Food Control 67 (2016) 135e143

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

A DNA macro-array to simultaneously identify 32 meat species in food

samples
Geoffrey Cottenet a, *, Ve

ronique Sonnard a, Carine Blancpain a, Hui Zhen Ho b,
Hann Lum Leong b, Poh Fong Chuah b
a
Food Safety & Quality, Nestec Ltd., Nestl

e Research Center, Vers-chez-les-Blanc, Lausanne, Switzerland
b
Nestl

e Quality Assurance Center, Quality Road, Republic of Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Considering the recent cases of meat adulteration and fraud, efficient and accurate analytical methods
Received 7 January 2016 are needed for the specific identification of meat species as a key importance to maintain consumers'
Received in revised form trust and to comply with various local labelling legislations. As a reference approach, the application of
24 February 2016
PCR to meat speciation is always limited to few species, whereas there is a strong need to broaden the
Accepted 25 February 2016
Available online 2 March 2016
number of identified meat species. PCR was here associated with a commercial DNA macro-array spotted
with meat-specific capture probes, allowing the simultaneous identification of 32 meat species on 8
samples per chip support. Tested on pure meat samples, spiked samples, proficiency test samples, and
Keywords:
Meat species
processed samples, the method showed high specificity on the targeted species and allowed a sensitivity
Screening down to 1% (w/w). Easy-to-use, fast and cost-efficient, this multi-screening approach is fit-for-purpose as
Identification a tool to detect meat substitution or adulteration for meat testing laboratories.
PCR © 2016 Elsevier Ltd. All rights reserved.
Array

1. Introduction the acceptable limit of general meat adulteration.

Following the horse meat crisis which spread throughout
Europe in 2013, food fraud and adulteration have been in the spot-
light and are identified as a top priority to be addressed by au-
thorities, regulators and food industries (Elliott, 2014). Economi-
cally motivated adulteration presents many challenges because
perpetrators are specifically seeking to avoid detection and
circumvent existing regulatory systems or testing methodologies
(Everstine, Spink, & Kennedy, 2013).
Various food labelling legislations require the description of the
meat species used in a meat-based product, and the recent Euro-
pean Recommendation (European Commission, 2013) considers
the presence of non-declared horse meat above 1% (w/w) as an
adulterated meat sample. This 1% threshold was set as a pragmatic
level to distinguish between gross contamination/adulteration and
trace levels coming from natural cross-contamination in the supply
chain or the slaughterhouses. Although specifically focussing on the
presence of horse meat, this 1% threshold has been applied to other
meat species (Food Standards Agency, 2014) and is considered as
* Corresponding author.
E-mail address: geoffrey.cottenet@rdls.nestle.com (G. Cottenet).
To comply with these legislations, and to maintain consumer's
trust, effective and accurate analytical methods are needed for the
identification of meat species. To verify meat species authenticity
and traceability, many technologies have been described, such as 2-
dimensional electrophoresis (Martinez & Friis, 2004), isoelectric
focussing (Scarpeid, Kraal, & Hildrum, 1998), protein capillary
electrophoresis (Vallejo-Cordoba, Gonzalez-Cordoba, Mazorra-
Manzano, & Rodriguez-Ramirez, 2005), HPLC (Aristoy & Toldra,
2004), ELISA (Ascensio, Gonzalez, Garcia, & Martin, 2008). These
protein-based analytical tools are however susceptible to protein
denaturation due to physical or chemical industrial treatments
(Murugaiah et al., 2009). More recently, peptide biomarker mass
spectrometry was applied for meat speciation (Ruiz Orduna, Husby,
Yang, Ghosh, & Beaudry, 2015), but its use was limited to the
identification of few meat species only and achieved a low sensi-
tivity. Similarly, the profile of lipid-based biomarkers can be
modified through cooking steps (Rohman, Erwanto, & Man, 2011)
and the feeding diet (Garcia et al., 2008).
To overcome these limitations, the use of DNA-based methods
and especially Polymerase Chain Reaction (PCR) techniques are
preferred for both raw ingredients and processed food (Hird et al.,
2006). Contrary to some proteins and lipids molecules, DNA is
present in all types of tissue, and short DNA fragments are known to

