Food Control 16 (2005) 391–394

www.elsevier.com/locate/foodcont

Duplex polymerase chain reaction for detection of pork meat in

horse meat fresh sausages from Italian retail sources
A. Di Pinto *, V.T. Forte, M.C. Conversano, G.M. Tantillo
Dipartimento di Sanit

a e Benessere degli Animali, Universit
a degli Studi––Bari, Provinciale per Casamassima, km 3, 70010 Valenzano––Bari, Italy
Received 16 February 2004; received in revised form 5 April 2004; accepted 6 April 2004

Abstract
Species identification in meat products represents an important subject in the field of modern food control according to the
European Union, which has implemented a set of very strict procedures to label food. Thus, specific, sensitive and easy analytical
methods for the species detection of food are necessary in order to verify the compliance with labelling requirements. A PCR-based
assay for the detection of pork meat in horse fresh sausages was optimised and it was used to evaluate the presence of fraudulently
added pork meat. The developed assay showed the presence of pork meat in 6/30 and the total absence of horse meat in 1/30 of the
analyzed horse sausage samples.
Ó 2004 Elsevier Ltd. All rights reserved.

Keywords: D-PCR; Species identification; Horse; Pork; Sausages

1. Introduction immunoassay, based on the use of antibodies raised

The identification of meat products species is
important to detect adulteration or fraudulent substi-
tution and to preserve the consumers from the presence
of unknown, less desiderable meat species for economic,
religious and health reasons (Meyer, H€ ofelein, L€
uthy, &
Candrian, 1995). The European Union has implemented
a set of very strict procedures for the labelling of food.
Throughout the whole legislative procedure, the EU
ensures the European consumers’ right to be fully in-
formed. Thus analytical methods for the species detec-
tion of food are necessary in order to verify compliance
with labelling requirements. The control and detection
of such foodstuffs may be based on protein detection
tests using antibodies such as the enzyme-linked
immunosorbent assay (ELISA)-test or DNA-based
tests, such as polymerase chain reaction (PCR).
Methods based on protein detection such as electro-
phoresis, isoelectric focusing (IEF) are characterized by
inaccuracy and may fail in species detection of processed
meat products due to denaturation of soluble proteins
during food processing. Moreover, the analysis by
*
Corresponding author. Tel.: +39-080-5443970; fax: +39-080-
5443855.
E-mail address: a.dipinto@veterinaria.uniba.it (A. Di Pinto).
0956-7135/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2004.04.004
against a specific protein, often presents cross-reaction
with closely related species (Meyer, Candrian, & L€uthy,
1994).
The molecular methods based on PCR have been
proposed as useful tools for the detection of several
animal species (Colombo, Marchisio, Pizzini, & Can-
toni, 2002; Guoli, Mingguang, Zhijiang, Hongsheng, &
Qiang, 1998). They are highly specific, sensitive and
characterized by a rapid processing time and low costs.
However, the presence of inhibitors in foods, in partic-
ular in meat products, can prevent primer binding and
diminish amplification efficiency, so that the extreme
sensitivity achievable by PCR is often reduced when
food is tested (Bottero, Civera, Anastasio, Turi, & Ro-
sati, 2002; Calvo, Zaragoza, & Osta, 2001).
In this paper a Duplex PCR (D-PCR) assay for the
detection of pork meat in Italian horse fresh sausages, a
meat product which requires cooking, was optimised to
evaluate the presence of pork meat added fraudulently
and consequently to verify the concordance with labels.
The approach involves the extraction of DNA with a
procedure in which total DNA is bound on a silica
membrane followed by amplification of a fragment of
the cytochrome b gene of mitochondrial DNA (mt
DNA), a target used largely in species identification
(Hsieh et al., 2001; Lau et al., 1998).
392 A. Di Pinto et al. / Food Control 16 (2005) 391–394
2. Materials and methods
2.1. Controls
Horse (Equus caballus) and pig (Sus scrofa domesti-
cus) DNA (BIOTOOLS B&M Labs, Madrid, Spain)
were used as positive controls. Cow DNA (BIOTOOLS
B&M Labs, Madrid, Spain) was used as negative con-
