MEAT

SCIENCE
Meat Science 69 (2005) 4752
www.elsevier.com/locate/meatsci

Analysis of raw meats and fats of pigs using polymerase

chain reaction for Halal authentication
A.A. Aida a, Y.B. Che Man
a

a,*

, C.M.V.L. Wong b, A.R. Raha b, R. Son

Department of Food Technology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
Department of Biotechnology, Faculty of Food Science and Biotechnology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Received 18 February 2004; received in revised form 8 June 2004; accepted 8 June 2004

Abstract
A method for species identication from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved
region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted
using Qiagen DNeasy Tissue Kits and subjected to PCR amplication targeting the mt cyt b gene. The genomic DNA from lard
was found to be of good quality and produced clear PCR products on the amplication of the mt cyt b gene of approximately 360
base pairs. To distinguish between species, the amplied PCR products were cut with restriction enzyme BsaJI resulting in porcinespecic restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identication assay yielded excellent results for identication of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for
Halal authentication.
2004 Elsevier Ltd. All rights reserved.
Keywords: Pork; Lard; Cytochrome b; PCR; RFLP

1. Introduction
Consumers are concerned by a variety of issues, such
as food authenticity and adulteration. This has resulted
in increased awareness regarding the composition of
food products. The identity of the ingredients in processed or composite mixtures is not always readily apparent and verication that the components are authentic
and from sources acceptable to the consumers may be required (Lockley & Bardsley, 2000). In most countries,
food manufacturers choose to use lard as a substitute ingredient for oil because it is cheaper and easily available.
Pork and lard are serious matters in the view of some religions such as Islam and Judaism. Biological complications and health risks may be associated with daily
*

Corresponding author. Tel.: +60-3-89468413; fax: +60-389423552.

E-mail address: yaakub@fsb.upm.edu.my (Y.B. Che Man).
0309-1740/$ - see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.meatsci.2004.06.020

intake. In Islam, foods containing pig sources are Haram

(unlawful or prohibited) for Muslims to consume.
Hence, it is an important task for food control laboratories to be able to carry out species dierentiation of
raw materials to be used for industrial food preparation
and the detection of animal species in food products.
This is especially crucial for Halal (lawful or permitted)
authentication, of food products. In order to protect
consumers from fraud and adulteration several analytical approaches have been made to identify animal species in food products. Methods have been developed
based on electrophoresis (Babiker, Glover, & Lawrie,
1981; Kim & Shelef, 1986), isoelectric focusing (Jaussen,
Hagele, Voorpostel, & de Baaij, 1990; King, 1984), chromatography (Ashoor, Moute, & Stiles, 1988; Saeed, Ali,
Abdul Rahman, & Sawaya, 1989), deoxyribonucleic
acid (DNA) hybridization (Chikuni, Ozutsumi,
Koishikawa, & Kato, 1990; Ebbehj, 1991), polymerase
chain reaction (PCR) (Meyer, Hofelein, Luthy, &

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A.A. Aida et al. / Meat Science 69 (2005) 4752

Candrain, 1995) and enzyme-linked immunosorbent assay (ELISA) (Chen, Hsieh, & Brigdman, 1998; Hsieh,
Johnson, Wetzstein, & Green, 1996).
In addition to protein-based techniques, DNA techniques have become very important and are widely used
nowadays. It cannot be denied that there are several advantages of DNA analysis methods: DNA is a relatively
stable molecule allowing analysis of processed and heattreated food products (Beneke & Hagen, 1998; Unseld,
Beyermann, Brandt, & Hiesel, 1995). DNA carries an
organisms genetic information, and the information
content of DNA is greater than protein due to the degeneracy of the genetic code as one goes from DNA to
protein (Wolf, Burgener, Hubner, & Luthy, 2000).
DNA is a remarkably stable molecule allowing its extraction from all kinds of tissue due to the ubiquity of
DNA in every type of cell (Wolf & Luthy, 2001; Wolf
et al., 2000).
PCR proved to be an adequate technique for detection of small amounts of DNA, specically amplifying
a target region of template DNA in a rapid and sensitive manner (Saiki et al., 1988). Many sequences of mitochondrial (mt) (e.g. cytochrome b (cyt b) gene)
(Burgener & Hubner, 1998; Matsunaga et al., 1999) or
genomic DNA have been analysed for various animals
like sh (Cespedes et al., 1999; Quinteiro et al., 1998),
game species (Wolf, Rentsch, & Hubner, 1999b) and
several domestic animals and from caviar (Birstein,
Doukakis, Sorkin, & DeSalle, 1998; Wolf, Hubner, &
Luthy, 1999a) by using either specic or universal primers for inter- and intraspecies relationships in order to
establish the molecular phylogeny (Birstein & Desakke,
1998).
Compared to other techniques for species identication by DNA-based methods, polymerase chain
reaction-restriction fragment length polymorphism
(PCR-RFLP) analysis of mt-DNA has oered the greatest advantage (Bellagamba, Moretti, Comincini, & Valfre`, 2001). The advantages of PCR-RFLP are many: one
universal PCR-primer system in combination with a few
restriction enzymes (RE) can be sucient for species
identication (Meyer et al., 1995). No references are necessary once the restriction patterns of the species of interest have been determined. A careful selection of RE
prevents ambiguous results caused by intraspecies polymorphisms (Wolf et al., 1999b). PCR-RFLP constitutes
a simpler alternative to sequencing for the identication
of genetic variation between and within species (Borgo,
Souly-Crosset, Bouchon, & Gomot, 1996). The analysis
of PCR-RFLP of cyt b fragments has already been successfully applied for species dierentiation in heated and
processed meat products e.g. sausages (Meyer et al.,
1995).
In this study, mt-DNA was chosen as the target of investigations. The objective of this study is to develop a
method for species identication from pork and lard