http://dx.doi.org/10.1016/j.foodcont.2016.02.042
0956-7135/© 2016 Elsevier Ltd. All rights reserved.
136 G. Cottenet et al. / Food Control 67 (2016) 135e143
be stable in processed samples (Ali, Razzak, & Hamid, 2014).
PCR also offers the possibility to simultaneously analyse several
species in the same reaction thanks to multiplexing (Ali, Razzak, &
Abd Hamid, 2014). Multiplex detection PCR methods have been
rising during these last ten years, allowing a reduction of the
analytical time and cost, and conserving precious sample material.
However the described PCR methods still present the same limi-
tation: the majority of them always target the usual meat species,
like beef, pork, horse, chicken and turkey, and/or are limited to
maximum 5e6 meat species (Fajardo, Gonzalez, Rojas, Garcia, &
Martin, 2010; Ali et al., 2014). Taking into account the latest cases
of meat adulteration in China where donkey and lamb meat were
found to contain fox and rat meat, respectively (Reuters, 2014;
Bejing, 2013), and the example of dried meat from South Africa
containing giraffe and kangaroo instead of beef (D'Amato, Alechine,
Cloete, Davison, & Corach, 2013), there is a strong need to expand
the range of detectable meat species. In addition, meat substitution
has been shown to be a quite frequent phenomena (Di Pinto et al.,
2015; Kane & Hellberg, 2016).
DNA macro-array, and more specifically a Meat Low Cost and
Density (LCD) Array, has already been evaluated by previous studies
and showed efficient and reliable meat identification with easy and
quick handling procedures (Iwobi, Huber, Hauner, Miller, & Busch,
2011; Cawthorn, Steinman, & Hoffman, 2013; Yosef, Al-Julaifi, &
Al-Rizqi, 2014). This Meat LCD Array approach is based on classical
PCR followed by a LCD array hybridisation. The PCR step amplifies a
fragment ranging from 115 to 125 base pair (bp) from the verte-
brates' 16S rRNA mitochondrial gene, then amplicons are hybri-
dised on a macro-array spotted with capture probes specific to 16
different meat species. Although considered as a high screening
capability, this Meat LCD Array has been upgraded to allow the
simultaneous detection of 32 meat species and cover a broader
range of meat species.
This study describes the evaluation of this 32-Meat LCD Array
version on pure meat species, spiked samples, proficiency tests, and
processed samples such as cooked meat and meat powder. The
specificity of the different capture probes was evaluated, as well as
the capability of the method to detect a 1% adulterant species, as
required by the EU recommendation (European Commission, 2013),
to detect potential economically motivated adulteration by meat
Fig. 1. Schematic representation of a slide, composed of 8 individual Meat LCD Arrays, spotted with 32 species-specific capture probes.
substitution or admixture.
2. Materials and methods
2.1. Samples
The following species were used during this study:
1 Meat species: beef (Bos taurus), pork (Sus scrofa), horse (Equus
caballus), donkey (Equus asinus), zebra (Equus quagga), sheep
(Ovis aries), goat (Capra hircus), bison (Bison bison), water buffalo
(Bubalus bubalis), hare (Lepus europaeus), rabbit (Oryctolagus
cuniculus), fallow deer (Dama dama), red deer (Cervus elaphus),
reindeer (Rangifer tarandus), roe deer (Capreolus capreolus),
chicken (Gallus gallus), turkey (Meleagris gallopavo), guinea fowl
(Numida meleagris), muscovy duck (Cairina moschata), mallard
duck (Anas platyrhyncos), goose (Anser anser), pigeon (Columba
livia), emu (Dromaius novaehollandiae), ostrich (Struthio cam-
elus), camel (Camelus dromedarius), lama (Lama guanicoe),
giraffe (Giraffa camelopardalis), springbok (Antidorcas marsu-
pialis), kudu (Tragelaphus strepsiceros), kangaroo (Macropus
rufus), red fox (Vulpes vulpes), cat (Felis catus), dog (Canis
familiaris), brown rat (Rattus norvegicus), badger (Meles meles),
oryx (Oryx gazella), pheasant (Phasianus colchicus), gnu (Con-
nochaetes taurinus), daw (Corvus monedula), elk (Alces alces),
impala (Aepyceros melampus), ermine (Mustela ermine), croco-
dile (Crocodylus niloticus), king quail (Coturnix coturnix), snail
(Helix pomatia), muskrat (Ondatra zibethicus), frog (Limnonectes
macrodon).
2 Fish species: chum salmon (Oncorhynchus keta), coho salmon (O.
kisutsch), rainbow trout (O. mykiss), Atlantic salmon (Salmo
salar), Atlantic halibut (Hippoglossus hippoglossus), pollock
(Pollachius pollachius), whiting (Merlangius merlangus), cod
(Gadus morhua), skipjack tuna (Katsuwonus pelamis), bigeye
tuna (Thunnus obesus), albacore tuna (T. alalunga), yellowfin
tuna (T. albacares), prawn (Pandalus borealis).
3 Plant species: maize (Zea mays), soya (Glycine max), wheat
(Triticum aestivum), rye (Secale cereal), barley (Hordeum vulgare),
rice (Oryza sativa), tomato (Solanum lycopersicum), cotton
 G. Cottenet et al. / Food Control 67 (2016) 135e143
Fig. 2. Gel electrophoresis of the Chipron PCR amplicons obtained on plants, fish and
meat samples. From 1 to 26: maize, soya, wheat, rye, barley, rice, tomato, cotton, po-
tato, rapeseed, chum salmon, coho salmon, Atlantic halibut, pollock, rainbow trout,
Atlantic salmon, whiting, cod, skipjack tuna, bigeye tuna, albacore tuna, yellowfin tuna,
beef, lama, zebra, pork. The 100-bp molecular ladder and the negative PCR control are
indicated by (M) and (), respectively. The beef sample was used as the PCR positive
control.
(Gossypium hirsutum), potato (Solanum tuberosum), rapeseed
(Brassica napus).
All samples were purchased from local markets, meat and fish
species were confirmed by DNA sequencing prior to further usage
(Handy et al., 2011). When not available as meat samples, certified
reference DNAs (CRD) were purchased from Coring System Diag-
nostix GmbH (Gernsheim, Germany) and Eurofins GeneScan
(Freiburg, Germany).
In addition, meat powder samples from beef, pork, chicken and
turkey were obtained from industrial suppliers.
Finally, proficiency test samples were acquired from the Food
and Environment Research Agency (York, United Kingdom) and
LGC Standards (Teddington, United Kingdom).
Fig. 3. Scan of a Meat LCD Array chip on which PCR negative control (A), beef (B), pork (C), horse (D), donkey (E), sheep (F), goat (G) and water buffalo (H) samples were analysed.
The dots present on 3 corners of each array correspond to hybridisation controls (red circles). Each species-specific probe is spotted in duplicate on each array (green circles). (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
137
2.2. Mixtures
To verify that the method is able to detect meat species down to
1% (w/w), meat admixtures were prepared by adding 10 ± 2 mg of
an adulterant meat species to 990 ± 5 mg of a matrix species. Taking
into account the uncertainty of weighing, the final mixtures con-
tained between 0.8 and 1.2% (w/w) which was considered as
acceptable for our purpose.
To evaluate the applicability of the method on processed sam-
ples, twenty different cuts of beef were spiked with 1% pork and 1%
reindeereconsidered to be amongst the less sensitive probes of the
array-and cooked 270 min at 100
C to mimic thermal industrial
processes of prepared meals.
2.3. DNA extraction and preparation
Samples, 1 g, were incubated in 3 mL of CTAB lysis buffer
(Applichem GmbH, Darmstadt, Germany), 660 mg/mL proteinase K
(Promega, Dübendorf, Switzerland), and 400 mg/mL RNAse A (Sig-
maeAldrich Chemie GmbH, Buchs, Switzerland) at 65
C for 1 h.
After maceration, the samples were centrifuged at 3000 g for 2 min.
The supernatant, 500 mL, was transferred into a new tube, mixed
with an equal volume of chloroform, and centrifuged at 15000 g for
2 min. The supernatant was added to an equal volume of PB binding
buffer (QIAgen, Hilden, Germany), mixed, loaded onto a QIAquick
column (QIAgen) and centrifuged at 15000 g for 2 min. The column
was washed with 700 mL of PE buffer (QIAgen), dried at 15000 g for
1 min, and placed in a new centrifuge tube. The retained DNA was
eluted by centrifugation for 1 min at 15000 g with 60 mL of EB
elution buffer (QIAgen) after 1 min of incubation. Following the
measurement of the DNA concentration using a Nanodrop (Thermo
Fischer Scientific, Waltham, USA), DNA extracts were finally diluted
at 20 ng/mL in EB buffer.
CRD available as lyophilised DNA were reconstituted at 20 ng/mL
with EB buffer.
Extracted DNA and reconstituted DNA were stored at 20
C
until further use.
2.4. Meat PCR
PCR runs were performed in a TGradient thermocycler (Bio-
metra GmbH, Go€ttingen, Germany) using the Platinum Taq
138 G. Cottenet et al. / Food Control 67 (2016) 135e143