trol. A 100% pork fresh sausage, a 100% horse fresh
sausage and sausages with different percentage of pork
and horse meat, according to Matsunaga et al. (1999)
were used as positive controls to optimise D-PCR.
2.2. DNA extraction
The sausage samples were subjected to DNA extrac-
tion using Tissue mini kit (QIAGEN, Hilden, Ger-
many). 500 mg of fresh sausage were incubated with 10
ml of lysis buffer ATL (QIAGEN, Hilden, Germany)
with 1 ml proteinase K (20 mg/ml) (QIAGEN, Hilden,
Germany) at 56 °C overnight and then for 1 h at 70 °C
with 10 ml Buffer AL (QIAGEN, Hilden, Germany).
The mixture was centrifuged at 4000g for 2 min and
ethanol was added to the transferred supernatant. The
resulting mixture was applied to the QIAamp DNA spin
column (QIAGEN, Hilden, Germany). The DNA
bound to the column was washed in two centrifugation
steps using two different wash buffers to improve the
purity of the eluted DNA. The purified DNA was then
eluted from the column in 80 ll of Elution Buffer
(QIAGEN, Hilden, Germany). The DNA concentration
and the purity of the eluate were measured by absor-
bance at 260 nm and by calculating the ratio of absor-
bance at 260 nm to absorbance at 280 nm using a
spectrophotometer DU-600 (Beckman, Fullerton, CA).
The DNA eluted was used as template in the PCR assay.
2.3. Oligonucleotide primers
Primers SIM 50 -GAC CTC CCA GCT CCA TCA
AAC ATC TCA TCT TGA TGA AA-30 , PIG 50 -GCT
GAT AGT AGA TTT GTG ATG ACC GTA-30 and
HOR 50 -CTC AGA TTC ACT CGA CGA GGG TAG
TA-30 , previously described and used in this study, were
targeted a 398 bp fragment in pig and a 439 bp fragment
in horse of mitochondrial cytochrome b genes (Matsu-
naga et al., 1999).
2.4. Duplex-PCR (D-PCR) assay
The reaction was performed in a final volume of 25 ll
using 12.5 ll of HotStarTaq Master Mix (QIAGEN,
Hilden, Germany and 0.5 lM of each primer and 20 ng
of DNA. The D-PCR was processed in a Mastercycler
5332 (Eppendorf) with an initial denaturation step of 95
°C for 15 min, followed by 35 cycles of denaturation at
94 °C for 30 s, annealing at 58 °C for 30 s and extension
of 30 s at 72 °C and a final extension at 72 °C for 5 min.
2.5. PCR assay
The PCR assay was carried out to confirm the D-
PCR results. The reaction was performed in a final
volume of 25 ll using 12.5 ll of HotStarTaq Master Mix
(QIAGEN, Hilden, Germany), 0.5 lM of each pig
primers and 20 ng of DNA. The PCR was processed in a
Mastercycler 5332 (Eppendorf) using D-PCR program.
The same reaction was carried out using horse primers.
2.6. Amplified product detection
PCR amplified products were analyzed by electro-
phoresis on 2.5% (w=v) agarose NA (Pharmacia, Upp-
sala, Sweden) gel in 1X TBE buffer containing 0.89 M
Tris, 0.89 M boric acid, 0.02 M EDTA, pH 8.0 (USB,
Cleveland, OH) and visualized by ethidium bromide
staining and a UV transilluminator. A Gene Rulere 100
bp DNA Ladder Plus (MBI Fermentas, Vilnius, Lithu-
ania) consisting of DNA fragments ranging in size from
3000 to 100 bp, was used as marker.
2.7. Testing samples
The procedures were used to test 30 fresh sausage
declared made of pure horse meat collected from dif-
ferent markets in Apulia. The samples, stored at 4 °C,
were transported to the laboratory and processed
immediately. Samples found positive in PCR were sub-
jected to sequence analysis to verify and confirm the
specificity of the PCR products. Sequence analysis of
PCR products was carried out by ABI PRISM 3100
(Applied Biosystem, Rome, Italy).
3. Results
3.1. DNA extraction
The approach followed for DNA extraction has al-
lowed to extract DNA suitable for PCR amplification.
The DNA extraction method, based on the binding of
DNA to a silica matrix in presence of chaotropic agents,
was considered effective and able to remove inhibitors,
which could interfere with PCR reaction.
3.2. D-PCR
The study confirmed the primer specificity and
detection limit according to Matsunaga et al. (1999).
None of the bovine DNA samples gave positive results.
D-PCR procedure to amplify mitochondrial cytochrome
b gene from pig and horse DNA indicates the presence
 A. Di Pinto et al. / Food Control 16 (2005) 391–394 393

of 439 and 398 bp specific amplicons only. These results

refer to ten experiments and did not show any significant
variability.

