Journal of the Science of Food and Agriculture J Sci Food Agric 87:569–572 (2007)

Detection of pig derivatives in

food products for halal authentication by
polymerase chain reaction–restriction
fragment length polymorphism
Azmi A Aida,1 Yaakob B Che Man,2,3∗ Abdul R Raha1 and Radu Son3
1 Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor
2 Instituteof Halal Food, Universiti Putra Malaysia, 43400 Serdang, Selangor
3 Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor

Abstract: A method for detection of the presence of pig derivatives in three types of food products – sausages
and casings, bread and biscuits – using polymerase chain reaction–restriction fragment length polymorphism
(PCR-RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed.
Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food
products were found to be of good quality for the sausages and produced clear PCR products on the amplification
of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing
samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene
was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were
digested with restriction enzyme (RE) BsaJI, resulting in species-specific RFLP. The cyt b PCR-RFLP species
identification assay gave excellent results for detection of pork adulteration in food products and is a potentially
reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication.
 2007 Society of Chemical Industry

Keywords: pork derivatives; food products; cytochrome b; PCR-RFLP

INTRODUCTION can allow the analysis of processed and heat-treated

In the last few years, revelations concerning pork and
lard being mixed in food products have surfaced.
There is an increasing trend in some countries to
mix pork and lard in their food products for the
purposes of adulteration to gain extra profit. Food
products containing pork and lard are of great concern
especially for Islamic religions, as the consumption
of pig sources are haram (unlawful or prohibited).
Consumers nowadays demand higher protection from
falsely labeled food products for economic, ethical or
religious and public health reasons.1
The high demand for transparency in the food
industry has enhanced the development of methods for
the analysis of food ingredients.2 A variety of analyti-
cal methods have been used to detect animal species
in food products, which is crucial for halal authen-
tication. These techniques include deoxyribonucleic
acid (DNA) hybridization,3 – 5 polymerase chain reac-
tion (PCR),6 – 8 DNA sequencing9 and immunological
techniques, as well as isoelectric focusing.10
PCR-based assays have been demonstrated to be
highly specific, sensitive and useful tools for the
detection of animal species when compared with
protein-based assay. They can detect small amounts
of DNA, which is a relatively stable molecule, and
food products.11,12 They are often characterized by
having rapid processing time and low cost.13 Proteins,
however, are thermo-sensitive molecules and are
therefore degraded during the processing of food.14
In our previous study, polymerase chain reac-
tion–restriction fragment length polymorphism (PCR-
RFLP) was shown to be able to detect and differentiate
raw meats and fats of pig samples from other animal
species,15 and the profile can be used as a reference.
The aim of the present study was to develop a method
for detection of pig derivatives in food products using
PCR-RFLP analysis of a conserved region in the mt
cyt b gene which was chosen as target of investigation.
MATERIALS AND METHODS
Samples
Three types of food products – sausages and casings,
bread and biscuits – were used. For sausages, three
types of chicken sausages, three types of beef sausages,
two types of pork sausages, two types of unknown
sausages (non-labeled product) and three types of
unknown casings (non-labeled product) were studied.
Pork sausages were utilized as controls in this study.
For bread four types of bread were used, and for

∗
Correspondence to: Yaakob B Che Man, Institute of Halal Food, Universiti Putra Malaysia, 43400 Serdang, Selangor
E-mail: yaakub@food.upm.edu.my
(Received 9 September 2005; revised version received 7 April 2006; accepted 30 August 2006)
Published online 15 January 2007; DOI: 10.1002/jsfa.2699