Table 1
Selectivity of the 32 species-specific targets on various pure meat, seafood and plant samples. Hybridisation signals and weak cross-reactions are indicated with an X and
within brackets, respectively. When not available as real meat samples, CRD were used (*).

Species-specific targets on the Meat LCD Array

Tested sample Cattle Pork Equine Sheep Goat Bison Water Buffalo Hare Rabbit Fallow deer Red deer Reindeer Roe deer Chicken

Beef X
Pork X
Horse, donkey*, zebra X
Sheep X
Goat X
Bison X X
Water buffalo X
Hare X
Rabbit X
Fallow deer X
Red deer X
Reindeer (x) X
Roe deer X
Chicken X
Turkey
Guinea fowl
Muscovy duck
Mallard duck *
Goose
Pigeon
Emu
Ostrich
Camel *
Lama
Giraffe
Springbok
Kudu *
Kangaroo
Red Fox *
Cat *
Dog
Brown rat *
Elk * (x)
Badger *
Oryx *
Pheasant
Gnu *
Daw *
Muskrat *
Ermine *
Crocodile
Impala *
Snail
King quail *
Frog
Seafood samples
Plant samples

Polymerase kit (Life Technologies, Carlsbad, CA, USA). Each PCR
reaction contained 25 mL of an amplification mix consisting in 2 mL
of sample DNA at 20 ng/mL, 1X PCR buffer, 2 mM MgCl2, 1.5 U of
Platinum Taq polymerase, 0.4 mM of each dNTP (Promega) and
1.5 mL of the MEAT PCR primers supplied in the Meat LCD Array kit
(Chipron GmbH, Berlin, Germany). Sequences of the PCR primers
were kept proprietary.
Following an activation of the hot-start DNA Taq polymerase for
2 min at 94
C, the specific thermocycling consisted of 35 cycles of a
denaturation step of 15 s at 94
C, an annealing step of 15 s at 57
C,
and an elongation step of 30 s at 72
C. A final step of 3 min at 72
C
ended the PCR program.
PCR products were then visualised by electrophoresis on 1.2%
agarose gel cassettes (Lonza, Basel, Switzerland).
Array, spotted with 32 species-specific capture probes (Fig. 1)
allowing the simultaneous detection of 32 meat species on 8
samples. The 32 targets were selected based on: i) the most com-
mon meat species in international food trade (beef, pork, sheep,
chicken …), ii) closely-related species used in food industry such as
poultry (chicken, turkey, duck, …) and venison species (deer spe-
cies), iii) previous cases of meat adulteration even exotic ones
(horse, fox, kangaroo …), and iv) cheap or easily accessible meat
although unconventional animal species (springbok, kudu, camel,
lama …) (Hoffman & Cawthorn, 2012).
The capture probes are between 20 and 32 nucleotides long, and
carry an immobilisation tag at their 50 end (sequences of the cap-
ture probes were kept proprietary). The probes were spotted as
duplicates in a 9  9 pattern, with average spot diameters of
350 mm.

