is not possible with
this kit (Kniel and Moser 2006; R-Biopharm 2006).
Table 3. Comparison of limits of detection.
Sample
Detection limit [mg kg
1
]
LPA Real-time PCR ELISA
Peanut chocolate 5 5 5
Hazelnut chocolate 5 10 10
Peanut-spiked cookie 5 0.5 5
Pesto cashew 5 2 *
Walnut cookies with
peanut, hazelnut,
pecan, macadamia
1000 (peanut, pecan,
macadamia)
100 (hazelnut)
1 (peanut)
10 (hazelnut)
100 (peanut)
1 (hazelnut)
*No method available.
Table 4. Analysis of commercially available samples by LPA.
Product
Declared allergenic
ingredients Precautionary labeling Detection
Spreads
Organic mixed nut butter Peanuts, hazelnuts,
Cashews, almonds
– Peanut, cashew, hazel-
nut, almond
Nutella Hazelnut 13% – hazelnut
Sausages
Original Thuringian sausage Walnuts – n.d.
Mortadella with pistachios Pistachios 1% – Pistachio
Convenience food dressings
Pesto alla Genovese Cashew nuts – Cashew
Sate
´dressing Peanuts 21%, peanut
flavor
– Peanut
Pesto goutweed (Aegopodium
podagraria)
Macadamia nut – Macadamia nut
Dairy products
Yoghurt with almonds Almonds 2% – Almond
Milkshake pistachio-cocos Pistachio pulp – Pistachio
Sweets/Cookies
Chocolate bar Hazelnut 5% Traces of almond,
peanut and other nuts
Hazelnut
Hazelnut bar Hazelnut mark 3.4% Traces of nuts and other
seeds
Hazelnut
Hazelnut bar with honey Hazelnut 66% Traces of peanut, sesame
or other nuts
Hazelnut, peanut
Peanut bar with honey Peanut 68% Traces of sesame or nuts Peanut, hazelnut
Sesame seed bar with honey Sesame 68% Traces of peanut or
other nuts
Sesame
Praline hazelnut Hazelnut 30.5%,
almonds
Traces pistachio Hazelnut, almond
Praline pistachio Almonds 12.5%,
pistachio 6.5%
Traces hazelnut Almond, pistachio
Praline coconut Almonds 5% Traces egg, hazelnut,
pistachio
Almond, pistachio
Gingerbread Hazelnuts, walnuts,
almonds, cashew nuts,
apricot kernels
Traces of other nuts and
kernels
Cashew, hazelnut,
almond, walnut
Food Additives and Contaminants 415
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Analysis of retail samples
A variety of commercial foods were tested for the
presence of the LPA targets. A total of 39 samples
from different food categories were analyzed. All DNA
extracts were checked for PCR inhibition and amplifia-
bility of DNA prior to LPA analysis by amplification
of non-coding regions of chloroplast DNA (Taberlet
et al. 1991). Results obtained for spreads, sausages,
dressings, dairy products and sweets are shown in
Table 4.
The results obtained for the samples ‘‘organic
mixed nut butter’’ and ‘‘gingerbread’’ demonstrate
the advantages of the developed multi-target LPA
method. In both cases, simultaneous detection of the
allergenic ingredients declared on the label could be
achieved. Figure 1b shows the results obtained for the
sample ‘‘organic mixed nut butter’’.
Except for walnuts in a Thuringian sausage, the
presence of ingredients declared on the label could be
confirmed by LPA analysis in all samples. In addition,
information given as part of a precautionary labeling
in some of the retail samples could be confirmed or
specified. Considering the described reduction in
sensitivity for targets contained as trace amounts in
the presence of a high amount of one of the other LPA
targets, the detection of undeclared allergenic ingre-
dients may be even further improved by analyzing
samples with probe mixes that exclude the probes that
would amplify the quantitatively dominating and
declared allergenic ingredients.
A limitation of the method is the inability to
differentiate almond from apricot, nectarine, peach
and plum. This became obvious for the ‘‘gingerbread’’
sample: the signal detected for almond could not be
assigned unequivocally to almond because apricot
kernels had also been declared in the list of ingredients.