 2007 Society of Chemical Industry. J Sci Food Agric 0022–5142/2007/$30.00

AA Aida et al.
biscuits five types of biscuits were used, three of them
being homemade biscuits with 1%, 50% and 100%
lard. The samples were purchased from supermarkets
and hypermarkets in Sri Serdang and Sri Petaling,
Selangor, Malaysia, respectively. The sausage and
casing samples were stored at −20 ◦ C until further
use for the extraction of DNA in order to prevent the
enzymatic degradation of DNA.
DNA extraction
DNA extraction from sausages and the casings samples
were carried out using the DNeasy Tissue Kit
(Qiagen, Hilden, Germany). Meanwhile, for bread
and biscuits samples, the DNeasy Plant Mini
Kit (Qiagen) was used. Both extraction processes
were conducted according to the manufacturer’s
instructions provided.
For extraction of DNA from sausage and casing
samples, approximately 25 mg of each sample was
blended using a blender (Braun AG, Frankfurt,
Germany) and incubated with 180 µL ATL buffer with
20 µL Proteinase K at 55 ◦ C in a water bath to disperse
the sample overnight until the tissue was completely
lysed. The following day, 4 µL RNase A (100 mg
mL−1 ) was added and incubated for 2 min at room
temperature. The mixture was mixed by vortexing for
15 s. A total of 200 µL AL buffer was then added to
the sample and mixed thoroughly by vortexing before
incubation at 70 ◦ C for 10 min. The mixture was
then added to 200 µL ethanol (96–100%) and mixed
by vortexing to yield a homogeneous solution. The
homogeneous solution was pipetted into the DNeasy
mini column placed in a 2 mL collection tube and
subjected to centrifugation at 12 000 × g for 1 min.
The purity of the eluted DNA was improved by two
centrifugation steps of washing the DNA bound to the
column using 500 µL of AW1 buffer and AW2 buffer.
Then, the purified DNA bound to the column was
eluted with 150 µL AE buffer and stored at 4 ◦ C until
further application.
For DNA extraction from bread and biscuit
samples, approximately 20 mg of each sample was
added with 400 µL AP1 buffer and 4 µL RNase A
(100 mg mL−1 ) and mixed by vortexing vigorously.
The mixture was then incubated for 10 min at 65 ◦ C
and was mixed two to three times during incubation
by inverting the tube. The lysate was then added with
130 µL AP2 buffer, mixed and incubated for 5 min on
ice. The lysate was then applied to the QIAshredder
mini spin column (lilac) placed in a 2 mL collection
tube and centrifuged for 2 min at 20 000 × g. The flow-
through fraction was transferred to a new tube without
disturbing the cell debris pellet. The cleared lysate was
added with 1.5 volumes AP3/E buffer and mixed by
pipetting. A total of 650 µL of the mixture was applied
to the DNeasy mini spin column placed in a 2 mL
collection tube and centrifuged for 1 min at 6000 × g.
The DNeasy mini spin column was placed in a new
2 mL collection tube, added with 500 µL AW buffer,
and centrifuged for 1 min at 6000 × g. The DNeasy
570
mini spin column was then added with 500 µL AW
buffer and centrifuged for 2 min at 20 000 × g to dry
the membrane. The DNeasy mini spin column was
then transferred to a 1.5 mL microcentrifuge tube.
A total of 100 µL AE buffer was pipetted directly
onto the DNeasy membrane, incubated for 5 min at
room temperature and then centrifuged for 1 min at
6000 × g to elute.
Oligonucleotide primers
The PCR primers CYTb1 (5 -CCA TCC AAC
ATC TCA GCA TGA TGA AA-3 ) and CYTb2
(5 -GCC CCT CAG AAT GAT ATT TGT CCT
CA-3 ), published by Kocher et al.,16 were used in this
study. The primers targeted an approximately 360 bp
fragment in the mt cyt b gene of pig.
PCR amplification of the cytochrome b gene
Amplification of the targeted gene was performed
in a final volume of 25 µL containing 30 ng of
extracted DNA, 1× PCR reaction buffer (50 mmol
L−1 KCl, 10 mmol L−1 Tris-HCl, pH 8.3), 25 mmol
L−1 MgCl2 , 10 mmol L−1 dNTPs, 10 pmol of each
primer and 5 units µL−1 of Taq DNA polymerase
(Finnzymes, Espoo, Finland). Amplification was
performed with a PerkinElmer (Gene Amp PCR
system 2400) thermal cycler according to the following
PCR step-cycle program: pre-denaturation at 94 ◦ C
for 2 min to completely denature the DNA template,
followed by 35 cycles of denaturation at 94 ◦ C for
5 s, annealing at 55 ◦ C for 30 s, and extension at
72 ◦ C for 40 s. Final extension at 72 ◦ C for 2 min
followed the final cycle for complete synthesis of
elongation DNA molecules. PCR products (10 µL)
were electrophoresed at constant voltage (74 V) on 2%
agarose gel (Promega, Madison, WI, USA) for about
an hour in 1× TAE buffer, pH 8.0, and stained with
ethidium bromide. A 100 bp DNA ladder (Promega,
Madison, WI, USA) was used as size reference. The gel
photo was taken using the Syngene gel documentation
system.
RFLP analysis
RE BsaJI (5 units µL−1 ) (New England Biolabs,
Beverly, MA, USA) was applied to 15 µL of amplified
DNA in a final volume of 20 µL digestion mixture
(containing 1× reaction buffer (10 mmol L−1 Tris-
HCl, 50–100 mmol L−1 NaCl, 10 mmol L−1 MgCl2
and 1 mmol L−1 dithiothreitol)) and incubated at
60 ◦ C overnight for optimal results. Digested samples
(10 µL) were electrophoresed at constant voltage
(100 V) on 2% agarose gel (Promega, Madison, WI,
USA) for about an hour in 1× TAE buffer, pH 8.0,
and stained with ethidium bromide. A 100 bp DNA
marker (Promega, Madison, WI, USA) was used as
size reference. The gel photo was taken using the
Syngene gel documentation system.
J Sci Food Agric 87:569–572 (2007)
DOI: 10.1002/jsfa
 Detection of pig derivatives in food products by PCR-RFLP