2.5. Design of the meat LCD array

Each macro-array slide is composed of 8 individual Meat LCD

 G. Cottenet et al. / Food Control 67 (2016) 135e143
Species-specific targets on the Meat LCD Array
Turkey Guinea fowl Muscovy duck Mallard duck Goose Pigeon Emu Ostrich Camel Lama Giraffe Springbok Kudu Kangaroo Red fox Cat Dog Brown rat
X
X
X
X
X
X
X
X
X
2.6. Analysis on the meat LCD array
The identification of meat species on the Meat LCD Array was
performed according to the manufacturer's instructions (Chirpon
GmbH). It consists in 4 main steps: 1) Hybridisation of PCR
amplicons to the species-specific capture probes on the array, 2)
Linkage of a streptavidineperoxidase conjugate to the biotin-
labelled PCR primers, 3) Staining after incubation with the perox-
idase substrate, 4) Visualisation of the dark precipitate using a LCD-
array slide scanner PF7250u (Chipron GmbH) and the SlideReader
version 12 analysis software (Chipron GmbH). The detection cut-off
threshold was set at 2000 pixel value (pv) in the software, knowing
that strong precipitates showed values close to 50000 pv.
2.7. Selectivity and sensitivity trials
In order to evaluate the specificity of the Meat LCD Array and
139
X
X
X
X
X
X
X
X
X
observe potential cross-reactions, pure meat, fish and plant species
were tested. Based on the EU recommendation (European
Commission, 2013), a detection threshold of 1% (w/w) was tar-
geted, and admixtures containing 1% adulterants were prepared
and analysed. In addition, to take into consideration the variability
of DNA content depending on the composition of meat tissues
(Ballin, Vogensen, & Karlsson, 2009; Floren, Wiedemann, Brenig,
Schütz, & Beck, 2015), different meat cuts were used as spiked
matrices: pieces of ribs, loins, sirloins, chucks, flanks and rounds
were selected for beef whereas breasts, tenderloins, thighs, drum-
sticks, wings and necks were selected for duck. Since the limit of
detection (LOD) is defined as the amount of analyte at which the
analytical method detects the presence of the analyte at least 95% of
the time (Codex Committee on Methods of Analysis and Sampling,
2010), twenty different beef cuts were spiked with 1% horse, pork,
reindeer, bison and water buffalo. Horse was selected due to the
recent European horsemeat crisis. Pork and reindeer have been
140 G. Cottenet et al. / Food Control 67 (2016) 135e143

Table 2
Signal values (pv) obtained on different adulterant species spiked at 1% in beef(B), duck(D), or chicken(C) matrices. Twenty replicates of each mixture were prepared from raw
meat, cooked meat or meat powder. Signal values below the detection threshold of 2000 pv are indicated with an asterisk (*).

Raw meat mixtures Processed meat mixtures

Cooked Powder
(B) (B) (B) (B) (B) (D) (D) (B) (B)
Horse Pork Reindeer Bison Water buffalo Chicken Guinea fowl Pork Reindeer Pork (B) Turkey (C)

1 17123 15551 17496 51864 59312 40670 8349 6558 20836 60336 28597
2 56332 29764 41140 59436 60108 29556 15984 3112 8304 59624 44320
3 57259 21402 40398 59254 58283 45138 30134 9204 20217 60670 48838
4 52462 40073 43104 54219 57360 49773 53439 29760 44994 54508 29155
5 54898 3844 36268 6746 58687 37550 37775 6367 6477 46243 32982
6 54101 47730 44849 48176 57884 36430 12276 3263 8694 58471 38754
7 38750 6571 39016 56640 59176 43350 33382 1881* 2442 53086 52831
8 58942 54196 33383 18704 59739 21168 14222 49746 34288 46280 44972
9 56686 52084 44111 44537 57862 30583 7851 51066 28830 43060 28352
10 53664 54253 7252 56013 58448 35154 8088 32592 19976 51786 43696
11 52073 17738 43034 12850 59952 35029 29242 2788 2501 37166 20607
12 51754 48986 48476 28055 56060 24616 47262 24407 18136 51478 31998
13 45851 46026 54324 26450 54234 30647 51800 26316 10936 47638 51420
14 42104 41179 40396 25678 55922 29473 54598 8698 4588 49396 51421
15 48196 13110 52008 12187 53800 36196 56871 9276 14667 42505 58067
16 55484 13360 48164 42382 57206 46523 49342 36542 47744 49186 48330
17 57920 7822 19781 3320 57968 19130 58108 11052 9842 51106 44806
18 57285 10706 42912 20912 51572 43844 55411 2726 3424 52694 40172
19 58004 9186 15050 52832 54672 42648 54376 10297 29018 48868 50488
20 53742 10108 40957 41859 50402 37435 27452 9782 9961 46694 26246