Quantitative conclusions on the contamination
found in the retail samples are difficult, but concentra-
tions at the lower ppm range are likely.
Conclusions
The LPA system for the detection of food allergens was
shown to be a specific and sensitive method suitable for
the simultaneous detection of peanut, cashew, pecan
nut, pistachio nut, hazelnut, sesame seeds, macadamia
nut, walnut and brazil nut in the lower mg kg
1
range.
The modular system allows extension to further target
sequences of interest. LPA analysis of different food
matrices resulted in LODs in the lower ppm range,
thus confirming the suitability of the method for
allergen detection (Goodwin 2004; Poms and Anklam
2004; Poms et al. 2004). The specificities of the probes
targeting almond DNA have to be improved to be
capable of differentiating almond from phylogeneti-
cally related species.
The lack of certified reference materials in the field
of allergen detection makes development and valida-
tion of appropriate methods more difficult. Further
reference materials with certified concentrations of
analytes are required to gain more information on
individual limits of detection of the LPA method in
actual food products.
References
Arlorio M, Cereti E, Coisson JD, Travaglia F, Martelli A.
2007. Detection of hazelnut (Corylus spp.) in processed
foods using real-time PCR. Food Control. 18(2):140–148.
Barbieri G, Frigeri G. 2006. Identification of hidden
allergens: detection of pistachio traces in mortadella.
Food Addit Contam. 23(12):1260–1264.
Belgrader P, Barany F, Lubin M. 1997. Detection of nucleic
acid sequence differences using coupled ligase detection
and polymerase chain reactions. PCT Internation Patent
Application Number 9745559:1–158.
Breiteneder H, Ebner C. 2000. Molecular and biochemical
classification of plant-derived food allergens. J Allergy
Clin Immunol. 106(1):27–36.
Brzezinski JL. 2006. Detection of cashew nut DNA in spiked
baked goods using a real-time polymerase chain reaction
method. J AOAC Int. 89(4):1035–1038.
Brezna B, Hudekova L, Kuchta T. 2005. A novel real-time
polymerase chain reaction (PCR) method for the detection
of walnuts in food. Eur Food Res Technol. DOI 10.1007/
s00217-005-0214-8.
Brezna B, Kuchta T. 2007. A novel real-time polymerase
chain reaction method for the detection of pecan nuts in
food. Eur Food Res Technol. DOI 10.1007/s00217-007-
0639-3.
Carrino JJ. 1996. Multiplex ligations-dependent amplifica-
tion using split probe reagents containing common primer
binding sites. PCT International Patent Application
Number 9615271:1–40.
Codex Alimentarius Commission 2007. Food Labelling.
Complete Texts. Joint FAO/WHO Food Standards
Program.
Congen. 2006a. Kit instruction. Art. No.: S3103, Version 1.1.
Berlin: Congen.
Congen. 2006b. Kit instruction. Art. No.: S3102, Version 1.0.
Berlin: Congen.
Dahinden I, Von Buren M, Luthy J. 2001. A quantitative
competitive PCR system to detect contamination of wheat,
barley or rye in gluten-free food for coeliac patients. Eur
Food Res Technol. 212(2):228–233.
Dovicovicova L, Olexova L, Pangallo D, Siekel P, Kuchta T.
2004. Polymerase chain reaction (PCR) for the detection of
celery (Apium graveolens) in food. Eur Food Res Technol.
218(5):493–495.
EC 2000. Directive 2000/13/EC of the European Parliament
and of the Council of 20 March 2000 on the approxima-
tion of the laws of the Member States relating to the
labelling, presentation and advertising of foodstuffs.
Off J Eur Commun. L109:29–42.
EC 2007. Commission Directive 2007/68/EC of the
European Parliament and of the Council of 27
416 A. Ehlert et al.
Downloaded By: [Bayerische Staatsbibliothek] At: 08:18 13 May 2009

November 2007 amending Annex IIIa to Directive 2000/
13/EC as regards certain food ingredients. Off J Eur
Commun. L310:11–14.