3.3. PCR assay

The PCR assay carried out to confirm the specificity

of D-PCR products gave the expected results (Fig. 1).

3.4. Testing samples

The survey showed the presence of pork meat in 6/30

(20%) and the total absence of horse meat in 1/30 (3.3%)
of the analyzed horse sausage samples (Fig. 2). The
samples without horse genome presented the 398 bp
amplicon only. The observed intensity of the 439 bp and
398 bp amplicons presented a low variability in all six
positive samples. The D-PCR results were confirmed by
PCR assay. Then, the sequence analysis, compared to
published sequences, showed the specificity of PCR
products.
4. Discussion
Recently the need of further information about the
composition of food products, in particular of meat
products, is increased, so identifying the species of origin
of meat products represents a considerable target for
food inspection, underlined by the enforcement of
community laws. The species identification in foods of
Fig. 1. Electrophoretic profile of PCR products of a horse sausage
sample carried out using pig and horse primer separately. Lane 1: 100
pb DNA Ladder; Lane 2: sample with pig primer, Lane 3: pig positive
control, Lane 4: sample with horse primer, Lane 5: horse positive
control, Lane 6: cow DNA control, Lane 7: negative (no DNA) con-
trol.
Fig. 2. Electrophoretic profile of D-PCR products carried out on
sausage samples. Lane 1: 100 pb DNA Ladder; Lane 2: pig sausage
sample; Lane 3: horse sausage sample; Lane 4: horse and pig sausage
sample; Lane 5: D-PCR positive control; Lane 6: negative (no DNA)
control.
animal origin represents an important subject in the field
of modern food control and new analytical methodol-
ogies are necessary. Actually stringent control measures
require methods that ensure an efficient species identi-
fication in meat products. PCR has been successfully
applied for the differentiation of species in meat prod-
ucts, including cooked products, thanks to its high
specificity and sensitivity as well as to its rapid pro-
cessing time, but it is mainly limited by the presence of
inhibitory substances such as collagen (Rajapaksha,
Thilakaratne, Chandrasiri, & Niroshan, 2002; Sun &
Lin, 2002; Verkaar, Nijman, Boutaga, & Lenstra, 2002).
The study highlights the efficiency of the extraction ap-
proach based on the use of a silica matrix for DNA
extraction guarantying the removal of meat compounds
inhibiting PCR assay and consequently the DNA
amplification. The extraction method is less time-con-
suming and technically demanding than the one previ-
ously described (Matsunaga et al., 1999). The specificity
of the PCR products, confirmed by sequence analysis,
indicated a high specificity of the PCR approach in
accordance with the results obtained by Matsunaga
et al. (1999).
Moreover, the absence of variability of the results
highlighted a high method reproducibility. The sensi-
tivity, the high specificity and the reproducibility of the
approach suggested that it is feasible and ideal for
routine analysis on sausages and meat products. The
results obtained in this study from sausages samples
394 A. Di Pinto et al. / Food Control 16 (2005) 391–394
highlighted that the substitution of meat species is rather
frequent and probably due to both unavoidable con-
tamination and intentional admixture for economic
reasons.
The optimised procedure represents a valid PCR-
based method to test fresh meat products and processed
meat products for fast, accurate results. Although the
PCR system allows a qualitative detection of meat ori-
gin, the developed method offers a valid screening ap-
proach for the meat product controls. It is proved to be
simple and reliable for the species identification and
appears an important tool for the European law
enforcement. Therefore the developments and the
availability of specific quantitative PCR-based methods
for identification of small amounts of DNA are neces-
sary as a support of an efficient surveillance system for
species substitution lacking nowadays. Finally, the
enforcements of legislation guidelines to guarantee
public health associated to the improvements of detec-
tion methodologies (Wolf & L€ uthy, 2001) appear to be
necessary to differentiate between technically inevitable
contamination or intentional admixture.
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