choseneapart from the great importance of pork as a frequently
found adulterantebecause the array probes for both species consist
of mixtures of several sequences to compensate for single nucleo-
tide polymorphisms (SNPs) present within the target region of
their 16S rRNA genes and therefore these species had to be
considered as slightly less sensitive targets present on the Meat LCD
Array. As a very close-related species to beef, bison could lead to
potential cross-reactions. Finally, water buffalo was also used as an
adulterant, since water buffalo meat (named carabeef) is well-
known to be a beef substitute in South Asian countries. Similarly,
twenty different replicates of duck spiked at 1% with chicken and
guinea fowl-cheaper poultry meats in Asiaewere prepared, and
analysed.
In addition, to evaluate the detection capability on processed
samples, twenty different cuts of beef were spiked with 1% pork
and 1% reindeer, and cooked 270 min at 100
C to mimic an
intensive industrial process which can be applied on culinary
products and prepared meals.
3. Results and discussion
3.1. Selectivity
The PCR step is dedicated to amplify a short fragment of all
vertebrates' 16S rRNA gene, whereas the selectivity of the described
method is brought by the species-specific probes spotted on the
array.
To evaluate the selectivity of the method and verify the absence
of potential cross-reactivity, pure samples from all the meat species
present on the array were tested as well as other meat species.
Similarly, several fish species were tested since the 16S rRNA gene is
present in all animals (Yang et al., 2014). To confirm the correct

Table 3
Detection of beef, pork, horse, chicken and sheep in proficiency test samples using the Meat LCD Array (X). The consensus presence of these 5 meat species were described in
the final p-test report (grey cells).