samples using PCR-RFLP analysis of a conserved region in the mt cyt b gene.

2. Materials and methods

2.1. Samples
Meat and fat samples from sheep, cow, chicken and
pig were used. Sheep, cow and chicken samples were
utilized as controls in this study. The samples were
purchased from a wet market in Sri Kembangan, Petaling Street and Pasar Borong Selangor, Malaysia. The
samples were stored at
20 C before the extraction
of DNA to prevent the enzymatic degradation of
DNA.
2.2. DNA extraction
DNA was extracted from 25 mg of meat and fat samples using the DNeasy Protocol for animal tissue provided with the DNeasy Tissue Kit (Qiagen, Hilden,
Germany). Approximately 25 mg of meat and fat samples was blended using a blender (Braun AG, Frankfurt,
Germany), placed in a 1.5 ml microcentrifuge tube. One
hundred and eighty microlitres ATL buer and 20 ll
Proteinase K were added and mixed by vortexing. The
mixture was incubated at 55 C in a water bath to disperse the sample overnight until the tissue was completely
lysed. The following day, 4 ll of RNase A (100 mg/ml)
was added and incubated for 2 min at room temperature. The mixture was mixed by vortexing for 15 s.
Two hundred microlitres AL buer was added to the
sample, mixed thoroughly by vortexing and incubated
at 70 C for 10 min. Two hundred microlitres ethanol
(96100%) was added to mixture and mixed by vortexing to yield a homogenous solution. The homogenous
solution was pipetted into the DNeasy mini column sitting in a 2 ml collection tube. The homogenous solution
was spun at 12,000g for 1 min. The ow-through and
collection tube was discarded and the DNeasy mini
column was placed in a new 2 ml collection tube. Five
hundred microlitres AW1 buer was added and spun
at 12,000g for 1 min. The ow-through and collection
tube was discarded and the DNeasy mini column was
placed in another 2 ml collection tube. Five hundred
microlitres AW2 buer was added and spun at full speed
for 3 min to dry the DNeasy membrane and then the
ow-through and collection tube was discarded. The
DNeasy mini column was placed in a clean 1.5 ml
microcentrifuge tube. One hundred and fty microlitres
AE buer was pipetted directly onto the DNeasy membrane and incubated at room temperature at 1 min. This
was then spun at 12,000g for 1 min to elute. The DNA
solution was stored at 4 C.

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49

2.3. Oligonucleotide primers

2.5. Restriction fragment length polymorphism analysis

A pair of primers was employed in PCR reaction. The

PCR primers used were CYTb1 (5 0 -CCA TCC AAC
ATC TCA GCA TGA TGA AA-3 0 ) and CYTb2 (5 0 GCC CCT CAG AAT GAT ATT TGT CCT CA-3 0 ),
which were published by Kocher et al. (1989).

Five units/ll of RE BsaJI (New England Biolabs,

Beverly, USA) were applied to 15 ll of amplied
DNA in a nal volume of 20 ll digestion mixture [containing 1x reaction buer (10 mM TrisHCl, 50100
mM NaCl, 10 mM MgCl2 and 1 mM dithiothreitol)]
and were incubated at 60 C overnight for optimal result. Ten microlitres of the digested samples were electrophoresed at constant voltage (100 V) on 2% agarose
gel (Promega, Madison, USA) for about an hour in 1x
TAE buer, pH 8.0 and stained by ethidium bromide.
A 100 bp (Promega, Madison, USA) and 1 kb plus
DNA ladder (New England Biolabs, Beverly, USA)
was used as size reference. The gel photo was taken using the Syngene gel documentation system.