Ehlert A, Hupfer C, Demmel A, Engel KH, Busch U. 2008a.
Detection of cashew nut in foods by a specific real-time
PCR method. Food Anal Methods. 1(2):136–143.
Ehlert A, Moreano F, Busch U, Engel KH. 2008b. Use of
ligation-dependent probe amplification for the simulta-
neous detection/screening of seven different targets of
genetically modified organisms. Eur Food Res Technol.
227(3):805–812.
Eldering E, Spek CA, Aberson HL, Grummels A, Derks IA,
de Vos AF, McElgunn CJ, Schouten JP. 2003. Expression
profiling via novel multiplex assay allows rapid assessment
of gene regulation in defined signalling pathways. Nucleic
Acids Res. 31(23):e153.
Ellman LK, Chatchatee P, Sicherer SH, Sampson HA. 2002.
Food hypersensitivity in two groups of children and young
adults with atopic dermatitis evaluated a decade apart.
Pedriatr Allergy Immunol. 13:295–298.
Germini A, Scaravelli E, Lesignoli F, Sforza S, Corradini R,
Marchelli R. 2005. Polymerase chain reaction coupled with
peptide nucleic acid high-performance liquid chromato-
graphy for the sensitive detection of traces of potentially
allergenic hazelnut in foodstuffs. Eur Food Res Technol.
220(5/6):619–624.
Gille JJP, Hogervorst FBL, Pals G, Wijnen JT, van
Schooten RJ, Dommering CJ, Meijer GA, Craanen ME,
Nederlof PM, de Jong D, et al. 2002. Genomic deletions
of MSH2 and MLH1 in colorectal cancer families detected
by a novel mutation detection approach. Br J Cancer.
87(8):892–897.
Goodwin PR. 2004. Food allergen detection methods: a
coordinated approach. J AOAC Int. 87(6):1383–1390.
Herman L, De Block J, Viane R. 2003. Detection of hazelnut
DNA traces in chocolate by PCR. Int J Food Sci Technol.
38(6):633–640.
Hernandez M, Esteve T, Pla M. 2005. Real-time polymerase
chain reaction based assays for quantitative detection
of barley, rice, sunflower, and wheat. J Agric Food Chem.
53(18):7003–7009.
Hird H, Lloyd J, Goodier R, Brown J, Reece P. 2003.
Detection of peanut using real-time polymerase chain
reaction. Eur Food Res Technol. 217(3):265–268.
Hogervorst FBL, Nederlof PM, Gille JJP, McElgunn CJ,
Grippeling M, Pruntel R, Regnerus R, Van Welsem T,
Van Spaendonk R, Menko FH, et al. 2003. Large genomic
deletions and duplications in the BRCA1 gene identified
by a novel quantitative method. Cancer Res. 63(7):
1449–1453.
Holzhauser T, Wangorsch A, Vieths S. 2000. Polymerase
chain reaction (PCR) for detection of potentially allergenic
hazelnut residues in complex food matrixes. Eur Food Res
Technol. 211(5):360–365.
Hsuih TC, Park YN, Zaretsky C, Wu F, Tyagi S,
Kramer FR, Sperling R, Zhang DY. 1996. Novel,
ligation-dependent PCR assay for detection of hepatitis
C in serum. J Clin Microbiol. 34(3):501–507.
Hupfer C, Waiblinger HU, Busch U. 2006. Development and
validation of a real-time PCR detection method for celery
in food. Eur Food Res Technol. 225(3/4):329–335.
Kniel B, Moser M. 2006. Nachweis allergener
Haselnußspuren in Backwaren und in den dafu
¨r verwen-
deten Rohstoffen und Zwischenprodukten.
Getreidetechnologie. 60(6):357–365.
Magnuson VL, Ally DS, Nylund SJ, Karanjawala ZE,
Rayman JB, Knapp JI, Lowe AL, Ghosh S, Collins FS.
1996. Substrate nucleotide-determined non-templated
addition of adenine by Taq DNA polymerase: implications
for PCR-based genotyping and cloning. Biotechniques.