Samples Matrix Species

Raw meat Powder Cooked Canned Beef Pork Horse Chicken Sheep

LGC MT202 749-1 C X

LGC MT202 749-2 C X
LGC MT202 749-3 C X X
LGC MT202 749-4 C X X
LGC MT202 749-5 C X X
LGC MT202 749-6 C X
FAPAS T2950QC C X X X
FAPAS T2951AQC C C C X X
FAPAS T2951BQC C C C X X
FAPAS T2955QC C X X X
FAPAS T2957QC C X X X
FAPAS 2956 A C X X X
FAPAS 2956 B C X
FAPAS 2956C C X
FAPAS 2956 D C X
FAPAS 2956 E C X X
FAPAS 2958 A C X X X X
FAPAS 2958 B C X X
FAPAS 2962 C X X X X
 G. Cottenet et al. / Food Control 67 (2016) 135e143
identity of meat and fish samples before use, their species were
verified by DNA sequencing (Handy et al., 2011) using the Barcode
Of Life database (Ratnasingham & Hebert, 2007); amplicons of
approximately 650 bp were obtained on all samples and the ex-
pected species were successfully confirmed (data not shown),
which ensured the correct identity of the collected samples. In
addition, plant species commonly present in culinary products like
wheat, corn and rice were also used, while their identity have been
previously verified (Cottenet, Blancpain, Sonnard, & Chuah, 2013).
As expected, all the tested meat and fish samples led to 16S rRNA
PCR amplifications of 115e125 bp whereas the plant samples
randomly led to unspecific amplicons (Fig. 2). When meat, fish and
plant PCR products were further hybridised on the Meat LCD Array,
only meat species gave hybridisation signals on their respective
species-specific capture probes (Fig. 3), whereas fish and plant
samples did not lead to any hybridisation dots (Table 1). However,
elk and reindeer led to a weak cross-reactivity on roe deer and red
deer probes, respectively. Such weak cross-reactions between deer
species have already been observed using a real-time PCR approach
(Fajardo et al., 2008). As already reported, Cervidae species show a
very high genetic similarity of their mitochondrial DNA which
makes challenging the design of deer-species-specific identification
methods (Hoffmann, Johannesen, & Griebeler, 2014).
Bison led to strong signals on both bison and cattle probes
which were designed to target Bison bison and the Bos genus,
respectively. Although Bison corresponds to a whole genus, some
taxonomists would place bison in its very close-related Bos genus
(Wilson & Ruff, 1999) still leading to ambiguous naming of bison as
Bison bison or Bos bison. Vice versa, beef randomly led to very weak
signals on the bison probe, but regularly below the detection
threshold. The bison probe was thus considered as specific to bison
only.
Besides these weak interactions, no false positives were
observed. Aves didn't show any cross-reactions, even between
poultry species. Chicken, turkey, goose, guinea fowl and the two
duck targets were specific to their meat species.
In addition, amongst all the runs performed, no hybridisation
signals have ever been observed on negative controls.
These data show that the species-specific probes spotted on the
Meat LCD Array allow specific signals when testing the corre-
sponding meat species only.
3.2. Sensitivity
Based on the EU recommendation (European Commission,
2013), a detection threshold of 1% (w/w) was targeted, and ad-
mixtures containing 1% adulterants were prepared and analysed.
Since DNA content is known to vary depending on the composition
of meat tissues (muscles, tendons, fat… (Ballin et al., 2009; Floren
et al., 2015)), twenty different beef cuts and duck pieces were
used as matrices and spiked with 1% pork, reindeer, horse, bison,
water buffalo, chicken and guinea fowl meats (Table 2). Pork and
reindeer are the most critical species to be tested since they are
considered as the less sensitive targets on the array. Their array
probes actually consist of a mixture of multiple probes to
compensate for single nucleotide polymorphisms (SNPs) present
within the target region of their 16S rRNA genes. Indeed, pork has
already been shown to exhibit a higher mitochondrial genetic
variability within its population than other livestock (Zhang &
Plastow, 2011), while reindeers have been characterised by a high
level of mitochondrial genetic diversity despite their low popula-
tion (Baranova, Kholodova, Davydov, & Rozhkov, 2012).
All the adulterant species, including pork and reindeer, were
successfully detected in all replicates with different signal values,
varying from 50000 to 2500 pv, just above the detection cut-off of
141
2000 pv. This successful detection was also valid for those species
which are represented by capture probes containing multiple se-
quences due to SNPs within the assays target region (pork and
reindeer) as well as for bison which had to be distinguished from its
very close relative B. taurus. The variation of signal intensity
certainly comes from the variability of tissue composition which
brings more or less DNA amount in the sample.
When further diluted down to 0.1% in either beef or chicken, the
detection rate dropped to 65% and below (data not shown). Since
the limit of detection (LOD) is defined as the amount of analyte at
which the analytical method detects the presence of the analyte at
least 95% of the time (Codex Committee on Methods of Analysis and
Sampling, 2010), the LOD of the method can thus be estimated
at  1% (w/w), which is aligned with the European requirement
(European Commission, 2013). This sensitivity corresponds to what
PCR-based methods can usually reach (Kumar et al., 2015), knowing
that for some specific purpose (like Halal or Kosher certifications),
detection of certain meat species like pork at traces level can be
required. The current evaluation focused on a detection at 1% level
to assess potential meat adulteration, and was not dedicated to
detect the absence of certain meat species at low level. However,
the sensitivity of this Meat LCD Array approach could be improved
by increasing the number of PCR cycles or increasing the DNA input
in the PCR reaction. Of course, a more sensitive method would then
detect natural cross-contamination coming from the supply chain
or the slaughterhouses but would not indicate voluntarily adul-
terated meat.
3.3. Applicability on processed samples