2.4. Polymerase chain reaction amplication of the

cytochrome b gene
Amplication of the mt cyt b gene was performed in a
nal volume of 25 ll containing 30 ng of extracted DNA
which was measured by a spectrophotometer (A 260
nm) (Beckman Coulter, CA, USA), 1x PCR reaction
buer (50 mM KCl, 10 mM TrisHCl, pH 8.3), 25
mM MgCl2, 10 mM dNTPs, 10 pmol of each primer
and 5 units/ll of Taq DNA polymerase (Finnzymes,
Espoo, Finland). Amplication was performed with a
PerkinElmer (Gene Amp PCR system 2400) thermal
cycler according to the following PCR step-cycle program: pre-denaturation of 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 nal cycle for complete
synthesis of elongated DNA molecules. Ten microlitres
of PCR products were electrophoresed at constant voltage (74 V) on 2% agarose gel (Promega, Madison, USA)
for about an hour in 1x TAE buer, pH 8.0 and stained
by ethidium bromide. A 100 bp DNA ladder (Promega,
Madison, USA) was used as size reference. The gel
photo was taken using the Syngene gel documentation
system.

3. Results and discussion

We employed a three step analysis to determine the
identity of meat and fat samples: (i) genomic and mtDNA isolation (ii) mt-DNA is subjected to PCR amplication of the cyt b gene and (iii) the cyt b amplicon is
cut with RE to reveal the RE cutting pattern so that
the identity of the meat and fat source can be revealed.
Additionally, we wanted to determine if enough
DNA could be obtained from fats to carry out PCR amplication.
The quality of the extracted DNA from 25 mg of
meat and fat samples using the DNeasy Protocol for
animal tissue provided with the DNeasy Tissue Kit
(Qiagen) was examined by electrophoretic analysis
through a 1.2% agarose gel (Promega). A band of high
intensity appeared in the lanes (Fig. 1). This showed that

Fig. 1. Electrophoresis analysis of DNA extraction from meat and fat samples. M-1 kb plus DNA ladder; 1, mutton; 2, beef; 3, chicken meat; 4,5,6
and 7, pork; 8, mutton fat; 9, beef fat; 10, chicken fat; 11, 12, 13 and 14, lard.

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A.A. Aida et al. / Meat Science 69 (2005) 4752

a signicantly high yield genomic and mt-DNA extracted

from meat and fat samples can be used as template for
PCR amplication of the cyt b.
Agarose gel electrophoresis of the PCR amplied
products (Fig. 2) from the samples resolved a band of
approximately 360 bp for the detection of cyt b gene.
The result shows that the meat and fat samples produced enough mt-DNA for PCR amplication of the
cyt b gene. The amplication of an approximately
360 bp fragment is consistent with the results reported
by Meyer et al. (1995).
The CYTb1/CYTb2 primers employed are considered to be universal (Kocher et al., 1989) and were
shown to amplify DNA from the sample, which comprises a considerable span of vertebrate evolutionary

diversity. An advantage in employing universal primers

is that it obviates the requirement for an internal control, which is otherwise used to monitor the success of
DNA amplication (Partis et al., 2000).
PCR is a highly sensitive method, which had been
proven by the successful amplication of amplicons.
The primers used were very specic amplifying only a
single band of expected size regardless of the concentration of template mt-DNA extracted from meats and
fats. Negative controls (PCR reaction mixtures without
template DNA) were always employed to make sure
there was no contamination in the PCR system.
Discrimination between species can be achieved by
RE digestion of PCR products, which may generate
RE fragments of dierent sizes which are unique to

Fig. 2. Electrophoresis analysis of cytochrome b PCR products amplied from meat and fat samples. M-100 bp DNA ladder; 1, mutton; 2, beef; 3,
chicken meat; 4,5,6 and 7, pork; 8, mutton fat; 9, cow fat; 10, chicken fat; 11,12,13 and 14, lard; N-negative control (no DNA).

Fig. 3. BsaJI restriction prole of cytochrome b PCR products amplied from meat and fat samples. M1-100 bp DNA ladder; 1, mutton; 2, beef; 3,
chicken meat; 4,5,6 and 7, pork; 10, chicken fat; 11,12,13 and 14, lard; M2-1 kb plus DNA ladder.

A.A. Aida et al. / Meat Science 69 (2005) 4752

the animal type (Kocher et al., 1989; Meyer et al., 1995).

In this study, the PCR products from pork and lard
were digested with RE BsaJI, which generates the expected fragments of 131 and 228 bp (Fig. 3). Thus, the
standard restriction pattern for pork can be generated
by PCR-RFLP using pork meat.

4. Conclusion
A method suitable for routine extraction of genomic
DNA from meat and fat samples has been obtained with
the DNeasy Tissue Kit (Qiagen). Using this kit, the genomic DNA was found to be suitable as PCR templates.
This study shows that the mt-DNA is good enough for
routine detection of cyt b. In comparison of mt-DNA
between meats and fats, both mt-DNA are good enough
as PCR templates. These results indicate that PCRRFLP analysis of cyt b represents a powerful and easy
method for identication of species. It is a potentially reliable technique for detection of pig meat and fat from
other animals for Halal authentication.

Acknowledgements
The authors thank the Universiti Putra Malaysia
(UPM) for providing the funding (IRPA No. 03-0304-0172 EA 001) for this study.

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