21(4):700–709.
Moreano F, Ehlert A, Busch U, Engel KH. 2006. Ligation-
dependent probe amplification for the simultaneous
event-specific detection and relative quantification of
DNA from two genetically modified organisms. Eur
Food Res Technol. 222(5–6):479–485.
MRC Holland. Available: http://www.mrc-holland.com/
pages/support_desing_synthetic_probespag.html. (Accessed:
21 August 2008).
Pancaldi M, Paganellil A, Righini G, Carboni E, Salvi A,
Rainieri M, Villa C, Benda S. 2005. Molecular detection
of vegetable-derived food allergens. Ingred. Aliment.
4(18):21–27.
Poms RE, Anklam E. 2004. Polymerase chain reaction
techniques for food allergen detection. J AOAC Int.
87(6):1391–1397.
Poms RE, Klein CL, Anklam E. 2004. Methods for allergen
analysis in food: a review. Food Addit Contam.
21(1):1–31.
R-Biopharm. 2006. Kit Instruction. Art. No.:R6801,
03-11-18. Darmstadt: R-Biopharm.
R-Biopharm. 2007. Kit Instruction. Art. No.:R6201,
07-03-20. Darmstadt: R-Biopharm.
Ronning SB, Berdal KG, Boydler Andersen C,
Holst-Jensen A. 2006. Novel reference gene, PKABA1,
used in a duplex real-time polymerase chain reaction for
detection and quantitation of wheat and barley-derived
DNA. J Agric Food Chem. 54(3):682–687.
Rossi S, Scaravelli E, Germini A, Corradini R, Fogher C,
Marchelli R. 2005. A PNAarray platform for the detection
of hidden allergens in foodstuffs. Eur Food Res Technol.
DOI 10.1007/s00217-005-0034-x.
Sandberg M, Lundberg L, Ferm M, Yman IM. 2003.
Real-time PCR for the detection and discrimination of
cereal contamination on gluten free foods. Eur Food Res
Technol. 217(4):344–349.
Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D,
Diepvens F, Pals G. 2002. Relative quantification of
40 nucleic acid sequences by multiplex ligation-depen-
dent probe amplification. Nucleic Acids Res. 30(12):e57/
1–e57/13.
Sicherer SH, Sampson HA. 2006. Food allergy. J Allergy
Clin Immunol. 117(2-Suppl Mini-Primer):470–475.
Stephan O, Weisz N, Vieths S, Weiser T, Rabe B,
Vatterott W. 2004. Protein quantification, sandwich
ELISA, and real-time PCR used to monitor industrial
cleaning procedures for contamination with peanut and
celery allergens. J AOAC Int. 87(6):1448–1457.
Stephan O, Vieths S. 2004. Development of a real-time PCR
and a sandwich ELISA for detection of potentially
allergenic trace amounts of peanut (Arachis hypogaea)
in processed foods. J Agric Food Chem. 52(12):3754–3760.
Food Additives and Contaminants 417
Downloaded By: [Bayerische Staatsbibliothek] At: 08:18 13 May 2009

Taberlet P, Gielly L, Pautou G, Bouvet J. 1991. Universal
primers for amplification of three non-coding regions of
chloroplast DNA. Plant Mol Biol. 17(5):1105–1109.
Taylor CF, Charlton RS, Burn J, Sheridan E, Taylor GR.
2003. Genomic deletions in MSH2 or MLH1 are a
frequent cause of hereditary non-polyposis colorectal
cancer: identification of novel and recurrent deletions by
MLPA. Hum Mutat. 22(6):428–433.
Wenz H, Robertson JM, Menchen S, Oaks F, Demorest DM,
Scheibler D, Rosenblum BB, Wike C, Gilbert DA,
Efcavitch JW. 1998. High-precision genotyping by denatur-
ing capillary electrophoresis. Genome Res. 8(1):69–80.
418 A. Ehlert et al.
Downloaded By: [Bayerische Staatsbibliothek] At: 08:18 13 May 2009