Cooking and drying are one of the most common industrial
processes encountered in food industry. To take into consideration
these treatments, both cooked meat and meat powders were
tested.
Twenty different cuts of beef were spiked with 1% pork and 1%
reindeer, and cooked 270 min at 100
C to mimic an intensive in-
dustrial process which can be applied on culinary products and
prepared meals. Pork and reindeer signals were lower than the
ones previously obtained on raw meat, with some very close to the
detection threshold of 2000 pv (Table 2), which can be explained by
DNA fragmentation during the cooking treatment (Hird et al.,
2006). Although degraded, amplification of short mitochondrion
DNA fragments below 150 bp allowed the detection of 95% of the
tested replicates, showing that even with a long heating process,
the 1% meat adulterants-pork and reindeer-were still amplifiable
and detectable.
In addition to cooked meat, powder meat samples were also
analysed. Beef and turkey powders were spiked with 1% pork and
1% chicken powders, respectively and analysed in 20 replicates. In
addition to the beef and turkey signals, both pork and chicken
adulterants were successfully detected in all the replicates.
3.4. Applicability on proficiency test samples
Used as indicators to evaluate the analytical performance of a
method or a laboratory, p-test samples mimic real world samples.
To evaluate the reliability and the routine behaviour of the method,
18 p-test samples composed of raw meat, cooked meat, canned
meat or meat powder were analysed (Table 3). The majority of
them were beef-or chicken-based matrices, pure or spiked with
additional meat species at a level higher than 1% (w/w). Although
the samples did not include all the meat species targeted by the
Meat LCD Array, they allowed testing the detection performance of
the most common ones such as beef, pork, horse, chicken, and
sheep.
142 G. Cottenet et al. / Food Control 67 (2016) 135e143
Results obtained on these p-test samples were all satisfactory
and in agreement with the final p-tests reports. All the spiked meat
species were successfully identified, even when the sample con-
tained 3 or 4 species, which showed the capability of the Meat LCD
Array to simultaneously detect several species, even in the most
processed samples (cooked and canned meat samples). This suc-
cessful evaluation indicates that the described method is adapted
for a routine usage in a meat testing laboratory.
4. Conclusion
This Meat LCD Array now allows simultaneously identifying 32
meat species on 8 samples. It enables the detection of the usual
meat species like beef, pork, and chicken, and also includes more
exotic meat species like springbok, kangaroo, cat, and dog, some of
them recently implicated in economically motivated adulteration
and fraud cases. To our knowledge, this is the first method to allow
such a broad screening of meat species. In addition, the present
evaluation shows that this Meat LCD Array was able to detect ad-
mixtures down to 1% (w/w) in raw meat, in meat powder and
cooked meat, which is compliant with the recent EU Recommen-
dation (European Commission, 2013). Although not evaluated in
this study, this approach could also be applied on milk and milk-
based products, where adulteration and substitution between
cow, goat and buffalo milk have already been described (Cottenet,
Blancpain, & Golay, 2011; Gonçalves, Pereira, Amorim, & Van
Asch, 2012).
Fast, specific, sensitive and easy-to-use, this method is fit-for-
purpose for meat testing laboratories.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We would like to thank Volker Heiser from Chipron GmbH for
his technical and scientific support during our study.
References
Ali, M. E., Razzak, M. A., & Abd Hamid, A. B. (2014). Multiplex PCR in species
authentication: probability and prospectsea review. Food Analytical Methods, 7,
1933e1949.
Aristoy, M. C., & Toldra, F. (2004). Histidine dipeptides HPLC-based test for the
detection of mammalian origin proteins in feeds for ruminants. Meat Science,
67, 211e217.
Ascensio, L., Gonzalez, I., Garcia, T., & Martin, R. (2008). Determination of food
authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control, 19,
1e8.
Ballin, N. Z., Vogensen, F. K., & Karlsson, A. H. (2009). Species determinationeCan
we detect and quantify meat adulteration? Meat Science, 83, 165e174.
Baranova, A. I., Kholodova, M. V., Davydov, A. V., & Rozhkov, L. (2012). Poly-
morphism of the mtDNA control region in wild reindeer Rangifer tarandus
(Mammalia: Artiodactyla) from the European part of Russia. Genetika, 48,
1098e1104.
Bejing, A. P. (2013). Rat meat sold as lamb in latest China food scandal. http://news.
yahoo.com/rat-meat-sold-lamb-latest-china-food-scandal-182459173.html.
Cawthorn, D.-M., Steinman, H. A., & Hoffman, L. C. (2013). A high incidence of
species substitution and mislabeling detected in meat products sold in South
Africa. Food Control, 32, 440e449.
Codex Committee on Methods of Analysis and Sampling. (2010). Guidelines on
performance criteria and validation of methods for detection, identification and
quantification of specific DNA sequences and specific protein in foods. CAC/GL 74-
2010. Rome: Codex Alimentarius-FAO.
Cottenet, G., Blancpain, C., & Golay, P. A. (2011). Simultaneous detection of cow and
buffalo species in milk from China, India, and Pakistan using multiplex real-
time PCR. Journal of Dairy Science, 94, 3787e3793.
Cottenet, G., Blancpain, C., Sonnard, V., & Chuah, P. F. (2013). Development and
validation of a multiplex real-time PCR method to simultaneously detect 47
targets for the identification of genetically modified organisms. Analytical and
Bioanalytical Chemistry, 405, 6831e6844.
D'Amato, M. E., Alechine, E., Cloete, K. W., Davison, S., & Corach, D. (2013). Where is
the game ? Wild meat products authentication in South Africa: a case study.
Investigative Genetics, 4, 6.
Di Pinto, A., Bottaro, M., Bonerba, E., Boszzo, G., Ceci, E., Merchetti, P., et al. (2015).
Occurrence of mislabeling in meat products using DNA-based assay. Journal of