RESULTS AND DISCUSSION cyt b primer used is to amplify homologous segments

A rapid and simple method to detect and determine
the presence of pig derivatives in food products has
been developed in this study by employing a three-
step analysis. This involved genomic and mt DNA
isolation, PCR amplification of the mt cyt b gene,
followed by digestion of the cyt b amplicon with RE to
reveal the RE cutting pattern for species identification.
The quality of the extracted DNA from sausage and
the casing, bread and biscuit samples was examined
by electrophoretic analysis through a 1.2% agarose
gel (Promega). A band of high intensity appeared
in the lanes for all sausage samples, including the
unknown sausages (non-labeled product) which could
be used directly as a template for PCR amplification
of the cyt b. However, no genomic DNA was detected
in the casings, probably because the casings were
made of synthetic fibrous material. The genomic
DNA extracted from bread and biscuits was of low
yield and of poor quality, probably because the DNA
was degraded during food processing under high
temperature, but it was still sufficient for use as a
template for PCR amplification of the cyt b.
Agarose gel electrophoresis of the PCR-amplified
products (Fig. 1) from the sausage samples resolved
a band of approximately 360 bp for the detection of
cyt b gene. The result shows that the sausage samples
produced sufficient mt DNA for PCR amplification of
the cyt b gene. The amplification of an approximately
360 bp fragment is consistent with the results reported
by Meyer et al.17 However, no amplification of mt cyt
b gene was produced from bread and biscuit samples,
probably because of absence of the targeted gene (mt
cyt b gene) in the samples.
In this study, the bread samples used contain
vegetable fats. This is a plant-based product, and
hence there is no mt cyt b gene presence since the
of mt DNA from animal species only.16 In biscuits,
failure of the PCR amplification of the targeted gene
could be the result of degraded DNA, insufficient
target DNA or DNA contaminated by inhibitors such
as organic and phenolic compounds.18 Generally, no
DNA is detected in highly heat-treated food products,
hydrolyzed plant proteins, purified lecithin, starch
derivatives or refined oils.19
PCR is a highly specific and sensitive method,
which had been proven by the successful amplification
of minute amounts of template DNA. The primers
used were specific, amplifying only a single band of
the expected amplicon size regardless of the amount
of template mt DNA extracted from food products.
Negative controls (PCR mixtures without template
DNA) were always employed to ensure there was no
contamination in the PCR system.
Detection of genetic variation between species can
be achieved by RE digestion of PCR products, which
may generate RE-digested fragments with different
sizes that are unique to the animal type of the sample
source.16,17 In this study, the PCR products from food
samples which contain pig species digested with RE
BsaJI generated the expected fragments of 131 bp
and 228 bp, consistent with the results reported by
Aida et al.15 (Fig. 2). Thus, the standard restriction-
digested pattern for pork was shown to be ably
generated by PCR-RFLP using DNA extracted from
the pork sausage.
CONCLUSION
A suitable method for routine extraction of genomic
DNA from processed food such as sausages and
the casings, bread and biscuits has been obtained
with the Qiagen DNeasy Tissue Kit and Qiagen

M A1 A2 A3 D1 D2 D3 K1 K2 P1 P2 C1 C2 C3 R1 R2 R3 R4 B1 B2 B3 B4 B5 N

500 bp

≈ 360 bp

Figure 1. Electrophoresis analysis of cytochrome b PCR products amplified from food samples. M: 100 bp DNA ladder; A1, A2 and A3: chicken
sausages of different brands; D1, D2 and D3: beef sausages of different brands; K1 and K2, pork sausages of different brands; P1 and P2, unknown
sausages; C1, C2 and C3, unknown casings; R1, R2, R3 and R4, bread of different brands; B1 and B2, biscuits of different brands; B3: homemade
biscuit with 1% lard; B4: homemade biscuit with 50% lard; B5: homemade biscuit with 100% lard; N: negative control (no DNA).

J Sci Food Agric 87:569–572 (2007) 571

DOI: 10.1002/jsfa
AA Aida et al.
M A1 A2 A3 D1
500 bp
≈ 360 bp
Figure 2. BsaJI restriction profile of cytochrome b PCR products amplified from food samples. M: 100 bp DNA ladder; A1, A2 and A3: chicken
sausages of different brands; D1, D2 and D3: beef sausages with different brands; K1 and K2: pork sausages of different brands; P1 and P2:
unknown products (sausages).
DNeasy Plant Mini Kit, respectively. Using this
kit, the genomic DNA was found to be suitable as
PCR templates in a rapid processing time. This study
showed that the cyt b PCR-RFLP species identification
assay proved to be a suitable, powerful and simple
method for identification of species for sausages.
However, the lard content in bread and biscuit samples
could not be detected. Utilizing RE BsaJI, it is a
potentially reliable, fast and sensitive technique for the
detection of pig derivatives in food products for halal
authentication.
ACKNOWLEDGEMENTS
The authors thank the Universiti Putra Malaysia
(UPM) for providing the funding (IRPA no. 03-03-
04-0172 EA 001) for this study. The authors also
greatly indebted to Mr Cheah Yoke Kqueen and Ms
Lesley Maurice Bilung for their assistance.
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