Food Science and Technology, 52, 2479e2484.
Elliott, C. (2014). Elliott review into the integrity and assurance of food supply net-
works: final reportea national food crime prevention framework. Food Standards
Agency accessed 22.04.15 https://www.gov.uk/government/publications/elliott-
review-into-the-integrity-and-assurance-of-food-supply-networks-final-
report.
European Commission. (2013). Recommendation 2013/99/EU on a coordinated control
plan with a view to establish the prevalence of fraudulent practices in the mar-
keting of certain foods accessed 21.04.15 http://eur-lex.europa.eu/legal-content/
EN/TXT/PDF/?uri¼CELEX:32013H0099&qid¼1429713190676&from¼EN.
Everstine, K., Spink, J., & Kennedy, S. (2013). Economically motivated adulteration
(EMA) of food: common characteristics of EMA incidents. Journal of Food Pro-
tection, 76, 723e735.
Fajardo, V., Gonzalez, I., Martin, I., Rojas, M., Hernandez, P. E., Garcia, T., et al. (2008).
Real-time PCR for detection and quantification of red deer (Cervus elaphus),
fallow deer (Dama dama), and roe deer (Capreolus capreolus) in meat mixtures.
Meat Science, 79, 289e298.
Fajardo, V., Gonzalez, I., Rojas, M., Garcia, T., & Martin, R. (2010). A review of current
PCR-based methodologies for the authentication of meats from game animal
species. Trends in Food Science & Technology, 21, 408e421.
Floren, C., Wiedemann, I., Brenig, B., Schütz, E., & Beck, J. (2015). Species identifi-
cation and quantification in meat and meat products using droplet digital PCR
(ddPCR). Food Chemistry, 173, 1054e1058.
Food Standards Agency. (2014). Testing for the verification for the presence of unde-
clared meat species and allergens in lamb products from takeaway outlet. https://
www.food.gov.uk/sites/default/files/lamb-takeaway-finalreport%20-Jan%
202015v2.pdf accessed 05.02.2016.
Garcia, P. T., Pensel, N. A., Sancho, A. M., Latimori, N. J., Kloster, A. M., Amigone, M. A.,
et al. (2008). Beef lipids in relation to animal breed and nutrition in Argentina.
Journal of Meat Science, 79, 500e508.
Gonçalves, J., Pereira, F., Amorim, A., & Van Asch, B. (2012). New method for the
simultaneous identification of cow, sheep, goat and water buffalo in dairy
products by analysis of short species-specific mitochondrial DNA targets. Jour-
nal of Agricultural and Food Chemistry, 60, 10480e10485.
Handy, S. M., Deeds, J. R., Ivanova, N. V., Hebert, P. D., Hanner, R. H., Ormos, A., et al.
(2011). A single-laboratory validated method for the generation of DNA barc-
odes for the identification of fish for regulatory compliance. Journal of AOAC
International, 94, 201e210.
Hird, H., Chisholm, J., Sanchez, A., Hernandez, M., Goodier, R., Schneede, K., et al.
(2006). Effect of heat and pressure processing on DNA fragmentation and im-
plications for the detection of meat using a real-time polymerase chain reac-
tion. Food Additives & Contaminants, 23, 645e650.
Hoffman, L. C., & Cawthorn, D.-M. (2012). What is the role and contribution of meat
from wildlife in providing high quality protein for consumption. Animal Fron-
tiers, 2, 40e53.
Hoffmann, G. S., Johannesen, J., & Griebeler, E. M. (2014). Species cross-
amplification, identification and genetic variation of 17 species of deer (Cervi-
dae) with microsatellite and mitochondrial DNA from antlers. Molecular Biology
Reports, 42, 1059e1067.
Iwobi, A. N., Huber, I., Hauner, G., Miller, A., & Busch, U. (2011). Biochip technology
for the detection of animal species in meat products. Food Analytical Methods, 4,
389e398.
Kane, D. E., & Hellberg, R. S. (2016). Identification of species in ground meat
products sold on the US commercial market using DNA-based methods. Food
Control, 59, 158e163.
Kumar, A., Kumar, R. R., Sharma, B. D., Gokulakrishnan, P., Mendiratta, S. K., &
Sharma, D. (2015). Identification of species origin of meat and meat products on
the DNA basis: a review. Critical Reviews in Food Science and Nutrition, 10,
1340e1351.
Martinez, I., & Friis, T. J. (2004). Application of proteome analysis to seafood
authentication. Proteomics, 4, 347e354.
Murugaiah, C., Noor, Z. M., Mastakim, M., Bilung, L. M., Selamat, J., & Radu, S. (2009).
Meat species identification and Halal authentication analysis using mitochon-
drial DNA. Meat Science, 83, 57e61.
Ratnasingham, S., & Hebert, P. D. N. (2007). Bold: the Barcode of Life Data system.
Molecular Ecology Notes, 7, 355e364. www.barcodinglife.org.
Reuters. (2014). Wal-Mart recalls donkey product in China after fox meat scandal.
http://www.reuters.com/article/2014/01/02/us-walmart-china-
idUSBREA0103O20140102 accessed 05.01.14.
Rohman, A. S., Erwanto, Y., & Man, Y. B. C. (2011). Analysis of pork adulteration in
beef meatball using Fourier transform infrared (FTIR) spectroscopy. Meat Sci-
ence, 88, 91e95.
Ruiz Orduna, A., Husby, E., Yang, C. T., Ghosh, D., & Beaudry, F. (2015). Assessment of
meat authenticity using bioinformatics, targeted peptide biomarkers and high-
resolution mass spectrometry. Food Additives & Contaminants: Part A, 10,
1709e1717.
Scarpeid, H. J., Kraal, K., & Hildrum, K. I. (1998). Identification of animal species in
ground meat mixtures by multivariate analysis of isoelectric focusing protein
profiles. Electrophoresis, 19, 3103e3109.
Vallejo-Cordoba, B., Gonzalez-Cordoba, A., Mazorra-Manzano, M., & Rodriguez-
 G. Cottenet et al. / Food Control 67 (2016) 135e143 143

Ramirez, R. (2005). Capillary electrophoresis for the analysis of meat authen-
ticity. Journal of Separation Science, 28, 826e836.
Wilson, D. E., & Ruff, S. (1999). The Smithsonian book of North American mammals.
Washington DC: Smithsonian Institution press.
Yang, L., Tan, Z., Wang, D., Xue, L., Guan, M. X., Huang, T., et al. (2014). Species
identification through mitochondrial rRNA genetic analysis. Scientific Reports, 4,
4089e4099.
Yosef, T. A., Al-Julaifi, M. Z., & Al-Rizqi, A. M. (2014). Food forensics: using DNA-
based technology for the detection of animal species in meat products. Na-
ture and Science, 12, 82e90.
Zhang, C., & Plastow, G. (2011). Genomic diversity in pig (Sus scrofa) and its com-
parison with human and other livestock. Current Genomics, 12, 138e146.